mutant ryr2 r2474s channel  (Thermo Fisher)


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    Structured Review

    Thermo Fisher mutant ryr2 r2474s channel
    The figure indicates links of <t>ryanodine</t> <t>receptors</t> <t>RyR1–RyR3</t> to neurodegenerative diseases ( , – , , , , ), ventilator-induced diaphragmatic dysfunction ( – ), heart failure ( , , , , , , , , – , ), cardiac arrhythmias ( – , – , , ), skeletal myopathies ( – , , , , ), cancer-associated muscle weakness , age-dependent loss of muscle function , and diabetes . Adapted with permission from Biochimica et Biophysica Acta: Molecular Cell Research .
    Mutant Ryr2 R2474s Channel, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mutant ryr2 r2474s channel/product/Thermo Fisher
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    mutant ryr2 r2474s channel - by Bioz Stars, 2023-01
    86/100 stars

    Images

    1) Product Images from "Targeting ryanodine receptors to treat human diseases"

    Article Title: Targeting ryanodine receptors to treat human diseases

    Journal: The Journal of Clinical Investigation

    doi: 10.1172/JCI162891

    The figure indicates links of ryanodine receptors RyR1–RyR3 to neurodegenerative diseases ( , – , , , , ), ventilator-induced diaphragmatic dysfunction ( – ), heart failure ( , , , , , , , , – , ), cardiac arrhythmias ( – , – , , ), skeletal myopathies ( – , , , , ), cancer-associated muscle weakness , age-dependent loss of muscle function , and diabetes . Adapted with permission from Biochimica et Biophysica Acta: Molecular Cell Research .
    Figure Legend Snippet: The figure indicates links of ryanodine receptors RyR1–RyR3 to neurodegenerative diseases ( , – , , , , ), ventilator-induced diaphragmatic dysfunction ( – ), heart failure ( , , , , , , , , – , ), cardiac arrhythmias ( – , – , , ), skeletal myopathies ( – , , , , ), cancer-associated muscle weakness , age-dependent loss of muscle function , and diabetes . Adapted with permission from Biochimica et Biophysica Acta: Molecular Cell Research .

    Techniques Used:

    The reconstructions show that the CPVT mutant RyR2-R2474S puts the channel into a primed state partway between closed and open, and treatment with the Rycal ARM210 or calmodulin puts the channel back toward the closed state, preventing leak. ( A ) Models of open PKA-phosphorylated RyR2 (Protein Data Bank [PDB; https://www.rcsb.org/ ]: 7U9R; yellow) and closed PKA-phosphorylated RyR2 (PDB: 7U9Q; gray). Arrows show the cytosolic shell of the PKA-phosphorylated RyR2 shifting downward and outward when the channel goes from the closed to the open state. Only the front protomer is colored. The sarcoplasmic reticular membranes are black discs. Conditions include 10 mM ATP, 150 nM free Ca 2+ , and 500 mM xanthine. ( B ) Models of closed PKA-phosphorylated RyR2 (PDB: 7U9Q; gray) and primed PKA-phosphorylated RyR2-R2474S (PDB: 7U9X; magenta), with arrows showing the cytosolic shell of RyR2-R2474S shifting downward and outward compared with closed PKA-phosphorylated RyR2, similar to the structural changes observed for PKA-phosphorylated RyR2 when the channel goes from closed to open. We define this state between closed and open as the primed state. ( C ) Models of primed PKA-phosphorylated RyR2-R2474S (PDB: 7U9X; magenta) and closed PKA-phosphorylated RyR2-R2474S + ARM210 (PDB: 7UA1; cyan). Arrows show the cytosolic shell of PKA-phosphorylated RyR2-R2474S + ARM210 shifting upward and inward compared with RyR2-R2474S, reversing the primed state closer to the closed state. ( D ) Models of primed PKA-phosphorylated RyR2-R2474S (PDB: 7U9X; magenta) and closed PKA-phosphorylated RyR2-R2474S + calmodulin (CaM) (PDB: 7UA3; cyan). Similarly to the Rycal ARM210, CaM reverses the primed state back to the closed state. Reconstructions were adapted with permission from Structure .
    Figure Legend Snippet: The reconstructions show that the CPVT mutant RyR2-R2474S puts the channel into a primed state partway between closed and open, and treatment with the Rycal ARM210 or calmodulin puts the channel back toward the closed state, preventing leak. ( A ) Models of open PKA-phosphorylated RyR2 (Protein Data Bank [PDB; https://www.rcsb.org/ ]: 7U9R; yellow) and closed PKA-phosphorylated RyR2 (PDB: 7U9Q; gray). Arrows show the cytosolic shell of the PKA-phosphorylated RyR2 shifting downward and outward when the channel goes from the closed to the open state. Only the front protomer is colored. The sarcoplasmic reticular membranes are black discs. Conditions include 10 mM ATP, 150 nM free Ca 2+ , and 500 mM xanthine. ( B ) Models of closed PKA-phosphorylated RyR2 (PDB: 7U9Q; gray) and primed PKA-phosphorylated RyR2-R2474S (PDB: 7U9X; magenta), with arrows showing the cytosolic shell of RyR2-R2474S shifting downward and outward compared with closed PKA-phosphorylated RyR2, similar to the structural changes observed for PKA-phosphorylated RyR2 when the channel goes from closed to open. We define this state between closed and open as the primed state. ( C ) Models of primed PKA-phosphorylated RyR2-R2474S (PDB: 7U9X; magenta) and closed PKA-phosphorylated RyR2-R2474S + ARM210 (PDB: 7UA1; cyan). Arrows show the cytosolic shell of PKA-phosphorylated RyR2-R2474S + ARM210 shifting upward and inward compared with RyR2-R2474S, reversing the primed state closer to the closed state. ( D ) Models of primed PKA-phosphorylated RyR2-R2474S (PDB: 7U9X; magenta) and closed PKA-phosphorylated RyR2-R2474S + calmodulin (CaM) (PDB: 7UA3; cyan). Similarly to the Rycal ARM210, CaM reverses the primed state back to the closed state. Reconstructions were adapted with permission from Structure .

    Techniques Used: Mutagenesis

    Left: Negative stain electron microscopic images of the “feet structures” of junctional terminal cisternae vesicles and purified ryanodine receptors first visualized in 1987 (images reproduced with permission from the Journal of Biological Chemistry ; ref. ). By comparing the shape and size of the purified RyR to that of the densities protruding from the surface of the SR vesicles, Fleischer and colleagues concluded that RyR was the foot structure spanning the gap between the terminal cisternae of the SR and T-tubules. But it was not known until later when RyR1 was cloned and functionally expressed that it was indeed the Ca 2+ release channel required for muscle excitation-contraction coupling . Arrowheads denote individual RyR1 channels in the SR membrane; arrows denote purified, isolated individual RyR1 channels. Original magnification, ×140,000. Right: The cryo-EM structure of RyR1 at approximately 2.4 Å resolution; two opposing protomers are shown in the side view (adapted with permission from Structure ; ref. ).
    Figure Legend Snippet: Left: Negative stain electron microscopic images of the “feet structures” of junctional terminal cisternae vesicles and purified ryanodine receptors first visualized in 1987 (images reproduced with permission from the Journal of Biological Chemistry ; ref. ). By comparing the shape and size of the purified RyR to that of the densities protruding from the surface of the SR vesicles, Fleischer and colleagues concluded that RyR was the foot structure spanning the gap between the terminal cisternae of the SR and T-tubules. But it was not known until later when RyR1 was cloned and functionally expressed that it was indeed the Ca 2+ release channel required for muscle excitation-contraction coupling . Arrowheads denote individual RyR1 channels in the SR membrane; arrows denote purified, isolated individual RyR1 channels. Original magnification, ×140,000. Right: The cryo-EM structure of RyR1 at approximately 2.4 Å resolution; two opposing protomers are shown in the side view (adapted with permission from Structure ; ref. ).

    Techniques Used: Staining, Purification, Clone Assay, Isolation, Cryo-EM Sample Prep

    recombinant ryr2  (Thermo Fisher)


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    Thermo Fisher recombinant ryr2
    The figure indicates links of <t>ryanodine</t> <t>receptors</t> <t>RyR1–RyR3</t> to neurodegenerative diseases ( , – , , , , ), ventilator-induced diaphragmatic dysfunction ( – ), heart failure ( , , , , , , , , – , ), cardiac arrhythmias ( – , – , , ), skeletal myopathies ( – , , , , ), cancer-associated muscle weakness , age-dependent loss of muscle function , and diabetes . Adapted with permission from Biochimica et Biophysica Acta: Molecular Cell Research .
    Recombinant Ryr2, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/recombinant ryr2/product/Thermo Fisher
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    recombinant ryr2 - by Bioz Stars, 2023-01
    86/100 stars

    Images

    1) Product Images from "Targeting ryanodine receptors to treat human diseases"

    Article Title: Targeting ryanodine receptors to treat human diseases

    Journal: The Journal of Clinical Investigation

    doi: 10.1172/JCI162891

    The figure indicates links of ryanodine receptors RyR1–RyR3 to neurodegenerative diseases ( , – , , , , ), ventilator-induced diaphragmatic dysfunction ( – ), heart failure ( , , , , , , , , – , ), cardiac arrhythmias ( – , – , , ), skeletal myopathies ( – , , , , ), cancer-associated muscle weakness , age-dependent loss of muscle function , and diabetes . Adapted with permission from Biochimica et Biophysica Acta: Molecular Cell Research .
    Figure Legend Snippet: The figure indicates links of ryanodine receptors RyR1–RyR3 to neurodegenerative diseases ( , – , , , , ), ventilator-induced diaphragmatic dysfunction ( – ), heart failure ( , , , , , , , , – , ), cardiac arrhythmias ( – , – , , ), skeletal myopathies ( – , , , , ), cancer-associated muscle weakness , age-dependent loss of muscle function , and diabetes . Adapted with permission from Biochimica et Biophysica Acta: Molecular Cell Research .

    Techniques Used:

    The reconstructions show that the CPVT mutant RyR2-R2474S puts the channel into a primed state partway between closed and open, and treatment with the Rycal ARM210 or calmodulin puts the channel back toward the closed state, preventing leak. ( A ) Models of open PKA-phosphorylated RyR2 (Protein Data Bank [PDB; https://www.rcsb.org/ ]: 7U9R; yellow) and closed PKA-phosphorylated RyR2 (PDB: 7U9Q; gray). Arrows show the cytosolic shell of the PKA-phosphorylated RyR2 shifting downward and outward when the channel goes from the closed to the open state. Only the front protomer is colored. The sarcoplasmic reticular membranes are black discs. Conditions include 10 mM ATP, 150 nM free Ca 2+ , and 500 mM xanthine. ( B ) Models of closed PKA-phosphorylated RyR2 (PDB: 7U9Q; gray) and primed PKA-phosphorylated RyR2-R2474S (PDB: 7U9X; magenta), with arrows showing the cytosolic shell of RyR2-R2474S shifting downward and outward compared with closed PKA-phosphorylated RyR2, similar to the structural changes observed for PKA-phosphorylated RyR2 when the channel goes from closed to open. We define this state between closed and open as the primed state. ( C ) Models of primed PKA-phosphorylated RyR2-R2474S (PDB: 7U9X; magenta) and closed PKA-phosphorylated RyR2-R2474S + ARM210 (PDB: 7UA1; cyan). Arrows show the cytosolic shell of PKA-phosphorylated RyR2-R2474S + ARM210 shifting upward and inward compared with RyR2-R2474S, reversing the primed state closer to the closed state. ( D ) Models of primed PKA-phosphorylated RyR2-R2474S (PDB: 7U9X; magenta) and closed PKA-phosphorylated RyR2-R2474S + calmodulin (CaM) (PDB: 7UA3; cyan). Similarly to the Rycal ARM210, CaM reverses the primed state back to the closed state. Reconstructions were adapted with permission from Structure .
    Figure Legend Snippet: The reconstructions show that the CPVT mutant RyR2-R2474S puts the channel into a primed state partway between closed and open, and treatment with the Rycal ARM210 or calmodulin puts the channel back toward the closed state, preventing leak. ( A ) Models of open PKA-phosphorylated RyR2 (Protein Data Bank [PDB; https://www.rcsb.org/ ]: 7U9R; yellow) and closed PKA-phosphorylated RyR2 (PDB: 7U9Q; gray). Arrows show the cytosolic shell of the PKA-phosphorylated RyR2 shifting downward and outward when the channel goes from the closed to the open state. Only the front protomer is colored. The sarcoplasmic reticular membranes are black discs. Conditions include 10 mM ATP, 150 nM free Ca 2+ , and 500 mM xanthine. ( B ) Models of closed PKA-phosphorylated RyR2 (PDB: 7U9Q; gray) and primed PKA-phosphorylated RyR2-R2474S (PDB: 7U9X; magenta), with arrows showing the cytosolic shell of RyR2-R2474S shifting downward and outward compared with closed PKA-phosphorylated RyR2, similar to the structural changes observed for PKA-phosphorylated RyR2 when the channel goes from closed to open. We define this state between closed and open as the primed state. ( C ) Models of primed PKA-phosphorylated RyR2-R2474S (PDB: 7U9X; magenta) and closed PKA-phosphorylated RyR2-R2474S + ARM210 (PDB: 7UA1; cyan). Arrows show the cytosolic shell of PKA-phosphorylated RyR2-R2474S + ARM210 shifting upward and inward compared with RyR2-R2474S, reversing the primed state closer to the closed state. ( D ) Models of primed PKA-phosphorylated RyR2-R2474S (PDB: 7U9X; magenta) and closed PKA-phosphorylated RyR2-R2474S + calmodulin (CaM) (PDB: 7UA3; cyan). Similarly to the Rycal ARM210, CaM reverses the primed state back to the closed state. Reconstructions were adapted with permission from Structure .

    Techniques Used: Mutagenesis

    Left: Negative stain electron microscopic images of the “feet structures” of junctional terminal cisternae vesicles and purified ryanodine receptors first visualized in 1987 (images reproduced with permission from the Journal of Biological Chemistry ; ref. ). By comparing the shape and size of the purified RyR to that of the densities protruding from the surface of the SR vesicles, Fleischer and colleagues concluded that RyR was the foot structure spanning the gap between the terminal cisternae of the SR and T-tubules. But it was not known until later when RyR1 was cloned and functionally expressed that it was indeed the Ca 2+ release channel required for muscle excitation-contraction coupling . Arrowheads denote individual RyR1 channels in the SR membrane; arrows denote purified, isolated individual RyR1 channels. Original magnification, ×140,000. Right: The cryo-EM structure of RyR1 at approximately 2.4 Å resolution; two opposing protomers are shown in the side view (adapted with permission from Structure ; ref. ).
    Figure Legend Snippet: Left: Negative stain electron microscopic images of the “feet structures” of junctional terminal cisternae vesicles and purified ryanodine receptors first visualized in 1987 (images reproduced with permission from the Journal of Biological Chemistry ; ref. ). By comparing the shape and size of the purified RyR to that of the densities protruding from the surface of the SR vesicles, Fleischer and colleagues concluded that RyR was the foot structure spanning the gap between the terminal cisternae of the SR and T-tubules. But it was not known until later when RyR1 was cloned and functionally expressed that it was indeed the Ca 2+ release channel required for muscle excitation-contraction coupling . Arrowheads denote individual RyR1 channels in the SR membrane; arrows denote purified, isolated individual RyR1 channels. Original magnification, ×140,000. Right: The cryo-EM structure of RyR1 at approximately 2.4 Å resolution; two opposing protomers are shown in the side view (adapted with permission from Structure ; ref. ).

    Techniques Used: Staining, Purification, Clone Assay, Isolation, Cryo-EM Sample Prep

    ryr1  (Thermo Fisher)


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    Thermo Fisher ryr1
    The figure indicates links of <t>ryanodine</t> <t>receptors</t> <t>RyR1–RyR3</t> to neurodegenerative diseases ( , – , , , , ), ventilator-induced diaphragmatic dysfunction ( – ), heart failure ( , , , , , , , , – , ), cardiac arrhythmias ( – , – , , ), skeletal myopathies ( – , , , , ), cancer-associated muscle weakness , age-dependent loss of muscle function , and diabetes . Adapted with permission from Biochimica et Biophysica Acta: Molecular Cell Research .
    Ryr1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ryr1/product/Thermo Fisher
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    ryr1 - by Bioz Stars, 2023-01
    86/100 stars

    Images

    1) Product Images from "Targeting ryanodine receptors to treat human diseases"

    Article Title: Targeting ryanodine receptors to treat human diseases

    Journal: The Journal of Clinical Investigation

    doi: 10.1172/JCI162891

    The figure indicates links of ryanodine receptors RyR1–RyR3 to neurodegenerative diseases ( , – , , , , ), ventilator-induced diaphragmatic dysfunction ( – ), heart failure ( , , , , , , , , – , ), cardiac arrhythmias ( – , – , , ), skeletal myopathies ( – , , , , ), cancer-associated muscle weakness , age-dependent loss of muscle function , and diabetes . Adapted with permission from Biochimica et Biophysica Acta: Molecular Cell Research .
    Figure Legend Snippet: The figure indicates links of ryanodine receptors RyR1–RyR3 to neurodegenerative diseases ( , – , , , , ), ventilator-induced diaphragmatic dysfunction ( – ), heart failure ( , , , , , , , , – , ), cardiac arrhythmias ( – , – , , ), skeletal myopathies ( – , , , , ), cancer-associated muscle weakness , age-dependent loss of muscle function , and diabetes . Adapted with permission from Biochimica et Biophysica Acta: Molecular Cell Research .

    Techniques Used:

    The reconstructions show that the CPVT mutant RyR2-R2474S puts the channel into a primed state partway between closed and open, and treatment with the Rycal ARM210 or calmodulin puts the channel back toward the closed state, preventing leak. ( A ) Models of open PKA-phosphorylated RyR2 (Protein Data Bank [PDB; https://www.rcsb.org/ ]: 7U9R; yellow) and closed PKA-phosphorylated RyR2 (PDB: 7U9Q; gray). Arrows show the cytosolic shell of the PKA-phosphorylated RyR2 shifting downward and outward when the channel goes from the closed to the open state. Only the front protomer is colored. The sarcoplasmic reticular membranes are black discs. Conditions include 10 mM ATP, 150 nM free Ca 2+ , and 500 mM xanthine. ( B ) Models of closed PKA-phosphorylated RyR2 (PDB: 7U9Q; gray) and primed PKA-phosphorylated RyR2-R2474S (PDB: 7U9X; magenta), with arrows showing the cytosolic shell of RyR2-R2474S shifting downward and outward compared with closed PKA-phosphorylated RyR2, similar to the structural changes observed for PKA-phosphorylated RyR2 when the channel goes from closed to open. We define this state between closed and open as the primed state. ( C ) Models of primed PKA-phosphorylated RyR2-R2474S (PDB: 7U9X; magenta) and closed PKA-phosphorylated RyR2-R2474S + ARM210 (PDB: 7UA1; cyan). Arrows show the cytosolic shell of PKA-phosphorylated RyR2-R2474S + ARM210 shifting upward and inward compared with RyR2-R2474S, reversing the primed state closer to the closed state. ( D ) Models of primed PKA-phosphorylated RyR2-R2474S (PDB: 7U9X; magenta) and closed PKA-phosphorylated RyR2-R2474S + calmodulin (CaM) (PDB: 7UA3; cyan). Similarly to the Rycal ARM210, CaM reverses the primed state back to the closed state. Reconstructions were adapted with permission from Structure .
    Figure Legend Snippet: The reconstructions show that the CPVT mutant RyR2-R2474S puts the channel into a primed state partway between closed and open, and treatment with the Rycal ARM210 or calmodulin puts the channel back toward the closed state, preventing leak. ( A ) Models of open PKA-phosphorylated RyR2 (Protein Data Bank [PDB; https://www.rcsb.org/ ]: 7U9R; yellow) and closed PKA-phosphorylated RyR2 (PDB: 7U9Q; gray). Arrows show the cytosolic shell of the PKA-phosphorylated RyR2 shifting downward and outward when the channel goes from the closed to the open state. Only the front protomer is colored. The sarcoplasmic reticular membranes are black discs. Conditions include 10 mM ATP, 150 nM free Ca 2+ , and 500 mM xanthine. ( B ) Models of closed PKA-phosphorylated RyR2 (PDB: 7U9Q; gray) and primed PKA-phosphorylated RyR2-R2474S (PDB: 7U9X; magenta), with arrows showing the cytosolic shell of RyR2-R2474S shifting downward and outward compared with closed PKA-phosphorylated RyR2, similar to the structural changes observed for PKA-phosphorylated RyR2 when the channel goes from closed to open. We define this state between closed and open as the primed state. ( C ) Models of primed PKA-phosphorylated RyR2-R2474S (PDB: 7U9X; magenta) and closed PKA-phosphorylated RyR2-R2474S + ARM210 (PDB: 7UA1; cyan). Arrows show the cytosolic shell of PKA-phosphorylated RyR2-R2474S + ARM210 shifting upward and inward compared with RyR2-R2474S, reversing the primed state closer to the closed state. ( D ) Models of primed PKA-phosphorylated RyR2-R2474S (PDB: 7U9X; magenta) and closed PKA-phosphorylated RyR2-R2474S + calmodulin (CaM) (PDB: 7UA3; cyan). Similarly to the Rycal ARM210, CaM reverses the primed state back to the closed state. Reconstructions were adapted with permission from Structure .

    Techniques Used: Mutagenesis

    Left: Negative stain electron microscopic images of the “feet structures” of junctional terminal cisternae vesicles and purified ryanodine receptors first visualized in 1987 (images reproduced with permission from the Journal of Biological Chemistry ; ref. ). By comparing the shape and size of the purified RyR to that of the densities protruding from the surface of the SR vesicles, Fleischer and colleagues concluded that RyR was the foot structure spanning the gap between the terminal cisternae of the SR and T-tubules. But it was not known until later when RyR1 was cloned and functionally expressed that it was indeed the Ca 2+ release channel required for muscle excitation-contraction coupling . Arrowheads denote individual RyR1 channels in the SR membrane; arrows denote purified, isolated individual RyR1 channels. Original magnification, ×140,000. Right: The cryo-EM structure of RyR1 at approximately 2.4 Å resolution; two opposing protomers are shown in the side view (adapted with permission from Structure ; ref. ).
    Figure Legend Snippet: Left: Negative stain electron microscopic images of the “feet structures” of junctional terminal cisternae vesicles and purified ryanodine receptors first visualized in 1987 (images reproduced with permission from the Journal of Biological Chemistry ; ref. ). By comparing the shape and size of the purified RyR to that of the densities protruding from the surface of the SR vesicles, Fleischer and colleagues concluded that RyR was the foot structure spanning the gap between the terminal cisternae of the SR and T-tubules. But it was not known until later when RyR1 was cloned and functionally expressed that it was indeed the Ca 2+ release channel required for muscle excitation-contraction coupling . Arrowheads denote individual RyR1 channels in the SR membrane; arrows denote purified, isolated individual RyR1 channels. Original magnification, ×140,000. Right: The cryo-EM structure of RyR1 at approximately 2.4 Å resolution; two opposing protomers are shown in the side view (adapted with permission from Structure ; ref. ).

    Techniques Used: Staining, Purification, Clone Assay, Isolation, Cryo-EM Sample Prep

    ca v 1 1 ryr1 electromechanical coupling  (Thermo Fisher)


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    Structured Review

    Thermo Fisher ca v 1 1 ryr1 electromechanical coupling
    Muscle and Ca V 1.1 organization. A) Muscle to muscle cell perspective. ( left ) Morphology of a segment of a skeletal muscle fiber ( right ). Note the characteristic striated pattern of muscle fibers, which results from highly organized array between sarcolemma, sarcoplasmic reticulum (SR), contractile elements and cytoarchitecture of the fibers. B) Structure of the triad. The cartoon depicts the triad, a specialized membrane-organelle array formed by the T-tubule and two segments of the terminal junctional SR. The T-tubules are infoldings of the sarcolemma that propagate the action potential radially into the fiber. Ca V 1.1 (blue) are located at the T-tubules, working primarily as voltage sensors that initiate the early steps of EC coupling. The SR Ca 2+ release channel, <t>RyR1</t> (brown), is predominantly located on the junctional domain of the SR surface. Typical profiles of triads (cross-sections) contain only two rows of RyR1 associated with alternating tetrads . C) Detailed architecture of the triad with a focus on Ca V 1.1 tetrads and RyR1 arrays as shown in (b). About half of the total RyR1s do not associate with Ca V 1.1, resulting in an alternating pattern of “free” and Ca V 1.1-associated RyR1s. Note: In addition to Ca V 1.1 tetrad (blue) and RyR1 (yellow) Ca 2+ release channels, many other proteins form part of the T-tubule- junctional SR complex (e.g. junctophilin, triadin, junctin, calsequestrin, not indicated here).
    Ca V 1 1 Ryr1 Electromechanical Coupling, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ca v 1 1 ryr1 electromechanical coupling/product/Thermo Fisher
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    ca v 1 1 ryr1 electromechanical coupling - by Bioz Stars, 2023-01
    86/100 stars

    Images

    1) Product Images from "Advances in Ca V 1.1 gating: New insights into permeation and voltage-sensing mechanisms"

    Article Title: Advances in Ca V 1.1 gating: New insights into permeation and voltage-sensing mechanisms

    Journal: Channels

    doi: 10.1080/19336950.2023.2167569

    Muscle and Ca V 1.1 organization. A) Muscle to muscle cell perspective. ( left ) Morphology of a segment of a skeletal muscle fiber ( right ). Note the characteristic striated pattern of muscle fibers, which results from highly organized array between sarcolemma, sarcoplasmic reticulum (SR), contractile elements and cytoarchitecture of the fibers. B) Structure of the triad. The cartoon depicts the triad, a specialized membrane-organelle array formed by the T-tubule and two segments of the terminal junctional SR. The T-tubules are infoldings of the sarcolemma that propagate the action potential radially into the fiber. Ca V 1.1 (blue) are located at the T-tubules, working primarily as voltage sensors that initiate the early steps of EC coupling. The SR Ca 2+ release channel, RyR1 (brown), is predominantly located on the junctional domain of the SR surface. Typical profiles of triads (cross-sections) contain only two rows of RyR1 associated with alternating tetrads . C) Detailed architecture of the triad with a focus on Ca V 1.1 tetrads and RyR1 arrays as shown in (b). About half of the total RyR1s do not associate with Ca V 1.1, resulting in an alternating pattern of “free” and Ca V 1.1-associated RyR1s. Note: In addition to Ca V 1.1 tetrad (blue) and RyR1 (yellow) Ca 2+ release channels, many other proteins form part of the T-tubule- junctional SR complex (e.g. junctophilin, triadin, junctin, calsequestrin, not indicated here).
    Figure Legend Snippet: Muscle and Ca V 1.1 organization. A) Muscle to muscle cell perspective. ( left ) Morphology of a segment of a skeletal muscle fiber ( right ). Note the characteristic striated pattern of muscle fibers, which results from highly organized array between sarcolemma, sarcoplasmic reticulum (SR), contractile elements and cytoarchitecture of the fibers. B) Structure of the triad. The cartoon depicts the triad, a specialized membrane-organelle array formed by the T-tubule and two segments of the terminal junctional SR. The T-tubules are infoldings of the sarcolemma that propagate the action potential radially into the fiber. Ca V 1.1 (blue) are located at the T-tubules, working primarily as voltage sensors that initiate the early steps of EC coupling. The SR Ca 2+ release channel, RyR1 (brown), is predominantly located on the junctional domain of the SR surface. Typical profiles of triads (cross-sections) contain only two rows of RyR1 associated with alternating tetrads . C) Detailed architecture of the triad with a focus on Ca V 1.1 tetrads and RyR1 arrays as shown in (b). About half of the total RyR1s do not associate with Ca V 1.1, resulting in an alternating pattern of “free” and Ca V 1.1-associated RyR1s. Note: In addition to Ca V 1.1 tetrad (blue) and RyR1 (yellow) Ca 2+ release channels, many other proteins form part of the T-tubule- junctional SR complex (e.g. junctophilin, triadin, junctin, calsequestrin, not indicated here).

    Techniques Used:

    Ca V 1.1 structure. Heteromultimeric protein complex of Ca V 1.1. α 1S , β 1a , α 2 δ-1, γ subunits, and SH3 domains of Stac3 are colored in blue, green, Orange, yellow, and purple, respectively. B) Side and upper views of the α 1S subunit, each domain is shown in shades of blue. Red dots indicate Ca 2+ ions. Panels A and B were prepared with Chimera . Protein data bank (PDB) IDs: 5GJV (Ca V 1.1) and 6UY7 (Stac3). STAC3 orientation and position relative to α1 and the β subunits is unknown.
    Figure Legend Snippet: Ca V 1.1 structure. Heteromultimeric protein complex of Ca V 1.1. α 1S , β 1a , α 2 δ-1, γ subunits, and SH3 domains of Stac3 are colored in blue, green, Orange, yellow, and purple, respectively. B) Side and upper views of the α 1S subunit, each domain is shown in shades of blue. Red dots indicate Ca 2+ ions. Panels A and B were prepared with Chimera . Protein data bank (PDB) IDs: 5GJV (Ca V 1.1) and 6UY7 (Stac3). STAC3 orientation and position relative to α1 and the β subunits is unknown.

    Techniques Used:

    Topology of the voltage sensor and selectivity filter domain of Ca V 1.1 α 1S subunit. A) Cartoon of α 1S subunit topology shows four homologous but non-identical domains, each containing six transmembrane helices (S1-S6). S1-S4 represents the voltage sensing domain (VSD, blue) while S6-S6 represents the pore-forming domain (Pore, yellow). Each domain contains an S5-S6 loop (P-Loop) buried in the pore acting as a selectivity filter. Intracellular loops connecting each domain are variable in length. The I–II and II–III loops are critical for EC coupling. Deletion of Exon 29 induces a 19 amino acid shortening of the S3-S4IV extracellular loop (red). Positively charged lysine and arginine within S4 are indicated as a “+” while countercharges within S2 and S3 are indicated as “-“. B) Selectivity filter amino acid sequences of rabbit, human, and zebra fish Ca V 1.1 and human Ca V 2.2. Critical glutamate residues used for Ca 2+ selectivity are indicated in red, while zebra fish P-loop Ca 2+ non-conductive mutations N617D is indicated in cyan. Selectivity filter (SF) sequence is indicated in bold and yellow, while alpha helices within P-loop are indicated in green. C) Top view of rabbit Ca V 1.1 selectivity filter colored as in B. Alpha helices are illustrated as ribbon, while selectivity filter motif is shown as a stick. N617D mutation is indicated in blue as a stick. Two ions can bind the pore, each stabilized by P-loops from opposite domains. D) S4 helices amino acid sequences of rabbit, human, and zebra fish Ca V 1.1 and human Ca V 2.2. Conserved gating charges are indicated in yellow (alignment based on Ref.) . Positively charged amino acids not considered as gating charges are highlighted in pink. E) Side view of rabbit Ca V 1.1 voltage sensing domains. α-helices are illustrated as ribbons, while gating charges, countercharges, and gating charge transfer phenylalanine are illustrated as sticks (in blue, green, and Orange respectively). Note that some helices (i.e. S1) are not fully displayed to facility the visualization of other elements. Panels C and E were created with PyMol  from P07293. rCa V 1.1: uniport IDs P07293; hCa V 1.1: uniport IDs Q13698; zfCa V 1.1a: GenBank accession no. FJ76922; zfCa V 1.1b: GenBank accession no. AY49569; hCa V 2.2: Q00975.
    Figure Legend Snippet: Topology of the voltage sensor and selectivity filter domain of Ca V 1.1 α 1S subunit. A) Cartoon of α 1S subunit topology shows four homologous but non-identical domains, each containing six transmembrane helices (S1-S6). S1-S4 represents the voltage sensing domain (VSD, blue) while S6-S6 represents the pore-forming domain (Pore, yellow). Each domain contains an S5-S6 loop (P-Loop) buried in the pore acting as a selectivity filter. Intracellular loops connecting each domain are variable in length. The I–II and II–III loops are critical for EC coupling. Deletion of Exon 29 induces a 19 amino acid shortening of the S3-S4IV extracellular loop (red). Positively charged lysine and arginine within S4 are indicated as a “+” while countercharges within S2 and S3 are indicated as “-“. B) Selectivity filter amino acid sequences of rabbit, human, and zebra fish Ca V 1.1 and human Ca V 2.2. Critical glutamate residues used for Ca 2+ selectivity are indicated in red, while zebra fish P-loop Ca 2+ non-conductive mutations N617D is indicated in cyan. Selectivity filter (SF) sequence is indicated in bold and yellow, while alpha helices within P-loop are indicated in green. C) Top view of rabbit Ca V 1.1 selectivity filter colored as in B. Alpha helices are illustrated as ribbon, while selectivity filter motif is shown as a stick. N617D mutation is indicated in blue as a stick. Two ions can bind the pore, each stabilized by P-loops from opposite domains. D) S4 helices amino acid sequences of rabbit, human, and zebra fish Ca V 1.1 and human Ca V 2.2. Conserved gating charges are indicated in yellow (alignment based on Ref.) . Positively charged amino acids not considered as gating charges are highlighted in pink. E) Side view of rabbit Ca V 1.1 voltage sensing domains. α-helices are illustrated as ribbons, while gating charges, countercharges, and gating charge transfer phenylalanine are illustrated as sticks (in blue, green, and Orange respectively). Note that some helices (i.e. S1) are not fully displayed to facility the visualization of other elements. Panels C and E were created with PyMol from P07293. rCa V 1.1: uniport IDs P07293; hCa V 1.1: uniport IDs Q13698; zfCa V 1.1a: GenBank accession no. FJ76922; zfCa V 1.1b: GenBank accession no. AY49569; hCa V 2.2: Q00975.

    Techniques Used: Sequencing, Mutagenesis

    Allosteric model for L-type Ca 2+ current and RyR Ca 2+ release. A) Hypothetical structural representation of a Ca V 1.1 tetrad coupled to RyR1 homotetramer. Four α 1S subunits (blue) are opposed to a RyR homotetramer (yellow). Ca V 1.1 has four VSDs that alter their conformation in response to surface transmembranal voltage changes. RyR1 does not have an intrinsic voltage sensing mechanism and relay on the voltage sensing machinery of Ca V 1.1, via mechanical coupling, to release Ca 2+ . The α 1S -RyR1 organization depicted here is hypothetical but based on the model suggested by Samsó et al. . Side and upper views (left and right respectively) in Panel A were created with BioRender and Chimera , PDBs: 5GJW and 5TAL for Ca V 1.1 and RyR1, respectively. B) Allosteric scheme for voltage dependent Ca V 1.1 channel opening and RyR1 activation. Four distinct VSDs (VSDI-IV) within one Ca V 1.1 control Ca V 1.1's pore conformation from close (“C”) to open (“O”) with either all or some VSDs in active (“A”) or resting (“R”) state. In parallel, four distinct Ca V 1.1s, each with four VSDs (i.e. tetrads array) control RyR1 pore conformational change from close (“C”) to open (“O”) with either all or some VSDs in active (“A”) or resting (“R”) states. The probability of each state in VSDs (R or A) is under the influence of the membrane voltage (ΔV). Note that in principle, based on structural evidence [ , , ], it is likely that four independently functioning Ca V 1.1 channels (tetrads) are associated with one RyR giving four sets of four VSDs, requiring a total of 16 VSDs. However, recent fluorometric experiments [ , ] suggest some features and reconsiderations for this model: not all VSDs within Ca V 1.1 contribute equally to gate Cav1.1 pore opening and not all VSDs contribute equally to gate RyR1 Ca 2+ release. How many VSDs per tetrad and which of the four VSDs of Ca V 1.1 are needed for RyR1-mediated Ca 2+ release is unknown.
    Figure Legend Snippet: Allosteric model for L-type Ca 2+ current and RyR Ca 2+ release. A) Hypothetical structural representation of a Ca V 1.1 tetrad coupled to RyR1 homotetramer. Four α 1S subunits (blue) are opposed to a RyR homotetramer (yellow). Ca V 1.1 has four VSDs that alter their conformation in response to surface transmembranal voltage changes. RyR1 does not have an intrinsic voltage sensing mechanism and relay on the voltage sensing machinery of Ca V 1.1, via mechanical coupling, to release Ca 2+ . The α 1S -RyR1 organization depicted here is hypothetical but based on the model suggested by Samsó et al. . Side and upper views (left and right respectively) in Panel A were created with BioRender and Chimera , PDBs: 5GJW and 5TAL for Ca V 1.1 and RyR1, respectively. B) Allosteric scheme for voltage dependent Ca V 1.1 channel opening and RyR1 activation. Four distinct VSDs (VSDI-IV) within one Ca V 1.1 control Ca V 1.1's pore conformation from close (“C”) to open (“O”) with either all or some VSDs in active (“A”) or resting (“R”) state. In parallel, four distinct Ca V 1.1s, each with four VSDs (i.e. tetrads array) control RyR1 pore conformational change from close (“C”) to open (“O”) with either all or some VSDs in active (“A”) or resting (“R”) states. The probability of each state in VSDs (R or A) is under the influence of the membrane voltage (ΔV). Note that in principle, based on structural evidence [ , , ], it is likely that four independently functioning Ca V 1.1 channels (tetrads) are associated with one RyR giving four sets of four VSDs, requiring a total of 16 VSDs. However, recent fluorometric experiments [ , ] suggest some features and reconsiderations for this model: not all VSDs within Ca V 1.1 contribute equally to gate Cav1.1 pore opening and not all VSDs contribute equally to gate RyR1 Ca 2+ release. How many VSDs per tetrad and which of the four VSDs of Ca V 1.1 are needed for RyR1-mediated Ca 2+ release is unknown.

    Techniques Used: Activation Assay

    Functional site-directed fluorometry of Ca V 1.1 S4 signals evaluated in Xenopus Oocytes and muscle fibers. A) Ionic current recording from cut-open oocytes (black) with 2 mM Ba 2+ in the external and superimposed fluorometric signal from each VSDs. Note the overlap of the fluorometric and ionic signal for VSD-I. Mean voltage dependence of the fluorometric signal for each VSD from cut open oocytes voltage clamp in presence of 2 mM Ba 2+ and fitted with a Boltzmann function. Note the differences in voltage dependence and slope of each VSDs fluorometric signal. C) Normalized fluorometric signal recorded from muscle fibers in response to self-propagated action potential by field stimulation and its comparison with optically measured membrane voltage (AP, yellow), action potential-evoked charge movement (Q, gray), Ca 2+ transient (Ca 2+ , black), and estimated SR Ca 2+ release flux (Rel, olive). D) Overlay of normalized fluorometric signals presented in C and kinetics quantification. Note the differences in kinetics for fluorometric signals from different VSDs. Time to peak, rise time, and time to 50% are faster for VSD-II. Panels A and B, and C, reproduced with permission from Refs. . and , respectively. Panel D, unpublished analysis from Ref.
    Figure Legend Snippet: Functional site-directed fluorometry of Ca V 1.1 S4 signals evaluated in Xenopus Oocytes and muscle fibers. A) Ionic current recording from cut-open oocytes (black) with 2 mM Ba 2+ in the external and superimposed fluorometric signal from each VSDs. Note the overlap of the fluorometric and ionic signal for VSD-I. Mean voltage dependence of the fluorometric signal for each VSD from cut open oocytes voltage clamp in presence of 2 mM Ba 2+ and fitted with a Boltzmann function. Note the differences in voltage dependence and slope of each VSDs fluorometric signal. C) Normalized fluorometric signal recorded from muscle fibers in response to self-propagated action potential by field stimulation and its comparison with optically measured membrane voltage (AP, yellow), action potential-evoked charge movement (Q, gray), Ca 2+ transient (Ca 2+ , black), and estimated SR Ca 2+ release flux (Rel, olive). D) Overlay of normalized fluorometric signals presented in C and kinetics quantification. Note the differences in kinetics for fluorometric signals from different VSDs. Time to peak, rise time, and time to 50% are faster for VSD-II. Panels A and B, and C, reproduced with permission from Refs. . and , respectively. Panel D, unpublished analysis from Ref.

    Techniques Used: Functional Assay


    Figure Legend Snippet: Comparison of Ca V 1.1 fluorometric signals obtained using cut-open voltage clamp in Xenopus oocytes or field stimulation in mouse muscle fibers.

    Techniques Used: Microscopy, Construct, Labeling, Expressing, Injection

    ryr1  (Thermo Fisher)


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    Structured Review

    Thermo Fisher ryr1
    Muscle and Ca V 1.1 organization. A) Muscle to muscle cell perspective. ( left ) Morphology of a segment of a skeletal muscle fiber ( right ). Note the characteristic striated pattern of muscle fibers, which results from highly organized array between sarcolemma, sarcoplasmic reticulum (SR), contractile elements and cytoarchitecture of the fibers. B) Structure of the triad. The cartoon depicts the triad, a specialized membrane-organelle array formed by the T-tubule and two segments of the terminal junctional SR. The T-tubules are infoldings of the sarcolemma that propagate the action potential radially into the fiber. Ca V 1.1 (blue) are located at the T-tubules, working primarily as voltage sensors that initiate the early steps of EC coupling. The SR Ca 2+ release channel, <t>RyR1</t> (brown), is predominantly located on the junctional domain of the SR surface. Typical profiles of triads (cross-sections) contain only two rows of RyR1 associated with alternating tetrads . C) Detailed architecture of the triad with a focus on Ca V 1.1 tetrads and RyR1 arrays as shown in (b). About half of the total RyR1s do not associate with Ca V 1.1, resulting in an alternating pattern of “free” and Ca V 1.1-associated RyR1s. Note: In addition to Ca V 1.1 tetrad (blue) and RyR1 (yellow) Ca 2+ release channels, many other proteins form part of the T-tubule- junctional SR complex (e.g. junctophilin, triadin, junctin, calsequestrin, not indicated here).
    Ryr1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Advances in Ca V 1.1 gating: New insights into permeation and voltage-sensing mechanisms"

    Article Title: Advances in Ca V 1.1 gating: New insights into permeation and voltage-sensing mechanisms

    Journal: Channels

    doi: 10.1080/19336950.2023.2167569

    Muscle and Ca V 1.1 organization. A) Muscle to muscle cell perspective. ( left ) Morphology of a segment of a skeletal muscle fiber ( right ). Note the characteristic striated pattern of muscle fibers, which results from highly organized array between sarcolemma, sarcoplasmic reticulum (SR), contractile elements and cytoarchitecture of the fibers. B) Structure of the triad. The cartoon depicts the triad, a specialized membrane-organelle array formed by the T-tubule and two segments of the terminal junctional SR. The T-tubules are infoldings of the sarcolemma that propagate the action potential radially into the fiber. Ca V 1.1 (blue) are located at the T-tubules, working primarily as voltage sensors that initiate the early steps of EC coupling. The SR Ca 2+ release channel, RyR1 (brown), is predominantly located on the junctional domain of the SR surface. Typical profiles of triads (cross-sections) contain only two rows of RyR1 associated with alternating tetrads . C) Detailed architecture of the triad with a focus on Ca V 1.1 tetrads and RyR1 arrays as shown in (b). About half of the total RyR1s do not associate with Ca V 1.1, resulting in an alternating pattern of “free” and Ca V 1.1-associated RyR1s. Note: In addition to Ca V 1.1 tetrad (blue) and RyR1 (yellow) Ca 2+ release channels, many other proteins form part of the T-tubule- junctional SR complex (e.g. junctophilin, triadin, junctin, calsequestrin, not indicated here).
    Figure Legend Snippet: Muscle and Ca V 1.1 organization. A) Muscle to muscle cell perspective. ( left ) Morphology of a segment of a skeletal muscle fiber ( right ). Note the characteristic striated pattern of muscle fibers, which results from highly organized array between sarcolemma, sarcoplasmic reticulum (SR), contractile elements and cytoarchitecture of the fibers. B) Structure of the triad. The cartoon depicts the triad, a specialized membrane-organelle array formed by the T-tubule and two segments of the terminal junctional SR. The T-tubules are infoldings of the sarcolemma that propagate the action potential radially into the fiber. Ca V 1.1 (blue) are located at the T-tubules, working primarily as voltage sensors that initiate the early steps of EC coupling. The SR Ca 2+ release channel, RyR1 (brown), is predominantly located on the junctional domain of the SR surface. Typical profiles of triads (cross-sections) contain only two rows of RyR1 associated with alternating tetrads . C) Detailed architecture of the triad with a focus on Ca V 1.1 tetrads and RyR1 arrays as shown in (b). About half of the total RyR1s do not associate with Ca V 1.1, resulting in an alternating pattern of “free” and Ca V 1.1-associated RyR1s. Note: In addition to Ca V 1.1 tetrad (blue) and RyR1 (yellow) Ca 2+ release channels, many other proteins form part of the T-tubule- junctional SR complex (e.g. junctophilin, triadin, junctin, calsequestrin, not indicated here).

    Techniques Used:

    Allosteric model for L-type Ca 2+ current and RyR Ca 2+ release. A) Hypothetical structural representation of a Ca V 1.1 tetrad coupled to RyR1 homotetramer. Four α 1S subunits (blue) are opposed to a RyR homotetramer (yellow). Ca V 1.1 has four VSDs that alter their conformation in response to surface transmembranal voltage changes. RyR1 does not have an intrinsic voltage sensing mechanism and relay on the voltage sensing machinery of Ca V 1.1, via mechanical coupling, to release Ca 2+ . The α 1S -RyR1 organization depicted here is hypothetical but based on the model suggested by Samsó et al. . Side and upper views (left and right respectively) in Panel A were created with BioRender and Chimera , PDBs: 5GJW and 5TAL for Ca V 1.1 and RyR1, respectively. B) Allosteric scheme for voltage dependent Ca V 1.1 channel opening and RyR1 activation. Four distinct VSDs (VSDI-IV) within one Ca V 1.1 control Ca V 1.1's pore conformation from close (“C”) to open (“O”) with either all or some VSDs in active (“A”) or resting (“R”) state. In parallel, four distinct Ca V 1.1s, each with four VSDs (i.e. tetrads array) control RyR1 pore conformational change from close (“C”) to open (“O”) with either all or some VSDs in active (“A”) or resting (“R”) states. The probability of each state in VSDs (R or A) is under the influence of the membrane voltage (ΔV). Note that in principle, based on structural evidence [ , , ], it is likely that four independently functioning Ca V 1.1 channels (tetrads) are associated with one RyR giving four sets of four VSDs, requiring a total of 16 VSDs. However, recent fluorometric experiments [ , ] suggest some features and reconsiderations for this model: not all VSDs within Ca V 1.1 contribute equally to gate Cav1.1 pore opening and not all VSDs contribute equally to gate RyR1 Ca 2+ release. How many VSDs per tetrad and which of the four VSDs of Ca V 1.1 are needed for RyR1-mediated Ca 2+ release is unknown.
    Figure Legend Snippet: Allosteric model for L-type Ca 2+ current and RyR Ca 2+ release. A) Hypothetical structural representation of a Ca V 1.1 tetrad coupled to RyR1 homotetramer. Four α 1S subunits (blue) are opposed to a RyR homotetramer (yellow). Ca V 1.1 has four VSDs that alter their conformation in response to surface transmembranal voltage changes. RyR1 does not have an intrinsic voltage sensing mechanism and relay on the voltage sensing machinery of Ca V 1.1, via mechanical coupling, to release Ca 2+ . The α 1S -RyR1 organization depicted here is hypothetical but based on the model suggested by Samsó et al. . Side and upper views (left and right respectively) in Panel A were created with BioRender and Chimera , PDBs: 5GJW and 5TAL for Ca V 1.1 and RyR1, respectively. B) Allosteric scheme for voltage dependent Ca V 1.1 channel opening and RyR1 activation. Four distinct VSDs (VSDI-IV) within one Ca V 1.1 control Ca V 1.1's pore conformation from close (“C”) to open (“O”) with either all or some VSDs in active (“A”) or resting (“R”) state. In parallel, four distinct Ca V 1.1s, each with four VSDs (i.e. tetrads array) control RyR1 pore conformational change from close (“C”) to open (“O”) with either all or some VSDs in active (“A”) or resting (“R”) states. The probability of each state in VSDs (R or A) is under the influence of the membrane voltage (ΔV). Note that in principle, based on structural evidence [ , , ], it is likely that four independently functioning Ca V 1.1 channels (tetrads) are associated with one RyR giving four sets of four VSDs, requiring a total of 16 VSDs. However, recent fluorometric experiments [ , ] suggest some features and reconsiderations for this model: not all VSDs within Ca V 1.1 contribute equally to gate Cav1.1 pore opening and not all VSDs contribute equally to gate RyR1 Ca 2+ release. How many VSDs per tetrad and which of the four VSDs of Ca V 1.1 are needed for RyR1-mediated Ca 2+ release is unknown.

    Techniques Used: Activation Assay

    cam ryr2 recognition mechanism  (Thermo Fisher)


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    Thermo Fisher cam ryr2 recognition mechanism
    Tertiary and quaternary structure of CaM variants in complex with <t>RyR1/2</t> peptides. (A) Cartoon representation of CaM in three different conformational states, namely apo (PDB: 1DMO ), Ca 2+ -bound (PDB: 1CLL ) and complexed with the <t>RyR2</t> peptide used in this study (PDB: 6Y4O ). The N-terminal domain is colored in yellow, the linker region in grey, while the C-terminal domain in green. The RyR2 peptide is represented in magenta. Ca 2+ ions are represented as red spheres while the side chains of N97 and Q135 are represented in sticks with C atoms colored according to the structural region, O atoms in red and N atoms in blue. (B) Pairwise sequence alignment of the Calmodulin Binding Domain-2 of human RyR1 (Uniprot entry P21817) and RyR2 (Uniprot entry Q92736). The sequence relative to the two RyR peptides employed in this study is highlighted in yellow, the residues that are not identical in such region are represented in bold and colored in red. (C) Near-UV CD spectra (250–320 nm) of 50 μM CaM were collected in the presence of 500 µM EGTA (black dashed line) and after sequential additions of 1 mM Ca 2+ (black solid line, 500 µM free Ca 2+ ) and 100 μM RyR peptides (blue solid line for RyR1, red solid line for RyR2). The spectrum of sole buffer was considered as blank and subtracted; each curve represents the average of five accumulations. Temperature was set at 25°C and signal was normalized to protein concentration.
    Cam Ryr2 Recognition Mechanism, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Calmodulin variants associated with congenital arrhythmia impair selectivity for ryanodine receptors"

    Article Title: Calmodulin variants associated with congenital arrhythmia impair selectivity for ryanodine receptors

    Journal: Frontiers in Molecular Biosciences

    doi: 10.3389/fmolb.2022.1100992

    Tertiary and quaternary structure of CaM variants in complex with RyR1/2 peptides. (A) Cartoon representation of CaM in three different conformational states, namely apo (PDB: 1DMO ), Ca 2+ -bound (PDB: 1CLL ) and complexed with the RyR2 peptide used in this study (PDB: 6Y4O ). The N-terminal domain is colored in yellow, the linker region in grey, while the C-terminal domain in green. The RyR2 peptide is represented in magenta. Ca 2+ ions are represented as red spheres while the side chains of N97 and Q135 are represented in sticks with C atoms colored according to the structural region, O atoms in red and N atoms in blue. (B) Pairwise sequence alignment of the Calmodulin Binding Domain-2 of human RyR1 (Uniprot entry P21817) and RyR2 (Uniprot entry Q92736). The sequence relative to the two RyR peptides employed in this study is highlighted in yellow, the residues that are not identical in such region are represented in bold and colored in red. (C) Near-UV CD spectra (250–320 nm) of 50 μM CaM were collected in the presence of 500 µM EGTA (black dashed line) and after sequential additions of 1 mM Ca 2+ (black solid line, 500 µM free Ca 2+ ) and 100 μM RyR peptides (blue solid line for RyR1, red solid line for RyR2). The spectrum of sole buffer was considered as blank and subtracted; each curve represents the average of five accumulations. Temperature was set at 25°C and signal was normalized to protein concentration.
    Figure Legend Snippet: Tertiary and quaternary structure of CaM variants in complex with RyR1/2 peptides. (A) Cartoon representation of CaM in three different conformational states, namely apo (PDB: 1DMO ), Ca 2+ -bound (PDB: 1CLL ) and complexed with the RyR2 peptide used in this study (PDB: 6Y4O ). The N-terminal domain is colored in yellow, the linker region in grey, while the C-terminal domain in green. The RyR2 peptide is represented in magenta. Ca 2+ ions are represented as red spheres while the side chains of N97 and Q135 are represented in sticks with C atoms colored according to the structural region, O atoms in red and N atoms in blue. (B) Pairwise sequence alignment of the Calmodulin Binding Domain-2 of human RyR1 (Uniprot entry P21817) and RyR2 (Uniprot entry Q92736). The sequence relative to the two RyR peptides employed in this study is highlighted in yellow, the residues that are not identical in such region are represented in bold and colored in red. (C) Near-UV CD spectra (250–320 nm) of 50 μM CaM were collected in the presence of 500 µM EGTA (black dashed line) and after sequential additions of 1 mM Ca 2+ (black solid line, 500 µM free Ca 2+ ) and 100 μM RyR peptides (blue solid line for RyR1, red solid line for RyR2). The spectrum of sole buffer was considered as blank and subtracted; each curve represents the average of five accumulations. Temperature was set at 25°C and signal was normalized to protein concentration.

    Techniques Used: Sequencing, Binding Assay, Protein Concentration

    Investigation of secondary structure of CaM variants and their RyR1/2 peptide complexes. Far-UV CD spectra (200–250 nm) of 10 μM CaM variants alone (top panels, black), and incubated with 20 μM RyR1 (center panels, blue), or 20 μM RyR2 (bottom panels, red), were collected in the presence of 300 µM EGTA (dashed lines) and after the addition of 600 µM Ca 2+ (300 µM free Ca 2+ , solid lines). The spectrum of sole buffer was considered as blank and subtracted; each curve represents the average of five accumulations. Temperature was set at 25°C.
    Figure Legend Snippet: Investigation of secondary structure of CaM variants and their RyR1/2 peptide complexes. Far-UV CD spectra (200–250 nm) of 10 μM CaM variants alone (top panels, black), and incubated with 20 μM RyR1 (center panels, blue), or 20 μM RyR2 (bottom panels, red), were collected in the presence of 300 µM EGTA (dashed lines) and after the addition of 600 µM Ca 2+ (300 µM free Ca 2+ , solid lines). The spectrum of sole buffer was considered as blank and subtracted; each curve represents the average of five accumulations. Temperature was set at 25°C.

    Techniques Used: Incubation

    CaM affinity for RyR1 and RyR2 peptides assessed by fluorescence spectroscopy. (A) One micromolar RyR1 (left column) or RyR2 (right column) peptide was incubated with increasing amounts of WT (top row), N97I (middle row) and Q135P (bottom row) CaM in the presence of 100 µM Ca 2+ . Data are reported as a function of the peptide fraction bound to CaM (see Materials and Methods for details). Curves report the mean ± std of each point obtained in three technical replicas. Representative fitting to one-site saturation ligand binding curve is superposed to each titration set. (B) Scatter plot of replicates reporting the K D values calculated from fitting procedures in each individual dataset using a one-site binding model. Stars represent t -test statistical significance: * p -value ≤ 0.05, ** p -value ≤ 0.01.
    Figure Legend Snippet: CaM affinity for RyR1 and RyR2 peptides assessed by fluorescence spectroscopy. (A) One micromolar RyR1 (left column) or RyR2 (right column) peptide was incubated with increasing amounts of WT (top row), N97I (middle row) and Q135P (bottom row) CaM in the presence of 100 µM Ca 2+ . Data are reported as a function of the peptide fraction bound to CaM (see Materials and Methods for details). Curves report the mean ± std of each point obtained in three technical replicas. Representative fitting to one-site saturation ligand binding curve is superposed to each titration set. (B) Scatter plot of replicates reporting the K D values calculated from fitting procedures in each individual dataset using a one-site binding model. Stars represent t -test statistical significance: * p -value ≤ 0.05, ** p -value ≤ 0.01.

    Techniques Used: Fluorescence, Spectroscopy, Incubation, Ligand Binding Assay, Titration, Binding Assay

    Apparent affinities of CaM-RyR1/2 complexes assessed by fluorescence spectroscopy.
    Figure Legend Snippet: Apparent affinities of CaM-RyR1/2 complexes assessed by fluorescence spectroscopy.

    Techniques Used: Fluorescence, Spectroscopy

    Thermodynamics of CaM-RyR1/2 peptide interaction assessed by isothermal titration calorimetry. (A) Examples of ITC titration curves obtained for each CaM variant upon interaction with RyR1 or RyR2 peptides. Measurements were performed at 25°C using 20 mM Tris pH 7.5, 150 mM KCl, 5 mM Ca 2+ as working buffer and setting stirring at 750 rpm. Each titration consisted in thirty 1-µL injections of 125 µM RyR1 or RyR2 (into the titrant syringe) with 10 µM CaM variants. (B) Scatter plot of replicates summarizing the K D values calculated from the fitting using a one-site binding model (see Materials and Methods). Stars represent the p -values: * p ≤ 0.05, *** p ≤ 0.001. Data for WT CaM titration with RyR2 are from .
    Figure Legend Snippet: Thermodynamics of CaM-RyR1/2 peptide interaction assessed by isothermal titration calorimetry. (A) Examples of ITC titration curves obtained for each CaM variant upon interaction with RyR1 or RyR2 peptides. Measurements were performed at 25°C using 20 mM Tris pH 7.5, 150 mM KCl, 5 mM Ca 2+ as working buffer and setting stirring at 750 rpm. Each titration consisted in thirty 1-µL injections of 125 µM RyR1 or RyR2 (into the titrant syringe) with 10 µM CaM variants. (B) Scatter plot of replicates summarizing the K D values calculated from the fitting using a one-site binding model (see Materials and Methods). Stars represent the p -values: * p ≤ 0.05, *** p ≤ 0.001. Data for WT CaM titration with RyR2 are from .

    Techniques Used: Isothermal Titration Calorimetry, Titration, Variant Assay, Binding Assay

    Thermodynamics of CaM-RyR1/2 peptide association assessed by isothermal titration calorimetry.
    Figure Legend Snippet: Thermodynamics of CaM-RyR1/2 peptide association assessed by isothermal titration calorimetry.

    Techniques Used: Titration

    Kinetics of CaM-RyR1/2 peptide interaction investigated by surface plasmon resonance. (A) Sensorgrams collected by flowing different amounts of RyR1 and RyR2 (125 nM—2 µM) on immobilized His-CaM variants using 20 mM Tris pH 7.5, 150 mM KCl, 0.005% Tween 20, 5 mM Ca 2+ , 100 µM DTT as a running buffer. Association and dissociation phases were followed for 60 s and 300 s, respectively. Experimental curves (black solid lines) are shown together with theoretical curves (red or blue solid lines) according to a 1:1 Langmuir binding model; fitting for association and dissociation phases led to the rate constants ( k on and k off ) reported in each panel (mean ± s.e.m. of 8-20 independent binding curves). (B) Scatter plot of replicates and statistical analysis comparing rate constants for WT and each pathogenic CaM variant. Stars represent p -values: * p ≤ 0.05, ** p ≤ 0.01, **** p ≤ 0.0001.
    Figure Legend Snippet: Kinetics of CaM-RyR1/2 peptide interaction investigated by surface plasmon resonance. (A) Sensorgrams collected by flowing different amounts of RyR1 and RyR2 (125 nM—2 µM) on immobilized His-CaM variants using 20 mM Tris pH 7.5, 150 mM KCl, 0.005% Tween 20, 5 mM Ca 2+ , 100 µM DTT as a running buffer. Association and dissociation phases were followed for 60 s and 300 s, respectively. Experimental curves (black solid lines) are shown together with theoretical curves (red or blue solid lines) according to a 1:1 Langmuir binding model; fitting for association and dissociation phases led to the rate constants ( k on and k off ) reported in each panel (mean ± s.e.m. of 8-20 independent binding curves). (B) Scatter plot of replicates and statistical analysis comparing rate constants for WT and each pathogenic CaM variant. Stars represent p -values: * p ≤ 0.05, ** p ≤ 0.01, **** p ≤ 0.0001.

    Techniques Used: SPR Assay, Binding Assay, Variant Assay

    Backbone flexibility of CaM-RyR1/2 complexes. Cα-Root-Mean Square Fluctuation (RMSF) of CaM (top panels) and RyR1 or RyR2 peptides (bottom panels) calculated over 1.2 μs MD simulations of the respective complex (WT: black, N97I: blue, and Q135P: red).
    Figure Legend Snippet: Backbone flexibility of CaM-RyR1/2 complexes. Cα-Root-Mean Square Fluctuation (RMSF) of CaM (top panels) and RyR1 or RyR2 peptides (bottom panels) calculated over 1.2 μs MD simulations of the respective complex (WT: black, N97I: blue, and Q135P: red).

    Techniques Used:

    Effects of CaM variants on the connectivity of hub residues of CaM (top panels) and RyR1/2 peptides (bottom panels) . Hubs were defined as residues with degree ≥6 in the Protein Structure Network (PSN) of at least one variant. ∆Degree is calculated as the difference in hub degree between the variant (N97I: black, Q135P: red) and the WT.
    Figure Legend Snippet: Effects of CaM variants on the connectivity of hub residues of CaM (top panels) and RyR1/2 peptides (bottom panels) . Hubs were defined as residues with degree ≥6 in the Protein Structure Network (PSN) of at least one variant. ∆Degree is calculated as the difference in hub degree between the variant (N97I: black, Q135P: red) and the WT.

    Techniques Used: Variant Assay

    Robustness of intramolecular communication between EF-hands in CaM-RyR1/2 complexes. (A) Intramolecular communication among EF-hands in the CaM-RyR1 (left) and CaM-RyR2 (right) complexes. Communication robustness between EF-hands (EF1 to EF4, represented by their bidentate Glu residues) in CaM WT (black), N97I (red), and Q135P (green) variants in complex with RyR peptides. (B) Effects of specific RyR peptides on the intramolecular communication among EF-hands in CaM-RyR complexes. Communication robustness between EF-hands (EF1 to EF4, represented by their bidentate Glu residues) in CaM variants in complex with RyR1 (black) and RyR2 (red) peptides.
    Figure Legend Snippet: Robustness of intramolecular communication between EF-hands in CaM-RyR1/2 complexes. (A) Intramolecular communication among EF-hands in the CaM-RyR1 (left) and CaM-RyR2 (right) complexes. Communication robustness between EF-hands (EF1 to EF4, represented by their bidentate Glu residues) in CaM WT (black), N97I (red), and Q135P (green) variants in complex with RyR peptides. (B) Effects of specific RyR peptides on the intramolecular communication among EF-hands in CaM-RyR complexes. Communication robustness between EF-hands (EF1 to EF4, represented by their bidentate Glu residues) in CaM variants in complex with RyR1 (black) and RyR2 (red) peptides.

    Techniques Used:

    ryr2 gating  (Thermo Fisher)


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    Structured Review

    Thermo Fisher ryr2 gating
    Tertiary and quaternary structure of CaM variants in complex with <t>RyR1/2</t> peptides. (A) Cartoon representation of CaM in three different conformational states, namely apo (PDB: 1DMO ), Ca 2+ -bound (PDB: 1CLL ) and complexed with the <t>RyR2</t> peptide used in this study (PDB: 6Y4O ). The N-terminal domain is colored in yellow, the linker region in grey, while the C-terminal domain in green. The RyR2 peptide is represented in magenta. Ca 2+ ions are represented as red spheres while the side chains of N97 and Q135 are represented in sticks with C atoms colored according to the structural region, O atoms in red and N atoms in blue. (B) Pairwise sequence alignment of the Calmodulin Binding Domain-2 of human RyR1 (Uniprot entry P21817) and RyR2 (Uniprot entry Q92736). The sequence relative to the two RyR peptides employed in this study is highlighted in yellow, the residues that are not identical in such region are represented in bold and colored in red. (C) Near-UV CD spectra (250–320 nm) of 50 μM CaM were collected in the presence of 500 µM EGTA (black dashed line) and after sequential additions of 1 mM Ca 2+ (black solid line, 500 µM free Ca 2+ ) and 100 μM RyR peptides (blue solid line for RyR1, red solid line for RyR2). The spectrum of sole buffer was considered as blank and subtracted; each curve represents the average of five accumulations. Temperature was set at 25°C and signal was normalized to protein concentration.
    Ryr2 Gating, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86/100 stars

    Images

    1) Product Images from "Calmodulin variants associated with congenital arrhythmia impair selectivity for ryanodine receptors"

    Article Title: Calmodulin variants associated with congenital arrhythmia impair selectivity for ryanodine receptors

    Journal: Frontiers in Molecular Biosciences

    doi: 10.3389/fmolb.2022.1100992

    Tertiary and quaternary structure of CaM variants in complex with RyR1/2 peptides. (A) Cartoon representation of CaM in three different conformational states, namely apo (PDB: 1DMO ), Ca 2+ -bound (PDB: 1CLL ) and complexed with the RyR2 peptide used in this study (PDB: 6Y4O ). The N-terminal domain is colored in yellow, the linker region in grey, while the C-terminal domain in green. The RyR2 peptide is represented in magenta. Ca 2+ ions are represented as red spheres while the side chains of N97 and Q135 are represented in sticks with C atoms colored according to the structural region, O atoms in red and N atoms in blue. (B) Pairwise sequence alignment of the Calmodulin Binding Domain-2 of human RyR1 (Uniprot entry P21817) and RyR2 (Uniprot entry Q92736). The sequence relative to the two RyR peptides employed in this study is highlighted in yellow, the residues that are not identical in such region are represented in bold and colored in red. (C) Near-UV CD spectra (250–320 nm) of 50 μM CaM were collected in the presence of 500 µM EGTA (black dashed line) and after sequential additions of 1 mM Ca 2+ (black solid line, 500 µM free Ca 2+ ) and 100 μM RyR peptides (blue solid line for RyR1, red solid line for RyR2). The spectrum of sole buffer was considered as blank and subtracted; each curve represents the average of five accumulations. Temperature was set at 25°C and signal was normalized to protein concentration.
    Figure Legend Snippet: Tertiary and quaternary structure of CaM variants in complex with RyR1/2 peptides. (A) Cartoon representation of CaM in three different conformational states, namely apo (PDB: 1DMO ), Ca 2+ -bound (PDB: 1CLL ) and complexed with the RyR2 peptide used in this study (PDB: 6Y4O ). The N-terminal domain is colored in yellow, the linker region in grey, while the C-terminal domain in green. The RyR2 peptide is represented in magenta. Ca 2+ ions are represented as red spheres while the side chains of N97 and Q135 are represented in sticks with C atoms colored according to the structural region, O atoms in red and N atoms in blue. (B) Pairwise sequence alignment of the Calmodulin Binding Domain-2 of human RyR1 (Uniprot entry P21817) and RyR2 (Uniprot entry Q92736). The sequence relative to the two RyR peptides employed in this study is highlighted in yellow, the residues that are not identical in such region are represented in bold and colored in red. (C) Near-UV CD spectra (250–320 nm) of 50 μM CaM were collected in the presence of 500 µM EGTA (black dashed line) and after sequential additions of 1 mM Ca 2+ (black solid line, 500 µM free Ca 2+ ) and 100 μM RyR peptides (blue solid line for RyR1, red solid line for RyR2). The spectrum of sole buffer was considered as blank and subtracted; each curve represents the average of five accumulations. Temperature was set at 25°C and signal was normalized to protein concentration.

    Techniques Used: Sequencing, Binding Assay, Protein Concentration

    Investigation of secondary structure of CaM variants and their RyR1/2 peptide complexes. Far-UV CD spectra (200–250 nm) of 10 μM CaM variants alone (top panels, black), and incubated with 20 μM RyR1 (center panels, blue), or 20 μM RyR2 (bottom panels, red), were collected in the presence of 300 µM EGTA (dashed lines) and after the addition of 600 µM Ca 2+ (300 µM free Ca 2+ , solid lines). The spectrum of sole buffer was considered as blank and subtracted; each curve represents the average of five accumulations. Temperature was set at 25°C.
    Figure Legend Snippet: Investigation of secondary structure of CaM variants and their RyR1/2 peptide complexes. Far-UV CD spectra (200–250 nm) of 10 μM CaM variants alone (top panels, black), and incubated with 20 μM RyR1 (center panels, blue), or 20 μM RyR2 (bottom panels, red), were collected in the presence of 300 µM EGTA (dashed lines) and after the addition of 600 µM Ca 2+ (300 µM free Ca 2+ , solid lines). The spectrum of sole buffer was considered as blank and subtracted; each curve represents the average of five accumulations. Temperature was set at 25°C.

    Techniques Used: Incubation

    CaM affinity for RyR1 and RyR2 peptides assessed by fluorescence spectroscopy. (A) One micromolar RyR1 (left column) or RyR2 (right column) peptide was incubated with increasing amounts of WT (top row), N97I (middle row) and Q135P (bottom row) CaM in the presence of 100 µM Ca 2+ . Data are reported as a function of the peptide fraction bound to CaM (see Materials and Methods for details). Curves report the mean ± std of each point obtained in three technical replicas. Representative fitting to one-site saturation ligand binding curve is superposed to each titration set. (B) Scatter plot of replicates reporting the K D values calculated from fitting procedures in each individual dataset using a one-site binding model. Stars represent t -test statistical significance: * p -value ≤ 0.05, ** p -value ≤ 0.01.
    Figure Legend Snippet: CaM affinity for RyR1 and RyR2 peptides assessed by fluorescence spectroscopy. (A) One micromolar RyR1 (left column) or RyR2 (right column) peptide was incubated with increasing amounts of WT (top row), N97I (middle row) and Q135P (bottom row) CaM in the presence of 100 µM Ca 2+ . Data are reported as a function of the peptide fraction bound to CaM (see Materials and Methods for details). Curves report the mean ± std of each point obtained in three technical replicas. Representative fitting to one-site saturation ligand binding curve is superposed to each titration set. (B) Scatter plot of replicates reporting the K D values calculated from fitting procedures in each individual dataset using a one-site binding model. Stars represent t -test statistical significance: * p -value ≤ 0.05, ** p -value ≤ 0.01.

    Techniques Used: Fluorescence, Spectroscopy, Incubation, Ligand Binding Assay, Titration, Binding Assay

    Apparent affinities of CaM-RyR1/2 complexes assessed by fluorescence spectroscopy.
    Figure Legend Snippet: Apparent affinities of CaM-RyR1/2 complexes assessed by fluorescence spectroscopy.

    Techniques Used: Fluorescence, Spectroscopy

    Thermodynamics of CaM-RyR1/2 peptide interaction assessed by isothermal titration calorimetry. (A) Examples of ITC titration curves obtained for each CaM variant upon interaction with RyR1 or RyR2 peptides. Measurements were performed at 25°C using 20 mM Tris pH 7.5, 150 mM KCl, 5 mM Ca 2+ as working buffer and setting stirring at 750 rpm. Each titration consisted in thirty 1-µL injections of 125 µM RyR1 or RyR2 (into the titrant syringe) with 10 µM CaM variants. (B) Scatter plot of replicates summarizing the K D values calculated from the fitting using a one-site binding model (see Materials and Methods). Stars represent the p -values: * p ≤ 0.05, *** p ≤ 0.001. Data for WT CaM titration with RyR2 are from .
    Figure Legend Snippet: Thermodynamics of CaM-RyR1/2 peptide interaction assessed by isothermal titration calorimetry. (A) Examples of ITC titration curves obtained for each CaM variant upon interaction with RyR1 or RyR2 peptides. Measurements were performed at 25°C using 20 mM Tris pH 7.5, 150 mM KCl, 5 mM Ca 2+ as working buffer and setting stirring at 750 rpm. Each titration consisted in thirty 1-µL injections of 125 µM RyR1 or RyR2 (into the titrant syringe) with 10 µM CaM variants. (B) Scatter plot of replicates summarizing the K D values calculated from the fitting using a one-site binding model (see Materials and Methods). Stars represent the p -values: * p ≤ 0.05, *** p ≤ 0.001. Data for WT CaM titration with RyR2 are from .

    Techniques Used: Isothermal Titration Calorimetry, Titration, Variant Assay, Binding Assay

    Thermodynamics of CaM-RyR1/2 peptide association assessed by isothermal titration calorimetry.
    Figure Legend Snippet: Thermodynamics of CaM-RyR1/2 peptide association assessed by isothermal titration calorimetry.

    Techniques Used: Titration

    Kinetics of CaM-RyR1/2 peptide interaction investigated by surface plasmon resonance. (A) Sensorgrams collected by flowing different amounts of RyR1 and RyR2 (125 nM—2 µM) on immobilized His-CaM variants using 20 mM Tris pH 7.5, 150 mM KCl, 0.005% Tween 20, 5 mM Ca 2+ , 100 µM DTT as a running buffer. Association and dissociation phases were followed for 60 s and 300 s, respectively. Experimental curves (black solid lines) are shown together with theoretical curves (red or blue solid lines) according to a 1:1 Langmuir binding model; fitting for association and dissociation phases led to the rate constants ( k on and k off ) reported in each panel (mean ± s.e.m. of 8-20 independent binding curves). (B) Scatter plot of replicates and statistical analysis comparing rate constants for WT and each pathogenic CaM variant. Stars represent p -values: * p ≤ 0.05, ** p ≤ 0.01, **** p ≤ 0.0001.
    Figure Legend Snippet: Kinetics of CaM-RyR1/2 peptide interaction investigated by surface plasmon resonance. (A) Sensorgrams collected by flowing different amounts of RyR1 and RyR2 (125 nM—2 µM) on immobilized His-CaM variants using 20 mM Tris pH 7.5, 150 mM KCl, 0.005% Tween 20, 5 mM Ca 2+ , 100 µM DTT as a running buffer. Association and dissociation phases were followed for 60 s and 300 s, respectively. Experimental curves (black solid lines) are shown together with theoretical curves (red or blue solid lines) according to a 1:1 Langmuir binding model; fitting for association and dissociation phases led to the rate constants ( k on and k off ) reported in each panel (mean ± s.e.m. of 8-20 independent binding curves). (B) Scatter plot of replicates and statistical analysis comparing rate constants for WT and each pathogenic CaM variant. Stars represent p -values: * p ≤ 0.05, ** p ≤ 0.01, **** p ≤ 0.0001.

    Techniques Used: SPR Assay, Binding Assay, Variant Assay

    Backbone flexibility of CaM-RyR1/2 complexes. Cα-Root-Mean Square Fluctuation (RMSF) of CaM (top panels) and RyR1 or RyR2 peptides (bottom panels) calculated over 1.2 μs MD simulations of the respective complex (WT: black, N97I: blue, and Q135P: red).
    Figure Legend Snippet: Backbone flexibility of CaM-RyR1/2 complexes. Cα-Root-Mean Square Fluctuation (RMSF) of CaM (top panels) and RyR1 or RyR2 peptides (bottom panels) calculated over 1.2 μs MD simulations of the respective complex (WT: black, N97I: blue, and Q135P: red).

    Techniques Used:

    Effects of CaM variants on the connectivity of hub residues of CaM (top panels) and RyR1/2 peptides (bottom panels) . Hubs were defined as residues with degree ≥6 in the Protein Structure Network (PSN) of at least one variant. ∆Degree is calculated as the difference in hub degree between the variant (N97I: black, Q135P: red) and the WT.
    Figure Legend Snippet: Effects of CaM variants on the connectivity of hub residues of CaM (top panels) and RyR1/2 peptides (bottom panels) . Hubs were defined as residues with degree ≥6 in the Protein Structure Network (PSN) of at least one variant. ∆Degree is calculated as the difference in hub degree between the variant (N97I: black, Q135P: red) and the WT.

    Techniques Used: Variant Assay

    Robustness of intramolecular communication between EF-hands in CaM-RyR1/2 complexes. (A) Intramolecular communication among EF-hands in the CaM-RyR1 (left) and CaM-RyR2 (right) complexes. Communication robustness between EF-hands (EF1 to EF4, represented by their bidentate Glu residues) in CaM WT (black), N97I (red), and Q135P (green) variants in complex with RyR peptides. (B) Effects of specific RyR peptides on the intramolecular communication among EF-hands in CaM-RyR complexes. Communication robustness between EF-hands (EF1 to EF4, represented by their bidentate Glu residues) in CaM variants in complex with RyR1 (black) and RyR2 (red) peptides.
    Figure Legend Snippet: Robustness of intramolecular communication between EF-hands in CaM-RyR1/2 complexes. (A) Intramolecular communication among EF-hands in the CaM-RyR1 (left) and CaM-RyR2 (right) complexes. Communication robustness between EF-hands (EF1 to EF4, represented by their bidentate Glu residues) in CaM WT (black), N97I (red), and Q135P (green) variants in complex with RyR peptides. (B) Effects of specific RyR peptides on the intramolecular communication among EF-hands in CaM-RyR complexes. Communication robustness between EF-hands (EF1 to EF4, represented by their bidentate Glu residues) in CaM variants in complex with RyR1 (black) and RyR2 (red) peptides.

    Techniques Used:

    ryr2  (Thermo Fisher)


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    Structured Review

    Thermo Fisher ryr2
    Tertiary and quaternary structure of CaM variants in complex with <t>RyR1/2</t> peptides. (A) Cartoon representation of CaM in three different conformational states, namely apo (PDB: 1DMO ), Ca 2+ -bound (PDB: 1CLL ) and complexed with the <t>RyR2</t> peptide used in this study (PDB: 6Y4O ). The N-terminal domain is colored in yellow, the linker region in grey, while the C-terminal domain in green. The RyR2 peptide is represented in magenta. Ca 2+ ions are represented as red spheres while the side chains of N97 and Q135 are represented in sticks with C atoms colored according to the structural region, O atoms in red and N atoms in blue. (B) Pairwise sequence alignment of the Calmodulin Binding Domain-2 of human RyR1 (Uniprot entry P21817) and RyR2 (Uniprot entry Q92736). The sequence relative to the two RyR peptides employed in this study is highlighted in yellow, the residues that are not identical in such region are represented in bold and colored in red. (C) Near-UV CD spectra (250–320 nm) of 50 μM CaM were collected in the presence of 500 µM EGTA (black dashed line) and after sequential additions of 1 mM Ca 2+ (black solid line, 500 µM free Ca 2+ ) and 100 μM RyR peptides (blue solid line for RyR1, red solid line for RyR2). The spectrum of sole buffer was considered as blank and subtracted; each curve represents the average of five accumulations. Temperature was set at 25°C and signal was normalized to protein concentration.
    Ryr2, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ryr2/product/Thermo Fisher
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    ryr2 - by Bioz Stars, 2023-01
    86/100 stars

    Images

    1) Product Images from "Calmodulin variants associated with congenital arrhythmia impair selectivity for ryanodine receptors"

    Article Title: Calmodulin variants associated with congenital arrhythmia impair selectivity for ryanodine receptors

    Journal: Frontiers in Molecular Biosciences

    doi: 10.3389/fmolb.2022.1100992

    Tertiary and quaternary structure of CaM variants in complex with RyR1/2 peptides. (A) Cartoon representation of CaM in three different conformational states, namely apo (PDB: 1DMO ), Ca 2+ -bound (PDB: 1CLL ) and complexed with the RyR2 peptide used in this study (PDB: 6Y4O ). The N-terminal domain is colored in yellow, the linker region in grey, while the C-terminal domain in green. The RyR2 peptide is represented in magenta. Ca 2+ ions are represented as red spheres while the side chains of N97 and Q135 are represented in sticks with C atoms colored according to the structural region, O atoms in red and N atoms in blue. (B) Pairwise sequence alignment of the Calmodulin Binding Domain-2 of human RyR1 (Uniprot entry P21817) and RyR2 (Uniprot entry Q92736). The sequence relative to the two RyR peptides employed in this study is highlighted in yellow, the residues that are not identical in such region are represented in bold and colored in red. (C) Near-UV CD spectra (250–320 nm) of 50 μM CaM were collected in the presence of 500 µM EGTA (black dashed line) and after sequential additions of 1 mM Ca 2+ (black solid line, 500 µM free Ca 2+ ) and 100 μM RyR peptides (blue solid line for RyR1, red solid line for RyR2). The spectrum of sole buffer was considered as blank and subtracted; each curve represents the average of five accumulations. Temperature was set at 25°C and signal was normalized to protein concentration.
    Figure Legend Snippet: Tertiary and quaternary structure of CaM variants in complex with RyR1/2 peptides. (A) Cartoon representation of CaM in three different conformational states, namely apo (PDB: 1DMO ), Ca 2+ -bound (PDB: 1CLL ) and complexed with the RyR2 peptide used in this study (PDB: 6Y4O ). The N-terminal domain is colored in yellow, the linker region in grey, while the C-terminal domain in green. The RyR2 peptide is represented in magenta. Ca 2+ ions are represented as red spheres while the side chains of N97 and Q135 are represented in sticks with C atoms colored according to the structural region, O atoms in red and N atoms in blue. (B) Pairwise sequence alignment of the Calmodulin Binding Domain-2 of human RyR1 (Uniprot entry P21817) and RyR2 (Uniprot entry Q92736). The sequence relative to the two RyR peptides employed in this study is highlighted in yellow, the residues that are not identical in such region are represented in bold and colored in red. (C) Near-UV CD spectra (250–320 nm) of 50 μM CaM were collected in the presence of 500 µM EGTA (black dashed line) and after sequential additions of 1 mM Ca 2+ (black solid line, 500 µM free Ca 2+ ) and 100 μM RyR peptides (blue solid line for RyR1, red solid line for RyR2). The spectrum of sole buffer was considered as blank and subtracted; each curve represents the average of five accumulations. Temperature was set at 25°C and signal was normalized to protein concentration.

    Techniques Used: Sequencing, Binding Assay, Protein Concentration

    Investigation of secondary structure of CaM variants and their RyR1/2 peptide complexes. Far-UV CD spectra (200–250 nm) of 10 μM CaM variants alone (top panels, black), and incubated with 20 μM RyR1 (center panels, blue), or 20 μM RyR2 (bottom panels, red), were collected in the presence of 300 µM EGTA (dashed lines) and after the addition of 600 µM Ca 2+ (300 µM free Ca 2+ , solid lines). The spectrum of sole buffer was considered as blank and subtracted; each curve represents the average of five accumulations. Temperature was set at 25°C.
    Figure Legend Snippet: Investigation of secondary structure of CaM variants and their RyR1/2 peptide complexes. Far-UV CD spectra (200–250 nm) of 10 μM CaM variants alone (top panels, black), and incubated with 20 μM RyR1 (center panels, blue), or 20 μM RyR2 (bottom panels, red), were collected in the presence of 300 µM EGTA (dashed lines) and after the addition of 600 µM Ca 2+ (300 µM free Ca 2+ , solid lines). The spectrum of sole buffer was considered as blank and subtracted; each curve represents the average of five accumulations. Temperature was set at 25°C.

    Techniques Used: Incubation

    CaM affinity for RyR1 and RyR2 peptides assessed by fluorescence spectroscopy. (A) One micromolar RyR1 (left column) or RyR2 (right column) peptide was incubated with increasing amounts of WT (top row), N97I (middle row) and Q135P (bottom row) CaM in the presence of 100 µM Ca 2+ . Data are reported as a function of the peptide fraction bound to CaM (see Materials and Methods for details). Curves report the mean ± std of each point obtained in three technical replicas. Representative fitting to one-site saturation ligand binding curve is superposed to each titration set. (B) Scatter plot of replicates reporting the K D values calculated from fitting procedures in each individual dataset using a one-site binding model. Stars represent t -test statistical significance: * p -value ≤ 0.05, ** p -value ≤ 0.01.
    Figure Legend Snippet: CaM affinity for RyR1 and RyR2 peptides assessed by fluorescence spectroscopy. (A) One micromolar RyR1 (left column) or RyR2 (right column) peptide was incubated with increasing amounts of WT (top row), N97I (middle row) and Q135P (bottom row) CaM in the presence of 100 µM Ca 2+ . Data are reported as a function of the peptide fraction bound to CaM (see Materials and Methods for details). Curves report the mean ± std of each point obtained in three technical replicas. Representative fitting to one-site saturation ligand binding curve is superposed to each titration set. (B) Scatter plot of replicates reporting the K D values calculated from fitting procedures in each individual dataset using a one-site binding model. Stars represent t -test statistical significance: * p -value ≤ 0.05, ** p -value ≤ 0.01.

    Techniques Used: Fluorescence, Spectroscopy, Incubation, Ligand Binding Assay, Titration, Binding Assay

    Apparent affinities of CaM-RyR1/2 complexes assessed by fluorescence spectroscopy.
    Figure Legend Snippet: Apparent affinities of CaM-RyR1/2 complexes assessed by fluorescence spectroscopy.

    Techniques Used: Fluorescence, Spectroscopy

    Thermodynamics of CaM-RyR1/2 peptide interaction assessed by isothermal titration calorimetry. (A) Examples of ITC titration curves obtained for each CaM variant upon interaction with RyR1 or RyR2 peptides. Measurements were performed at 25°C using 20 mM Tris pH 7.5, 150 mM KCl, 5 mM Ca 2+ as working buffer and setting stirring at 750 rpm. Each titration consisted in thirty 1-µL injections of 125 µM RyR1 or RyR2 (into the titrant syringe) with 10 µM CaM variants. (B) Scatter plot of replicates summarizing the K D values calculated from the fitting using a one-site binding model (see Materials and Methods). Stars represent the p -values: * p ≤ 0.05, *** p ≤ 0.001. Data for WT CaM titration with RyR2 are from .
    Figure Legend Snippet: Thermodynamics of CaM-RyR1/2 peptide interaction assessed by isothermal titration calorimetry. (A) Examples of ITC titration curves obtained for each CaM variant upon interaction with RyR1 or RyR2 peptides. Measurements were performed at 25°C using 20 mM Tris pH 7.5, 150 mM KCl, 5 mM Ca 2+ as working buffer and setting stirring at 750 rpm. Each titration consisted in thirty 1-µL injections of 125 µM RyR1 or RyR2 (into the titrant syringe) with 10 µM CaM variants. (B) Scatter plot of replicates summarizing the K D values calculated from the fitting using a one-site binding model (see Materials and Methods). Stars represent the p -values: * p ≤ 0.05, *** p ≤ 0.001. Data for WT CaM titration with RyR2 are from .

    Techniques Used: Isothermal Titration Calorimetry, Titration, Variant Assay, Binding Assay

    Thermodynamics of CaM-RyR1/2 peptide association assessed by isothermal titration calorimetry.
    Figure Legend Snippet: Thermodynamics of CaM-RyR1/2 peptide association assessed by isothermal titration calorimetry.

    Techniques Used: Titration

    Kinetics of CaM-RyR1/2 peptide interaction investigated by surface plasmon resonance. (A) Sensorgrams collected by flowing different amounts of RyR1 and RyR2 (125 nM—2 µM) on immobilized His-CaM variants using 20 mM Tris pH 7.5, 150 mM KCl, 0.005% Tween 20, 5 mM Ca 2+ , 100 µM DTT as a running buffer. Association and dissociation phases were followed for 60 s and 300 s, respectively. Experimental curves (black solid lines) are shown together with theoretical curves (red or blue solid lines) according to a 1:1 Langmuir binding model; fitting for association and dissociation phases led to the rate constants ( k on and k off ) reported in each panel (mean ± s.e.m. of 8-20 independent binding curves). (B) Scatter plot of replicates and statistical analysis comparing rate constants for WT and each pathogenic CaM variant. Stars represent p -values: * p ≤ 0.05, ** p ≤ 0.01, **** p ≤ 0.0001.
    Figure Legend Snippet: Kinetics of CaM-RyR1/2 peptide interaction investigated by surface plasmon resonance. (A) Sensorgrams collected by flowing different amounts of RyR1 and RyR2 (125 nM—2 µM) on immobilized His-CaM variants using 20 mM Tris pH 7.5, 150 mM KCl, 0.005% Tween 20, 5 mM Ca 2+ , 100 µM DTT as a running buffer. Association and dissociation phases were followed for 60 s and 300 s, respectively. Experimental curves (black solid lines) are shown together with theoretical curves (red or blue solid lines) according to a 1:1 Langmuir binding model; fitting for association and dissociation phases led to the rate constants ( k on and k off ) reported in each panel (mean ± s.e.m. of 8-20 independent binding curves). (B) Scatter plot of replicates and statistical analysis comparing rate constants for WT and each pathogenic CaM variant. Stars represent p -values: * p ≤ 0.05, ** p ≤ 0.01, **** p ≤ 0.0001.

    Techniques Used: SPR Assay, Binding Assay, Variant Assay

    Backbone flexibility of CaM-RyR1/2 complexes. Cα-Root-Mean Square Fluctuation (RMSF) of CaM (top panels) and RyR1 or RyR2 peptides (bottom panels) calculated over 1.2 μs MD simulations of the respective complex (WT: black, N97I: blue, and Q135P: red).
    Figure Legend Snippet: Backbone flexibility of CaM-RyR1/2 complexes. Cα-Root-Mean Square Fluctuation (RMSF) of CaM (top panels) and RyR1 or RyR2 peptides (bottom panels) calculated over 1.2 μs MD simulations of the respective complex (WT: black, N97I: blue, and Q135P: red).

    Techniques Used:

    Effects of CaM variants on the connectivity of hub residues of CaM (top panels) and RyR1/2 peptides (bottom panels) . Hubs were defined as residues with degree ≥6 in the Protein Structure Network (PSN) of at least one variant. ∆Degree is calculated as the difference in hub degree between the variant (N97I: black, Q135P: red) and the WT.
    Figure Legend Snippet: Effects of CaM variants on the connectivity of hub residues of CaM (top panels) and RyR1/2 peptides (bottom panels) . Hubs were defined as residues with degree ≥6 in the Protein Structure Network (PSN) of at least one variant. ∆Degree is calculated as the difference in hub degree between the variant (N97I: black, Q135P: red) and the WT.

    Techniques Used: Variant Assay

    Robustness of intramolecular communication between EF-hands in CaM-RyR1/2 complexes. (A) Intramolecular communication among EF-hands in the CaM-RyR1 (left) and CaM-RyR2 (right) complexes. Communication robustness between EF-hands (EF1 to EF4, represented by their bidentate Glu residues) in CaM WT (black), N97I (red), and Q135P (green) variants in complex with RyR peptides. (B) Effects of specific RyR peptides on the intramolecular communication among EF-hands in CaM-RyR complexes. Communication robustness between EF-hands (EF1 to EF4, represented by their bidentate Glu residues) in CaM variants in complex with RyR1 (black) and RyR2 (red) peptides.
    Figure Legend Snippet: Robustness of intramolecular communication between EF-hands in CaM-RyR1/2 complexes. (A) Intramolecular communication among EF-hands in the CaM-RyR1 (left) and CaM-RyR2 (right) complexes. Communication robustness between EF-hands (EF1 to EF4, represented by their bidentate Glu residues) in CaM WT (black), N97I (red), and Q135P (green) variants in complex with RyR peptides. (B) Effects of specific RyR peptides on the intramolecular communication among EF-hands in CaM-RyR complexes. Communication robustness between EF-hands (EF1 to EF4, represented by their bidentate Glu residues) in CaM variants in complex with RyR1 (black) and RyR2 (red) peptides.

    Techniques Used:

    overlapping ryr2 sites  (Thermo Fisher)


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    Structured Review

    Thermo Fisher overlapping ryr2 sites
    Tertiary and quaternary structure of CaM variants in complex with <t>RyR1/2</t> peptides. (A) Cartoon representation of CaM in three different conformational states, namely apo (PDB: 1DMO ), Ca 2+ -bound (PDB: 1CLL ) and complexed with the <t>RyR2</t> peptide used in this study (PDB: 6Y4O ). The N-terminal domain is colored in yellow, the linker region in grey, while the C-terminal domain in green. The RyR2 peptide is represented in magenta. Ca 2+ ions are represented as red spheres while the side chains of N97 and Q135 are represented in sticks with C atoms colored according to the structural region, O atoms in red and N atoms in blue. (B) Pairwise sequence alignment of the Calmodulin Binding Domain-2 of human RyR1 (Uniprot entry P21817) and RyR2 (Uniprot entry Q92736). The sequence relative to the two RyR peptides employed in this study is highlighted in yellow, the residues that are not identical in such region are represented in bold and colored in red. (C) Near-UV CD spectra (250–320 nm) of 50 μM CaM were collected in the presence of 500 µM EGTA (black dashed line) and after sequential additions of 1 mM Ca 2+ (black solid line, 500 µM free Ca 2+ ) and 100 μM RyR peptides (blue solid line for RyR1, red solid line for RyR2). The spectrum of sole buffer was considered as blank and subtracted; each curve represents the average of five accumulations. Temperature was set at 25°C and signal was normalized to protein concentration.
    Overlapping Ryr2 Sites, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Calmodulin variants associated with congenital arrhythmia impair selectivity for ryanodine receptors"

    Article Title: Calmodulin variants associated with congenital arrhythmia impair selectivity for ryanodine receptors

    Journal: Frontiers in Molecular Biosciences

    doi: 10.3389/fmolb.2022.1100992

    Tertiary and quaternary structure of CaM variants in complex with RyR1/2 peptides. (A) Cartoon representation of CaM in three different conformational states, namely apo (PDB: 1DMO ), Ca 2+ -bound (PDB: 1CLL ) and complexed with the RyR2 peptide used in this study (PDB: 6Y4O ). The N-terminal domain is colored in yellow, the linker region in grey, while the C-terminal domain in green. The RyR2 peptide is represented in magenta. Ca 2+ ions are represented as red spheres while the side chains of N97 and Q135 are represented in sticks with C atoms colored according to the structural region, O atoms in red and N atoms in blue. (B) Pairwise sequence alignment of the Calmodulin Binding Domain-2 of human RyR1 (Uniprot entry P21817) and RyR2 (Uniprot entry Q92736). The sequence relative to the two RyR peptides employed in this study is highlighted in yellow, the residues that are not identical in such region are represented in bold and colored in red. (C) Near-UV CD spectra (250–320 nm) of 50 μM CaM were collected in the presence of 500 µM EGTA (black dashed line) and after sequential additions of 1 mM Ca 2+ (black solid line, 500 µM free Ca 2+ ) and 100 μM RyR peptides (blue solid line for RyR1, red solid line for RyR2). The spectrum of sole buffer was considered as blank and subtracted; each curve represents the average of five accumulations. Temperature was set at 25°C and signal was normalized to protein concentration.
    Figure Legend Snippet: Tertiary and quaternary structure of CaM variants in complex with RyR1/2 peptides. (A) Cartoon representation of CaM in three different conformational states, namely apo (PDB: 1DMO ), Ca 2+ -bound (PDB: 1CLL ) and complexed with the RyR2 peptide used in this study (PDB: 6Y4O ). The N-terminal domain is colored in yellow, the linker region in grey, while the C-terminal domain in green. The RyR2 peptide is represented in magenta. Ca 2+ ions are represented as red spheres while the side chains of N97 and Q135 are represented in sticks with C atoms colored according to the structural region, O atoms in red and N atoms in blue. (B) Pairwise sequence alignment of the Calmodulin Binding Domain-2 of human RyR1 (Uniprot entry P21817) and RyR2 (Uniprot entry Q92736). The sequence relative to the two RyR peptides employed in this study is highlighted in yellow, the residues that are not identical in such region are represented in bold and colored in red. (C) Near-UV CD spectra (250–320 nm) of 50 μM CaM were collected in the presence of 500 µM EGTA (black dashed line) and after sequential additions of 1 mM Ca 2+ (black solid line, 500 µM free Ca 2+ ) and 100 μM RyR peptides (blue solid line for RyR1, red solid line for RyR2). The spectrum of sole buffer was considered as blank and subtracted; each curve represents the average of five accumulations. Temperature was set at 25°C and signal was normalized to protein concentration.

    Techniques Used: Sequencing, Binding Assay, Protein Concentration

    Investigation of secondary structure of CaM variants and their RyR1/2 peptide complexes. Far-UV CD spectra (200–250 nm) of 10 μM CaM variants alone (top panels, black), and incubated with 20 μM RyR1 (center panels, blue), or 20 μM RyR2 (bottom panels, red), were collected in the presence of 300 µM EGTA (dashed lines) and after the addition of 600 µM Ca 2+ (300 µM free Ca 2+ , solid lines). The spectrum of sole buffer was considered as blank and subtracted; each curve represents the average of five accumulations. Temperature was set at 25°C.
    Figure Legend Snippet: Investigation of secondary structure of CaM variants and their RyR1/2 peptide complexes. Far-UV CD spectra (200–250 nm) of 10 μM CaM variants alone (top panels, black), and incubated with 20 μM RyR1 (center panels, blue), or 20 μM RyR2 (bottom panels, red), were collected in the presence of 300 µM EGTA (dashed lines) and after the addition of 600 µM Ca 2+ (300 µM free Ca 2+ , solid lines). The spectrum of sole buffer was considered as blank and subtracted; each curve represents the average of five accumulations. Temperature was set at 25°C.

    Techniques Used: Incubation

    CaM affinity for RyR1 and RyR2 peptides assessed by fluorescence spectroscopy. (A) One micromolar RyR1 (left column) or RyR2 (right column) peptide was incubated with increasing amounts of WT (top row), N97I (middle row) and Q135P (bottom row) CaM in the presence of 100 µM Ca 2+ . Data are reported as a function of the peptide fraction bound to CaM (see Materials and Methods for details). Curves report the mean ± std of each point obtained in three technical replicas. Representative fitting to one-site saturation ligand binding curve is superposed to each titration set. (B) Scatter plot of replicates reporting the K D values calculated from fitting procedures in each individual dataset using a one-site binding model. Stars represent t -test statistical significance: * p -value ≤ 0.05, ** p -value ≤ 0.01.
    Figure Legend Snippet: CaM affinity for RyR1 and RyR2 peptides assessed by fluorescence spectroscopy. (A) One micromolar RyR1 (left column) or RyR2 (right column) peptide was incubated with increasing amounts of WT (top row), N97I (middle row) and Q135P (bottom row) CaM in the presence of 100 µM Ca 2+ . Data are reported as a function of the peptide fraction bound to CaM (see Materials and Methods for details). Curves report the mean ± std of each point obtained in three technical replicas. Representative fitting to one-site saturation ligand binding curve is superposed to each titration set. (B) Scatter plot of replicates reporting the K D values calculated from fitting procedures in each individual dataset using a one-site binding model. Stars represent t -test statistical significance: * p -value ≤ 0.05, ** p -value ≤ 0.01.

    Techniques Used: Fluorescence, Spectroscopy, Incubation, Ligand Binding Assay, Titration, Binding Assay

    Apparent affinities of CaM-RyR1/2 complexes assessed by fluorescence spectroscopy.
    Figure Legend Snippet: Apparent affinities of CaM-RyR1/2 complexes assessed by fluorescence spectroscopy.

    Techniques Used: Fluorescence, Spectroscopy

    Thermodynamics of CaM-RyR1/2 peptide interaction assessed by isothermal titration calorimetry. (A) Examples of ITC titration curves obtained for each CaM variant upon interaction with RyR1 or RyR2 peptides. Measurements were performed at 25°C using 20 mM Tris pH 7.5, 150 mM KCl, 5 mM Ca 2+ as working buffer and setting stirring at 750 rpm. Each titration consisted in thirty 1-µL injections of 125 µM RyR1 or RyR2 (into the titrant syringe) with 10 µM CaM variants. (B) Scatter plot of replicates summarizing the K D values calculated from the fitting using a one-site binding model (see Materials and Methods). Stars represent the p -values: * p ≤ 0.05, *** p ≤ 0.001. Data for WT CaM titration with RyR2 are from .
    Figure Legend Snippet: Thermodynamics of CaM-RyR1/2 peptide interaction assessed by isothermal titration calorimetry. (A) Examples of ITC titration curves obtained for each CaM variant upon interaction with RyR1 or RyR2 peptides. Measurements were performed at 25°C using 20 mM Tris pH 7.5, 150 mM KCl, 5 mM Ca 2+ as working buffer and setting stirring at 750 rpm. Each titration consisted in thirty 1-µL injections of 125 µM RyR1 or RyR2 (into the titrant syringe) with 10 µM CaM variants. (B) Scatter plot of replicates summarizing the K D values calculated from the fitting using a one-site binding model (see Materials and Methods). Stars represent the p -values: * p ≤ 0.05, *** p ≤ 0.001. Data for WT CaM titration with RyR2 are from .

    Techniques Used: Isothermal Titration Calorimetry, Titration, Variant Assay, Binding Assay

    Thermodynamics of CaM-RyR1/2 peptide association assessed by isothermal titration calorimetry.
    Figure Legend Snippet: Thermodynamics of CaM-RyR1/2 peptide association assessed by isothermal titration calorimetry.

    Techniques Used: Titration

    Kinetics of CaM-RyR1/2 peptide interaction investigated by surface plasmon resonance. (A) Sensorgrams collected by flowing different amounts of RyR1 and RyR2 (125 nM—2 µM) on immobilized His-CaM variants using 20 mM Tris pH 7.5, 150 mM KCl, 0.005% Tween 20, 5 mM Ca 2+ , 100 µM DTT as a running buffer. Association and dissociation phases were followed for 60 s and 300 s, respectively. Experimental curves (black solid lines) are shown together with theoretical curves (red or blue solid lines) according to a 1:1 Langmuir binding model; fitting for association and dissociation phases led to the rate constants ( k on and k off ) reported in each panel (mean ± s.e.m. of 8-20 independent binding curves). (B) Scatter plot of replicates and statistical analysis comparing rate constants for WT and each pathogenic CaM variant. Stars represent p -values: * p ≤ 0.05, ** p ≤ 0.01, **** p ≤ 0.0001.
    Figure Legend Snippet: Kinetics of CaM-RyR1/2 peptide interaction investigated by surface plasmon resonance. (A) Sensorgrams collected by flowing different amounts of RyR1 and RyR2 (125 nM—2 µM) on immobilized His-CaM variants using 20 mM Tris pH 7.5, 150 mM KCl, 0.005% Tween 20, 5 mM Ca 2+ , 100 µM DTT as a running buffer. Association and dissociation phases were followed for 60 s and 300 s, respectively. Experimental curves (black solid lines) are shown together with theoretical curves (red or blue solid lines) according to a 1:1 Langmuir binding model; fitting for association and dissociation phases led to the rate constants ( k on and k off ) reported in each panel (mean ± s.e.m. of 8-20 independent binding curves). (B) Scatter plot of replicates and statistical analysis comparing rate constants for WT and each pathogenic CaM variant. Stars represent p -values: * p ≤ 0.05, ** p ≤ 0.01, **** p ≤ 0.0001.

    Techniques Used: SPR Assay, Binding Assay, Variant Assay

    Backbone flexibility of CaM-RyR1/2 complexes. Cα-Root-Mean Square Fluctuation (RMSF) of CaM (top panels) and RyR1 or RyR2 peptides (bottom panels) calculated over 1.2 μs MD simulations of the respective complex (WT: black, N97I: blue, and Q135P: red).
    Figure Legend Snippet: Backbone flexibility of CaM-RyR1/2 complexes. Cα-Root-Mean Square Fluctuation (RMSF) of CaM (top panels) and RyR1 or RyR2 peptides (bottom panels) calculated over 1.2 μs MD simulations of the respective complex (WT: black, N97I: blue, and Q135P: red).

    Techniques Used:

    Effects of CaM variants on the connectivity of hub residues of CaM (top panels) and RyR1/2 peptides (bottom panels) . Hubs were defined as residues with degree ≥6 in the Protein Structure Network (PSN) of at least one variant. ∆Degree is calculated as the difference in hub degree between the variant (N97I: black, Q135P: red) and the WT.
    Figure Legend Snippet: Effects of CaM variants on the connectivity of hub residues of CaM (top panels) and RyR1/2 peptides (bottom panels) . Hubs were defined as residues with degree ≥6 in the Protein Structure Network (PSN) of at least one variant. ∆Degree is calculated as the difference in hub degree between the variant (N97I: black, Q135P: red) and the WT.

    Techniques Used: Variant Assay

    Robustness of intramolecular communication between EF-hands in CaM-RyR1/2 complexes. (A) Intramolecular communication among EF-hands in the CaM-RyR1 (left) and CaM-RyR2 (right) complexes. Communication robustness between EF-hands (EF1 to EF4, represented by their bidentate Glu residues) in CaM WT (black), N97I (red), and Q135P (green) variants in complex with RyR peptides. (B) Effects of specific RyR peptides on the intramolecular communication among EF-hands in CaM-RyR complexes. Communication robustness between EF-hands (EF1 to EF4, represented by their bidentate Glu residues) in CaM variants in complex with RyR1 (black) and RyR2 (red) peptides.
    Figure Legend Snippet: Robustness of intramolecular communication between EF-hands in CaM-RyR1/2 complexes. (A) Intramolecular communication among EF-hands in the CaM-RyR1 (left) and CaM-RyR2 (right) complexes. Communication robustness between EF-hands (EF1 to EF4, represented by their bidentate Glu residues) in CaM WT (black), N97I (red), and Q135P (green) variants in complex with RyR peptides. (B) Effects of specific RyR peptides on the intramolecular communication among EF-hands in CaM-RyR complexes. Communication robustness between EF-hands (EF1 to EF4, represented by their bidentate Glu residues) in CaM variants in complex with RyR1 (black) and RyR2 (red) peptides.

    Techniques Used:

    gene exp ryr2 mm00465877 m1  (Thermo Fisher)


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    Structured Review

    Thermo Fisher gene exp ryr2 mm00465877 m1
    Comparative real-time PCR mRNA expression of 47 genes related to contractile function in the afferent arterioles from Notch3 −/− and wild-type littermates. Note that the expression of Cacna1h coding the α 1H subunit of the T-type Ca 2+ channel (Ca v 3.2) gene was strongly downregulated in Notch3 −/− ( p < 0.001 vs. wild-type). In contrast, Cacna1c coding the α 1C subunit of the L-type Ca 2+ channel was similarly expressed in the two strains (No. 26 on the list).
    Gene Exp Ryr2 Mm00465877 M1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Deletion of Notch3 Impairs Contractility of Renal Resistance Vessels Due to Deficient Ca 2+ Entry"

    Article Title: Deletion of Notch3 Impairs Contractility of Renal Resistance Vessels Due to Deficient Ca 2+ Entry

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms232416068

    Comparative real-time PCR mRNA expression of 47 genes related to contractile function in the afferent arterioles from Notch3 −/− and wild-type littermates. Note that the expression of Cacna1h coding the α 1H subunit of the T-type Ca 2+ channel (Ca v 3.2) gene was strongly downregulated in Notch3 −/− ( p < 0.001 vs. wild-type). In contrast, Cacna1c coding the α 1C subunit of the L-type Ca 2+ channel was similarly expressed in the two strains (No. 26 on the list).
    Figure Legend Snippet: Comparative real-time PCR mRNA expression of 47 genes related to contractile function in the afferent arterioles from Notch3 −/− and wild-type littermates. Note that the expression of Cacna1h coding the α 1H subunit of the T-type Ca 2+ channel (Ca v 3.2) gene was strongly downregulated in Notch3 −/− ( p < 0.001 vs. wild-type). In contrast, Cacna1c coding the α 1C subunit of the L-type Ca 2+ channel was similarly expressed in the two strains (No. 26 on the list).

    Techniques Used: Real-time Polymerase Chain Reaction, Expressing

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    Thermo Fisher mutant ryr2 r2474s channel
    The figure indicates links of <t>ryanodine</t> <t>receptors</t> <t>RyR1–RyR3</t> to neurodegenerative diseases ( , – , , , , ), ventilator-induced diaphragmatic dysfunction ( – ), heart failure ( , , , , , , , , – , ), cardiac arrhythmias ( – , – , , ), skeletal myopathies ( – , , , , ), cancer-associated muscle weakness , age-dependent loss of muscle function , and diabetes . Adapted with permission from Biochimica et Biophysica Acta: Molecular Cell Research .
    Mutant Ryr2 R2474s Channel, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Recombinant Ryr2, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher ryr1
    The figure indicates links of <t>ryanodine</t> <t>receptors</t> <t>RyR1–RyR3</t> to neurodegenerative diseases ( , – , , , , ), ventilator-induced diaphragmatic dysfunction ( – ), heart failure ( , , , , , , , , – , ), cardiac arrhythmias ( – , – , , ), skeletal myopathies ( – , , , , ), cancer-associated muscle weakness , age-dependent loss of muscle function , and diabetes . Adapted with permission from Biochimica et Biophysica Acta: Molecular Cell Research .
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    Thermo Fisher ca v 1 1 ryr1 electromechanical coupling
    Muscle and Ca V 1.1 organization. A) Muscle to muscle cell perspective. ( left ) Morphology of a segment of a skeletal muscle fiber ( right ). Note the characteristic striated pattern of muscle fibers, which results from highly organized array between sarcolemma, sarcoplasmic reticulum (SR), contractile elements and cytoarchitecture of the fibers. B) Structure of the triad. The cartoon depicts the triad, a specialized membrane-organelle array formed by the T-tubule and two segments of the terminal junctional SR. The T-tubules are infoldings of the sarcolemma that propagate the action potential radially into the fiber. Ca V 1.1 (blue) are located at the T-tubules, working primarily as voltage sensors that initiate the early steps of EC coupling. The SR Ca 2+ release channel, <t>RyR1</t> (brown), is predominantly located on the junctional domain of the SR surface. Typical profiles of triads (cross-sections) contain only two rows of RyR1 associated with alternating tetrads . C) Detailed architecture of the triad with a focus on Ca V 1.1 tetrads and RyR1 arrays as shown in (b). About half of the total RyR1s do not associate with Ca V 1.1, resulting in an alternating pattern of “free” and Ca V 1.1-associated RyR1s. Note: In addition to Ca V 1.1 tetrad (blue) and RyR1 (yellow) Ca 2+ release channels, many other proteins form part of the T-tubule- junctional SR complex (e.g. junctophilin, triadin, junctin, calsequestrin, not indicated here).
    Ca V 1 1 Ryr1 Electromechanical Coupling, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher cam ryr2 recognition mechanism
    Tertiary and quaternary structure of CaM variants in complex with <t>RyR1/2</t> peptides. (A) Cartoon representation of CaM in three different conformational states, namely apo (PDB: 1DMO ), Ca 2+ -bound (PDB: 1CLL ) and complexed with the <t>RyR2</t> peptide used in this study (PDB: 6Y4O ). The N-terminal domain is colored in yellow, the linker region in grey, while the C-terminal domain in green. The RyR2 peptide is represented in magenta. Ca 2+ ions are represented as red spheres while the side chains of N97 and Q135 are represented in sticks with C atoms colored according to the structural region, O atoms in red and N atoms in blue. (B) Pairwise sequence alignment of the Calmodulin Binding Domain-2 of human RyR1 (Uniprot entry P21817) and RyR2 (Uniprot entry Q92736). The sequence relative to the two RyR peptides employed in this study is highlighted in yellow, the residues that are not identical in such region are represented in bold and colored in red. (C) Near-UV CD spectra (250–320 nm) of 50 μM CaM were collected in the presence of 500 µM EGTA (black dashed line) and after sequential additions of 1 mM Ca 2+ (black solid line, 500 µM free Ca 2+ ) and 100 μM RyR peptides (blue solid line for RyR1, red solid line for RyR2). The spectrum of sole buffer was considered as blank and subtracted; each curve represents the average of five accumulations. Temperature was set at 25°C and signal was normalized to protein concentration.
    Cam Ryr2 Recognition Mechanism, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher ryr2 gating
    Tertiary and quaternary structure of CaM variants in complex with <t>RyR1/2</t> peptides. (A) Cartoon representation of CaM in three different conformational states, namely apo (PDB: 1DMO ), Ca 2+ -bound (PDB: 1CLL ) and complexed with the <t>RyR2</t> peptide used in this study (PDB: 6Y4O ). The N-terminal domain is colored in yellow, the linker region in grey, while the C-terminal domain in green. The RyR2 peptide is represented in magenta. Ca 2+ ions are represented as red spheres while the side chains of N97 and Q135 are represented in sticks with C atoms colored according to the structural region, O atoms in red and N atoms in blue. (B) Pairwise sequence alignment of the Calmodulin Binding Domain-2 of human RyR1 (Uniprot entry P21817) and RyR2 (Uniprot entry Q92736). The sequence relative to the two RyR peptides employed in this study is highlighted in yellow, the residues that are not identical in such region are represented in bold and colored in red. (C) Near-UV CD spectra (250–320 nm) of 50 μM CaM were collected in the presence of 500 µM EGTA (black dashed line) and after sequential additions of 1 mM Ca 2+ (black solid line, 500 µM free Ca 2+ ) and 100 μM RyR peptides (blue solid line for RyR1, red solid line for RyR2). The spectrum of sole buffer was considered as blank and subtracted; each curve represents the average of five accumulations. Temperature was set at 25°C and signal was normalized to protein concentration.
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    Thermo Fisher ryr2
    Tertiary and quaternary structure of CaM variants in complex with <t>RyR1/2</t> peptides. (A) Cartoon representation of CaM in three different conformational states, namely apo (PDB: 1DMO ), Ca 2+ -bound (PDB: 1CLL ) and complexed with the <t>RyR2</t> peptide used in this study (PDB: 6Y4O ). The N-terminal domain is colored in yellow, the linker region in grey, while the C-terminal domain in green. The RyR2 peptide is represented in magenta. Ca 2+ ions are represented as red spheres while the side chains of N97 and Q135 are represented in sticks with C atoms colored according to the structural region, O atoms in red and N atoms in blue. (B) Pairwise sequence alignment of the Calmodulin Binding Domain-2 of human RyR1 (Uniprot entry P21817) and RyR2 (Uniprot entry Q92736). The sequence relative to the two RyR peptides employed in this study is highlighted in yellow, the residues that are not identical in such region are represented in bold and colored in red. (C) Near-UV CD spectra (250–320 nm) of 50 μM CaM were collected in the presence of 500 µM EGTA (black dashed line) and after sequential additions of 1 mM Ca 2+ (black solid line, 500 µM free Ca 2+ ) and 100 μM RyR peptides (blue solid line for RyR1, red solid line for RyR2). The spectrum of sole buffer was considered as blank and subtracted; each curve represents the average of five accumulations. Temperature was set at 25°C and signal was normalized to protein concentration.
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    Thermo Fisher overlapping ryr2 sites
    Tertiary and quaternary structure of CaM variants in complex with <t>RyR1/2</t> peptides. (A) Cartoon representation of CaM in three different conformational states, namely apo (PDB: 1DMO ), Ca 2+ -bound (PDB: 1CLL ) and complexed with the <t>RyR2</t> peptide used in this study (PDB: 6Y4O ). The N-terminal domain is colored in yellow, the linker region in grey, while the C-terminal domain in green. The RyR2 peptide is represented in magenta. Ca 2+ ions are represented as red spheres while the side chains of N97 and Q135 are represented in sticks with C atoms colored according to the structural region, O atoms in red and N atoms in blue. (B) Pairwise sequence alignment of the Calmodulin Binding Domain-2 of human RyR1 (Uniprot entry P21817) and RyR2 (Uniprot entry Q92736). The sequence relative to the two RyR peptides employed in this study is highlighted in yellow, the residues that are not identical in such region are represented in bold and colored in red. (C) Near-UV CD spectra (250–320 nm) of 50 μM CaM were collected in the presence of 500 µM EGTA (black dashed line) and after sequential additions of 1 mM Ca 2+ (black solid line, 500 µM free Ca 2+ ) and 100 μM RyR peptides (blue solid line for RyR1, red solid line for RyR2). The spectrum of sole buffer was considered as blank and subtracted; each curve represents the average of five accumulations. Temperature was set at 25°C and signal was normalized to protein concentration.
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    93
    Thermo Fisher gene exp ryr2 mm00465877 m1
    Comparative real-time PCR mRNA expression of 47 genes related to contractile function in the afferent arterioles from Notch3 −/− and wild-type littermates. Note that the expression of Cacna1h coding the α 1H subunit of the T-type Ca 2+ channel (Ca v 3.2) gene was strongly downregulated in Notch3 −/− ( p < 0.001 vs. wild-type). In contrast, Cacna1c coding the α 1C subunit of the L-type Ca 2+ channel was similarly expressed in the two strains (No. 26 on the list).
    Gene Exp Ryr2 Mm00465877 M1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    The figure indicates links of ryanodine receptors RyR1–RyR3 to neurodegenerative diseases ( , – , , , , ), ventilator-induced diaphragmatic dysfunction ( – ), heart failure ( , , , , , , , , – , ), cardiac arrhythmias ( – , – , , ), skeletal myopathies ( – , , , , ), cancer-associated muscle weakness , age-dependent loss of muscle function , and diabetes . Adapted with permission from Biochimica et Biophysica Acta: Molecular Cell Research .

    Journal: The Journal of Clinical Investigation

    Article Title: Targeting ryanodine receptors to treat human diseases

    doi: 10.1172/JCI162891

    Figure Lengend Snippet: The figure indicates links of ryanodine receptors RyR1–RyR3 to neurodegenerative diseases ( , – , , , , ), ventilator-induced diaphragmatic dysfunction ( – ), heart failure ( , , , , , , , , – , ), cardiac arrhythmias ( – , – , , ), skeletal myopathies ( – , , , , ), cancer-associated muscle weakness , age-dependent loss of muscle function , and diabetes . Adapted with permission from Biochimica et Biophysica Acta: Molecular Cell Research .

    Article Snippet: First, we found that the mutant RyR2-R2474S channel linked to CPVT had an overall alteration in its structure as determined by cryo-EM ( ).

    Techniques:

    The reconstructions show that the CPVT mutant RyR2-R2474S puts the channel into a primed state partway between closed and open, and treatment with the Rycal ARM210 or calmodulin puts the channel back toward the closed state, preventing leak. ( A ) Models of open PKA-phosphorylated RyR2 (Protein Data Bank [PDB; https://www.rcsb.org/ ]: 7U9R; yellow) and closed PKA-phosphorylated RyR2 (PDB: 7U9Q; gray). Arrows show the cytosolic shell of the PKA-phosphorylated RyR2 shifting downward and outward when the channel goes from the closed to the open state. Only the front protomer is colored. The sarcoplasmic reticular membranes are black discs. Conditions include 10 mM ATP, 150 nM free Ca 2+ , and 500 mM xanthine. ( B ) Models of closed PKA-phosphorylated RyR2 (PDB: 7U9Q; gray) and primed PKA-phosphorylated RyR2-R2474S (PDB: 7U9X; magenta), with arrows showing the cytosolic shell of RyR2-R2474S shifting downward and outward compared with closed PKA-phosphorylated RyR2, similar to the structural changes observed for PKA-phosphorylated RyR2 when the channel goes from closed to open. We define this state between closed and open as the primed state. ( C ) Models of primed PKA-phosphorylated RyR2-R2474S (PDB: 7U9X; magenta) and closed PKA-phosphorylated RyR2-R2474S + ARM210 (PDB: 7UA1; cyan). Arrows show the cytosolic shell of PKA-phosphorylated RyR2-R2474S + ARM210 shifting upward and inward compared with RyR2-R2474S, reversing the primed state closer to the closed state. ( D ) Models of primed PKA-phosphorylated RyR2-R2474S (PDB: 7U9X; magenta) and closed PKA-phosphorylated RyR2-R2474S + calmodulin (CaM) (PDB: 7UA3; cyan). Similarly to the Rycal ARM210, CaM reverses the primed state back to the closed state. Reconstructions were adapted with permission from Structure .

    Journal: The Journal of Clinical Investigation

    Article Title: Targeting ryanodine receptors to treat human diseases

    doi: 10.1172/JCI162891

    Figure Lengend Snippet: The reconstructions show that the CPVT mutant RyR2-R2474S puts the channel into a primed state partway between closed and open, and treatment with the Rycal ARM210 or calmodulin puts the channel back toward the closed state, preventing leak. ( A ) Models of open PKA-phosphorylated RyR2 (Protein Data Bank [PDB; https://www.rcsb.org/ ]: 7U9R; yellow) and closed PKA-phosphorylated RyR2 (PDB: 7U9Q; gray). Arrows show the cytosolic shell of the PKA-phosphorylated RyR2 shifting downward and outward when the channel goes from the closed to the open state. Only the front protomer is colored. The sarcoplasmic reticular membranes are black discs. Conditions include 10 mM ATP, 150 nM free Ca 2+ , and 500 mM xanthine. ( B ) Models of closed PKA-phosphorylated RyR2 (PDB: 7U9Q; gray) and primed PKA-phosphorylated RyR2-R2474S (PDB: 7U9X; magenta), with arrows showing the cytosolic shell of RyR2-R2474S shifting downward and outward compared with closed PKA-phosphorylated RyR2, similar to the structural changes observed for PKA-phosphorylated RyR2 when the channel goes from closed to open. We define this state between closed and open as the primed state. ( C ) Models of primed PKA-phosphorylated RyR2-R2474S (PDB: 7U9X; magenta) and closed PKA-phosphorylated RyR2-R2474S + ARM210 (PDB: 7UA1; cyan). Arrows show the cytosolic shell of PKA-phosphorylated RyR2-R2474S + ARM210 shifting upward and inward compared with RyR2-R2474S, reversing the primed state closer to the closed state. ( D ) Models of primed PKA-phosphorylated RyR2-R2474S (PDB: 7U9X; magenta) and closed PKA-phosphorylated RyR2-R2474S + calmodulin (CaM) (PDB: 7UA3; cyan). Similarly to the Rycal ARM210, CaM reverses the primed state back to the closed state. Reconstructions were adapted with permission from Structure .

    Article Snippet: First, we found that the mutant RyR2-R2474S channel linked to CPVT had an overall alteration in its structure as determined by cryo-EM ( ).

    Techniques: Mutagenesis

    Left: Negative stain electron microscopic images of the “feet structures” of junctional terminal cisternae vesicles and purified ryanodine receptors first visualized in 1987 (images reproduced with permission from the Journal of Biological Chemistry ; ref. ). By comparing the shape and size of the purified RyR to that of the densities protruding from the surface of the SR vesicles, Fleischer and colleagues concluded that RyR was the foot structure spanning the gap between the terminal cisternae of the SR and T-tubules. But it was not known until later when RyR1 was cloned and functionally expressed that it was indeed the Ca 2+ release channel required for muscle excitation-contraction coupling . Arrowheads denote individual RyR1 channels in the SR membrane; arrows denote purified, isolated individual RyR1 channels. Original magnification, ×140,000. Right: The cryo-EM structure of RyR1 at approximately 2.4 Å resolution; two opposing protomers are shown in the side view (adapted with permission from Structure ; ref. ).

    Journal: The Journal of Clinical Investigation

    Article Title: Targeting ryanodine receptors to treat human diseases

    doi: 10.1172/JCI162891

    Figure Lengend Snippet: Left: Negative stain electron microscopic images of the “feet structures” of junctional terminal cisternae vesicles and purified ryanodine receptors first visualized in 1987 (images reproduced with permission from the Journal of Biological Chemistry ; ref. ). By comparing the shape and size of the purified RyR to that of the densities protruding from the surface of the SR vesicles, Fleischer and colleagues concluded that RyR was the foot structure spanning the gap between the terminal cisternae of the SR and T-tubules. But it was not known until later when RyR1 was cloned and functionally expressed that it was indeed the Ca 2+ release channel required for muscle excitation-contraction coupling . Arrowheads denote individual RyR1 channels in the SR membrane; arrows denote purified, isolated individual RyR1 channels. Original magnification, ×140,000. Right: The cryo-EM structure of RyR1 at approximately 2.4 Å resolution; two opposing protomers are shown in the side view (adapted with permission from Structure ; ref. ).

    Article Snippet: First, we found that the mutant RyR2-R2474S channel linked to CPVT had an overall alteration in its structure as determined by cryo-EM ( ).

    Techniques: Staining, Purification, Clone Assay, Isolation, Cryo-EM Sample Prep

    The figure indicates links of ryanodine receptors RyR1–RyR3 to neurodegenerative diseases ( , – , , , , ), ventilator-induced diaphragmatic dysfunction ( – ), heart failure ( , , , , , , , , – , ), cardiac arrhythmias ( – , – , , ), skeletal myopathies ( – , , , , ), cancer-associated muscle weakness , age-dependent loss of muscle function , and diabetes . Adapted with permission from Biochimica et Biophysica Acta: Molecular Cell Research .

    Journal: The Journal of Clinical Investigation

    Article Title: Targeting ryanodine receptors to treat human diseases

    doi: 10.1172/JCI162891

    Figure Lengend Snippet: The figure indicates links of ryanodine receptors RyR1–RyR3 to neurodegenerative diseases ( , – , , , , ), ventilator-induced diaphragmatic dysfunction ( – ), heart failure ( , , , , , , , , – , ), cardiac arrhythmias ( – , – , , ), skeletal myopathies ( – , , , , ), cancer-associated muscle weakness , age-dependent loss of muscle function , and diabetes . Adapted with permission from Biochimica et Biophysica Acta: Molecular Cell Research .

    Article Snippet: We expressed the human recombinant RyR2 in HEK293T cells and purified it for cryo-EM; this allowed us to also introduce the CPVT mutations into the channel.

    Techniques:

    The reconstructions show that the CPVT mutant RyR2-R2474S puts the channel into a primed state partway between closed and open, and treatment with the Rycal ARM210 or calmodulin puts the channel back toward the closed state, preventing leak. ( A ) Models of open PKA-phosphorylated RyR2 (Protein Data Bank [PDB; https://www.rcsb.org/ ]: 7U9R; yellow) and closed PKA-phosphorylated RyR2 (PDB: 7U9Q; gray). Arrows show the cytosolic shell of the PKA-phosphorylated RyR2 shifting downward and outward when the channel goes from the closed to the open state. Only the front protomer is colored. The sarcoplasmic reticular membranes are black discs. Conditions include 10 mM ATP, 150 nM free Ca 2+ , and 500 mM xanthine. ( B ) Models of closed PKA-phosphorylated RyR2 (PDB: 7U9Q; gray) and primed PKA-phosphorylated RyR2-R2474S (PDB: 7U9X; magenta), with arrows showing the cytosolic shell of RyR2-R2474S shifting downward and outward compared with closed PKA-phosphorylated RyR2, similar to the structural changes observed for PKA-phosphorylated RyR2 when the channel goes from closed to open. We define this state between closed and open as the primed state. ( C ) Models of primed PKA-phosphorylated RyR2-R2474S (PDB: 7U9X; magenta) and closed PKA-phosphorylated RyR2-R2474S + ARM210 (PDB: 7UA1; cyan). Arrows show the cytosolic shell of PKA-phosphorylated RyR2-R2474S + ARM210 shifting upward and inward compared with RyR2-R2474S, reversing the primed state closer to the closed state. ( D ) Models of primed PKA-phosphorylated RyR2-R2474S (PDB: 7U9X; magenta) and closed PKA-phosphorylated RyR2-R2474S + calmodulin (CaM) (PDB: 7UA3; cyan). Similarly to the Rycal ARM210, CaM reverses the primed state back to the closed state. Reconstructions were adapted with permission from Structure .

    Journal: The Journal of Clinical Investigation

    Article Title: Targeting ryanodine receptors to treat human diseases

    doi: 10.1172/JCI162891

    Figure Lengend Snippet: The reconstructions show that the CPVT mutant RyR2-R2474S puts the channel into a primed state partway between closed and open, and treatment with the Rycal ARM210 or calmodulin puts the channel back toward the closed state, preventing leak. ( A ) Models of open PKA-phosphorylated RyR2 (Protein Data Bank [PDB; https://www.rcsb.org/ ]: 7U9R; yellow) and closed PKA-phosphorylated RyR2 (PDB: 7U9Q; gray). Arrows show the cytosolic shell of the PKA-phosphorylated RyR2 shifting downward and outward when the channel goes from the closed to the open state. Only the front protomer is colored. The sarcoplasmic reticular membranes are black discs. Conditions include 10 mM ATP, 150 nM free Ca 2+ , and 500 mM xanthine. ( B ) Models of closed PKA-phosphorylated RyR2 (PDB: 7U9Q; gray) and primed PKA-phosphorylated RyR2-R2474S (PDB: 7U9X; magenta), with arrows showing the cytosolic shell of RyR2-R2474S shifting downward and outward compared with closed PKA-phosphorylated RyR2, similar to the structural changes observed for PKA-phosphorylated RyR2 when the channel goes from closed to open. We define this state between closed and open as the primed state. ( C ) Models of primed PKA-phosphorylated RyR2-R2474S (PDB: 7U9X; magenta) and closed PKA-phosphorylated RyR2-R2474S + ARM210 (PDB: 7UA1; cyan). Arrows show the cytosolic shell of PKA-phosphorylated RyR2-R2474S + ARM210 shifting upward and inward compared with RyR2-R2474S, reversing the primed state closer to the closed state. ( D ) Models of primed PKA-phosphorylated RyR2-R2474S (PDB: 7U9X; magenta) and closed PKA-phosphorylated RyR2-R2474S + calmodulin (CaM) (PDB: 7UA3; cyan). Similarly to the Rycal ARM210, CaM reverses the primed state back to the closed state. Reconstructions were adapted with permission from Structure .

    Article Snippet: We expressed the human recombinant RyR2 in HEK293T cells and purified it for cryo-EM; this allowed us to also introduce the CPVT mutations into the channel.

    Techniques: Mutagenesis

    Left: Negative stain electron microscopic images of the “feet structures” of junctional terminal cisternae vesicles and purified ryanodine receptors first visualized in 1987 (images reproduced with permission from the Journal of Biological Chemistry ; ref. ). By comparing the shape and size of the purified RyR to that of the densities protruding from the surface of the SR vesicles, Fleischer and colleagues concluded that RyR was the foot structure spanning the gap between the terminal cisternae of the SR and T-tubules. But it was not known until later when RyR1 was cloned and functionally expressed that it was indeed the Ca 2+ release channel required for muscle excitation-contraction coupling . Arrowheads denote individual RyR1 channels in the SR membrane; arrows denote purified, isolated individual RyR1 channels. Original magnification, ×140,000. Right: The cryo-EM structure of RyR1 at approximately 2.4 Å resolution; two opposing protomers are shown in the side view (adapted with permission from Structure ; ref. ).

    Journal: The Journal of Clinical Investigation

    Article Title: Targeting ryanodine receptors to treat human diseases

    doi: 10.1172/JCI162891

    Figure Lengend Snippet: Left: Negative stain electron microscopic images of the “feet structures” of junctional terminal cisternae vesicles and purified ryanodine receptors first visualized in 1987 (images reproduced with permission from the Journal of Biological Chemistry ; ref. ). By comparing the shape and size of the purified RyR to that of the densities protruding from the surface of the SR vesicles, Fleischer and colleagues concluded that RyR was the foot structure spanning the gap between the terminal cisternae of the SR and T-tubules. But it was not known until later when RyR1 was cloned and functionally expressed that it was indeed the Ca 2+ release channel required for muscle excitation-contraction coupling . Arrowheads denote individual RyR1 channels in the SR membrane; arrows denote purified, isolated individual RyR1 channels. Original magnification, ×140,000. Right: The cryo-EM structure of RyR1 at approximately 2.4 Å resolution; two opposing protomers are shown in the side view (adapted with permission from Structure ; ref. ).

    Article Snippet: We expressed the human recombinant RyR2 in HEK293T cells and purified it for cryo-EM; this allowed us to also introduce the CPVT mutations into the channel.

    Techniques: Staining, Purification, Clone Assay, Isolation, Cryo-EM Sample Prep

    The figure indicates links of ryanodine receptors RyR1–RyR3 to neurodegenerative diseases ( , – , , , , ), ventilator-induced diaphragmatic dysfunction ( – ), heart failure ( , , , , , , , , – , ), cardiac arrhythmias ( – , – , , ), skeletal myopathies ( – , , , , ), cancer-associated muscle weakness , age-dependent loss of muscle function , and diabetes . Adapted with permission from Biochimica et Biophysica Acta: Molecular Cell Research .

    Journal: The Journal of Clinical Investigation

    Article Title: Targeting ryanodine receptors to treat human diseases

    doi: 10.1172/JCI162891

    Figure Lengend Snippet: The figure indicates links of ryanodine receptors RyR1–RyR3 to neurodegenerative diseases ( , – , , , , ), ventilator-induced diaphragmatic dysfunction ( – ), heart failure ( , , , , , , , , – , ), cardiac arrhythmias ( – , – , , ), skeletal myopathies ( – , , , , ), cancer-associated muscle weakness , age-dependent loss of muscle function , and diabetes . Adapted with permission from Biochimica et Biophysica Acta: Molecular Cell Research .

    Article Snippet: Collaborating with Wayne Hendrickson and Joachim Frank, our group, along with two others ( , ), was able to solve the structure of the mammalian RyR1 using cryo-EM ( ).

    Techniques:

    The reconstructions show that the CPVT mutant RyR2-R2474S puts the channel into a primed state partway between closed and open, and treatment with the Rycal ARM210 or calmodulin puts the channel back toward the closed state, preventing leak. ( A ) Models of open PKA-phosphorylated RyR2 (Protein Data Bank [PDB; https://www.rcsb.org/ ]: 7U9R; yellow) and closed PKA-phosphorylated RyR2 (PDB: 7U9Q; gray). Arrows show the cytosolic shell of the PKA-phosphorylated RyR2 shifting downward and outward when the channel goes from the closed to the open state. Only the front protomer is colored. The sarcoplasmic reticular membranes are black discs. Conditions include 10 mM ATP, 150 nM free Ca 2+ , and 500 mM xanthine. ( B ) Models of closed PKA-phosphorylated RyR2 (PDB: 7U9Q; gray) and primed PKA-phosphorylated RyR2-R2474S (PDB: 7U9X; magenta), with arrows showing the cytosolic shell of RyR2-R2474S shifting downward and outward compared with closed PKA-phosphorylated RyR2, similar to the structural changes observed for PKA-phosphorylated RyR2 when the channel goes from closed to open. We define this state between closed and open as the primed state. ( C ) Models of primed PKA-phosphorylated RyR2-R2474S (PDB: 7U9X; magenta) and closed PKA-phosphorylated RyR2-R2474S + ARM210 (PDB: 7UA1; cyan). Arrows show the cytosolic shell of PKA-phosphorylated RyR2-R2474S + ARM210 shifting upward and inward compared with RyR2-R2474S, reversing the primed state closer to the closed state. ( D ) Models of primed PKA-phosphorylated RyR2-R2474S (PDB: 7U9X; magenta) and closed PKA-phosphorylated RyR2-R2474S + calmodulin (CaM) (PDB: 7UA3; cyan). Similarly to the Rycal ARM210, CaM reverses the primed state back to the closed state. Reconstructions were adapted with permission from Structure .

    Journal: The Journal of Clinical Investigation

    Article Title: Targeting ryanodine receptors to treat human diseases

    doi: 10.1172/JCI162891

    Figure Lengend Snippet: The reconstructions show that the CPVT mutant RyR2-R2474S puts the channel into a primed state partway between closed and open, and treatment with the Rycal ARM210 or calmodulin puts the channel back toward the closed state, preventing leak. ( A ) Models of open PKA-phosphorylated RyR2 (Protein Data Bank [PDB; https://www.rcsb.org/ ]: 7U9R; yellow) and closed PKA-phosphorylated RyR2 (PDB: 7U9Q; gray). Arrows show the cytosolic shell of the PKA-phosphorylated RyR2 shifting downward and outward when the channel goes from the closed to the open state. Only the front protomer is colored. The sarcoplasmic reticular membranes are black discs. Conditions include 10 mM ATP, 150 nM free Ca 2+ , and 500 mM xanthine. ( B ) Models of closed PKA-phosphorylated RyR2 (PDB: 7U9Q; gray) and primed PKA-phosphorylated RyR2-R2474S (PDB: 7U9X; magenta), with arrows showing the cytosolic shell of RyR2-R2474S shifting downward and outward compared with closed PKA-phosphorylated RyR2, similar to the structural changes observed for PKA-phosphorylated RyR2 when the channel goes from closed to open. We define this state between closed and open as the primed state. ( C ) Models of primed PKA-phosphorylated RyR2-R2474S (PDB: 7U9X; magenta) and closed PKA-phosphorylated RyR2-R2474S + ARM210 (PDB: 7UA1; cyan). Arrows show the cytosolic shell of PKA-phosphorylated RyR2-R2474S + ARM210 shifting upward and inward compared with RyR2-R2474S, reversing the primed state closer to the closed state. ( D ) Models of primed PKA-phosphorylated RyR2-R2474S (PDB: 7U9X; magenta) and closed PKA-phosphorylated RyR2-R2474S + calmodulin (CaM) (PDB: 7UA3; cyan). Similarly to the Rycal ARM210, CaM reverses the primed state back to the closed state. Reconstructions were adapted with permission from Structure .

    Article Snippet: Collaborating with Wayne Hendrickson and Joachim Frank, our group, along with two others ( , ), was able to solve the structure of the mammalian RyR1 using cryo-EM ( ).

    Techniques: Mutagenesis

    Left: Negative stain electron microscopic images of the “feet structures” of junctional terminal cisternae vesicles and purified ryanodine receptors first visualized in 1987 (images reproduced with permission from the Journal of Biological Chemistry ; ref. ). By comparing the shape and size of the purified RyR to that of the densities protruding from the surface of the SR vesicles, Fleischer and colleagues concluded that RyR was the foot structure spanning the gap between the terminal cisternae of the SR and T-tubules. But it was not known until later when RyR1 was cloned and functionally expressed that it was indeed the Ca 2+ release channel required for muscle excitation-contraction coupling . Arrowheads denote individual RyR1 channels in the SR membrane; arrows denote purified, isolated individual RyR1 channels. Original magnification, ×140,000. Right: The cryo-EM structure of RyR1 at approximately 2.4 Å resolution; two opposing protomers are shown in the side view (adapted with permission from Structure ; ref. ).

    Journal: The Journal of Clinical Investigation

    Article Title: Targeting ryanodine receptors to treat human diseases

    doi: 10.1172/JCI162891

    Figure Lengend Snippet: Left: Negative stain electron microscopic images of the “feet structures” of junctional terminal cisternae vesicles and purified ryanodine receptors first visualized in 1987 (images reproduced with permission from the Journal of Biological Chemistry ; ref. ). By comparing the shape and size of the purified RyR to that of the densities protruding from the surface of the SR vesicles, Fleischer and colleagues concluded that RyR was the foot structure spanning the gap between the terminal cisternae of the SR and T-tubules. But it was not known until later when RyR1 was cloned and functionally expressed that it was indeed the Ca 2+ release channel required for muscle excitation-contraction coupling . Arrowheads denote individual RyR1 channels in the SR membrane; arrows denote purified, isolated individual RyR1 channels. Original magnification, ×140,000. Right: The cryo-EM structure of RyR1 at approximately 2.4 Å resolution; two opposing protomers are shown in the side view (adapted with permission from Structure ; ref. ).

    Article Snippet: Collaborating with Wayne Hendrickson and Joachim Frank, our group, along with two others ( , ), was able to solve the structure of the mammalian RyR1 using cryo-EM ( ).

    Techniques: Staining, Purification, Clone Assay, Isolation, Cryo-EM Sample Prep

    Muscle and Ca V 1.1 organization. A) Muscle to muscle cell perspective. ( left ) Morphology of a segment of a skeletal muscle fiber ( right ). Note the characteristic striated pattern of muscle fibers, which results from highly organized array between sarcolemma, sarcoplasmic reticulum (SR), contractile elements and cytoarchitecture of the fibers. B) Structure of the triad. The cartoon depicts the triad, a specialized membrane-organelle array formed by the T-tubule and two segments of the terminal junctional SR. The T-tubules are infoldings of the sarcolemma that propagate the action potential radially into the fiber. Ca V 1.1 (blue) are located at the T-tubules, working primarily as voltage sensors that initiate the early steps of EC coupling. The SR Ca 2+ release channel, RyR1 (brown), is predominantly located on the junctional domain of the SR surface. Typical profiles of triads (cross-sections) contain only two rows of RyR1 associated with alternating tetrads . C) Detailed architecture of the triad with a focus on Ca V 1.1 tetrads and RyR1 arrays as shown in (b). About half of the total RyR1s do not associate with Ca V 1.1, resulting in an alternating pattern of “free” and Ca V 1.1-associated RyR1s. Note: In addition to Ca V 1.1 tetrad (blue) and RyR1 (yellow) Ca 2+ release channels, many other proteins form part of the T-tubule- junctional SR complex (e.g. junctophilin, triadin, junctin, calsequestrin, not indicated here).

    Journal: Channels

    Article Title: Advances in Ca V 1.1 gating: New insights into permeation and voltage-sensing mechanisms

    doi: 10.1080/19336950.2023.2167569

    Figure Lengend Snippet: Muscle and Ca V 1.1 organization. A) Muscle to muscle cell perspective. ( left ) Morphology of a segment of a skeletal muscle fiber ( right ). Note the characteristic striated pattern of muscle fibers, which results from highly organized array between sarcolemma, sarcoplasmic reticulum (SR), contractile elements and cytoarchitecture of the fibers. B) Structure of the triad. The cartoon depicts the triad, a specialized membrane-organelle array formed by the T-tubule and two segments of the terminal junctional SR. The T-tubules are infoldings of the sarcolemma that propagate the action potential radially into the fiber. Ca V 1.1 (blue) are located at the T-tubules, working primarily as voltage sensors that initiate the early steps of EC coupling. The SR Ca 2+ release channel, RyR1 (brown), is predominantly located on the junctional domain of the SR surface. Typical profiles of triads (cross-sections) contain only two rows of RyR1 associated with alternating tetrads . C) Detailed architecture of the triad with a focus on Ca V 1.1 tetrads and RyR1 arrays as shown in (b). About half of the total RyR1s do not associate with Ca V 1.1, resulting in an alternating pattern of “free” and Ca V 1.1-associated RyR1s. Note: In addition to Ca V 1.1 tetrad (blue) and RyR1 (yellow) Ca 2+ release channels, many other proteins form part of the T-tubule- junctional SR complex (e.g. junctophilin, triadin, junctin, calsequestrin, not indicated here).

    Article Snippet: Further refinement of the structures of the Ca V 1.1 and RyR1, ideally in their native membranes with cryo-EM and single-particle image reconstruction, is needed to identify the key determinants for Ca V 1.1-RyR1 electromechanical coupling.

    Techniques:

    Ca V 1.1 structure. Heteromultimeric protein complex of Ca V 1.1. α 1S , β 1a , α 2 δ-1, γ subunits, and SH3 domains of Stac3 are colored in blue, green, Orange, yellow, and purple, respectively. B) Side and upper views of the α 1S subunit, each domain is shown in shades of blue. Red dots indicate Ca 2+ ions. Panels A and B were prepared with Chimera . Protein data bank (PDB) IDs: 5GJV (Ca V 1.1) and 6UY7 (Stac3). STAC3 orientation and position relative to α1 and the β subunits is unknown.

    Journal: Channels

    Article Title: Advances in Ca V 1.1 gating: New insights into permeation and voltage-sensing mechanisms

    doi: 10.1080/19336950.2023.2167569

    Figure Lengend Snippet: Ca V 1.1 structure. Heteromultimeric protein complex of Ca V 1.1. α 1S , β 1a , α 2 δ-1, γ subunits, and SH3 domains of Stac3 are colored in blue, green, Orange, yellow, and purple, respectively. B) Side and upper views of the α 1S subunit, each domain is shown in shades of blue. Red dots indicate Ca 2+ ions. Panels A and B were prepared with Chimera . Protein data bank (PDB) IDs: 5GJV (Ca V 1.1) and 6UY7 (Stac3). STAC3 orientation and position relative to α1 and the β subunits is unknown.

    Article Snippet: Further refinement of the structures of the Ca V 1.1 and RyR1, ideally in their native membranes with cryo-EM and single-particle image reconstruction, is needed to identify the key determinants for Ca V 1.1-RyR1 electromechanical coupling.

    Techniques:

    Topology of the voltage sensor and selectivity filter domain of Ca V 1.1 α 1S subunit. A) Cartoon of α 1S subunit topology shows four homologous but non-identical domains, each containing six transmembrane helices (S1-S6). S1-S4 represents the voltage sensing domain (VSD, blue) while S6-S6 represents the pore-forming domain (Pore, yellow). Each domain contains an S5-S6 loop (P-Loop) buried in the pore acting as a selectivity filter. Intracellular loops connecting each domain are variable in length. The I–II and II–III loops are critical for EC coupling. Deletion of Exon 29 induces a 19 amino acid shortening of the S3-S4IV extracellular loop (red). Positively charged lysine and arginine within S4 are indicated as a “+” while countercharges within S2 and S3 are indicated as “-“. B) Selectivity filter amino acid sequences of rabbit, human, and zebra fish Ca V 1.1 and human Ca V 2.2. Critical glutamate residues used for Ca 2+ selectivity are indicated in red, while zebra fish P-loop Ca 2+ non-conductive mutations N617D is indicated in cyan. Selectivity filter (SF) sequence is indicated in bold and yellow, while alpha helices within P-loop are indicated in green. C) Top view of rabbit Ca V 1.1 selectivity filter colored as in B. Alpha helices are illustrated as ribbon, while selectivity filter motif is shown as a stick. N617D mutation is indicated in blue as a stick. Two ions can bind the pore, each stabilized by P-loops from opposite domains. D) S4 helices amino acid sequences of rabbit, human, and zebra fish Ca V 1.1 and human Ca V 2.2. Conserved gating charges are indicated in yellow (alignment based on Ref.) . Positively charged amino acids not considered as gating charges are highlighted in pink. E) Side view of rabbit Ca V 1.1 voltage sensing domains. α-helices are illustrated as ribbons, while gating charges, countercharges, and gating charge transfer phenylalanine are illustrated as sticks (in blue, green, and Orange respectively). Note that some helices (i.e. S1) are not fully displayed to facility the visualization of other elements. Panels C and E were created with PyMol  from P07293. rCa V 1.1: uniport IDs P07293; hCa V 1.1: uniport IDs Q13698; zfCa V 1.1a: GenBank accession no. FJ76922; zfCa V 1.1b: GenBank accession no. AY49569; hCa V 2.2: Q00975.

    Journal: Channels

    Article Title: Advances in Ca V 1.1 gating: New insights into permeation and voltage-sensing mechanisms

    doi: 10.1080/19336950.2023.2167569

    Figure Lengend Snippet: Topology of the voltage sensor and selectivity filter domain of Ca V 1.1 α 1S subunit. A) Cartoon of α 1S subunit topology shows four homologous but non-identical domains, each containing six transmembrane helices (S1-S6). S1-S4 represents the voltage sensing domain (VSD, blue) while S6-S6 represents the pore-forming domain (Pore, yellow). Each domain contains an S5-S6 loop (P-Loop) buried in the pore acting as a selectivity filter. Intracellular loops connecting each domain are variable in length. The I–II and II–III loops are critical for EC coupling. Deletion of Exon 29 induces a 19 amino acid shortening of the S3-S4IV extracellular loop (red). Positively charged lysine and arginine within S4 are indicated as a “+” while countercharges within S2 and S3 are indicated as “-“. B) Selectivity filter amino acid sequences of rabbit, human, and zebra fish Ca V 1.1 and human Ca V 2.2. Critical glutamate residues used for Ca 2+ selectivity are indicated in red, while zebra fish P-loop Ca 2+ non-conductive mutations N617D is indicated in cyan. Selectivity filter (SF) sequence is indicated in bold and yellow, while alpha helices within P-loop are indicated in green. C) Top view of rabbit Ca V 1.1 selectivity filter colored as in B. Alpha helices are illustrated as ribbon, while selectivity filter motif is shown as a stick. N617D mutation is indicated in blue as a stick. Two ions can bind the pore, each stabilized by P-loops from opposite domains. D) S4 helices amino acid sequences of rabbit, human, and zebra fish Ca V 1.1 and human Ca V 2.2. Conserved gating charges are indicated in yellow (alignment based on Ref.) . Positively charged amino acids not considered as gating charges are highlighted in pink. E) Side view of rabbit Ca V 1.1 voltage sensing domains. α-helices are illustrated as ribbons, while gating charges, countercharges, and gating charge transfer phenylalanine are illustrated as sticks (in blue, green, and Orange respectively). Note that some helices (i.e. S1) are not fully displayed to facility the visualization of other elements. Panels C and E were created with PyMol from P07293. rCa V 1.1: uniport IDs P07293; hCa V 1.1: uniport IDs Q13698; zfCa V 1.1a: GenBank accession no. FJ76922; zfCa V 1.1b: GenBank accession no. AY49569; hCa V 2.2: Q00975.

    Article Snippet: Further refinement of the structures of the Ca V 1.1 and RyR1, ideally in their native membranes with cryo-EM and single-particle image reconstruction, is needed to identify the key determinants for Ca V 1.1-RyR1 electromechanical coupling.

    Techniques: Sequencing, Mutagenesis

    Allosteric model for L-type Ca 2+ current and RyR Ca 2+ release. A) Hypothetical structural representation of a Ca V 1.1 tetrad coupled to RyR1 homotetramer. Four α 1S subunits (blue) are opposed to a RyR homotetramer (yellow). Ca V 1.1 has four VSDs that alter their conformation in response to surface transmembranal voltage changes. RyR1 does not have an intrinsic voltage sensing mechanism and relay on the voltage sensing machinery of Ca V 1.1, via mechanical coupling, to release Ca 2+ . The α 1S -RyR1 organization depicted here is hypothetical but based on the model suggested by Samsó et al. . Side and upper views (left and right respectively) in Panel A were created with BioRender and Chimera , PDBs: 5GJW and 5TAL for Ca V 1.1 and RyR1, respectively. B) Allosteric scheme for voltage dependent Ca V 1.1 channel opening and RyR1 activation. Four distinct VSDs (VSDI-IV) within one Ca V 1.1 control Ca V 1.1's pore conformation from close (“C”) to open (“O”) with either all or some VSDs in active (“A”) or resting (“R”) state. In parallel, four distinct Ca V 1.1s, each with four VSDs (i.e. tetrads array) control RyR1 pore conformational change from close (“C”) to open (“O”) with either all or some VSDs in active (“A”) or resting (“R”) states. The probability of each state in VSDs (R or A) is under the influence of the membrane voltage (ΔV). Note that in principle, based on structural evidence [ , , ], it is likely that four independently functioning Ca V 1.1 channels (tetrads) are associated with one RyR giving four sets of four VSDs, requiring a total of 16 VSDs. However, recent fluorometric experiments [ , ] suggest some features and reconsiderations for this model: not all VSDs within Ca V 1.1 contribute equally to gate Cav1.1 pore opening and not all VSDs contribute equally to gate RyR1 Ca 2+ release. How many VSDs per tetrad and which of the four VSDs of Ca V 1.1 are needed for RyR1-mediated Ca 2+ release is unknown.

    Journal: Channels

    Article Title: Advances in Ca V 1.1 gating: New insights into permeation and voltage-sensing mechanisms

    doi: 10.1080/19336950.2023.2167569

    Figure Lengend Snippet: Allosteric model for L-type Ca 2+ current and RyR Ca 2+ release. A) Hypothetical structural representation of a Ca V 1.1 tetrad coupled to RyR1 homotetramer. Four α 1S subunits (blue) are opposed to a RyR homotetramer (yellow). Ca V 1.1 has four VSDs that alter their conformation in response to surface transmembranal voltage changes. RyR1 does not have an intrinsic voltage sensing mechanism and relay on the voltage sensing machinery of Ca V 1.1, via mechanical coupling, to release Ca 2+ . The α 1S -RyR1 organization depicted here is hypothetical but based on the model suggested by Samsó et al. . Side and upper views (left and right respectively) in Panel A were created with BioRender and Chimera , PDBs: 5GJW and 5TAL for Ca V 1.1 and RyR1, respectively. B) Allosteric scheme for voltage dependent Ca V 1.1 channel opening and RyR1 activation. Four distinct VSDs (VSDI-IV) within one Ca V 1.1 control Ca V 1.1's pore conformation from close (“C”) to open (“O”) with either all or some VSDs in active (“A”) or resting (“R”) state. In parallel, four distinct Ca V 1.1s, each with four VSDs (i.e. tetrads array) control RyR1 pore conformational change from close (“C”) to open (“O”) with either all or some VSDs in active (“A”) or resting (“R”) states. The probability of each state in VSDs (R or A) is under the influence of the membrane voltage (ΔV). Note that in principle, based on structural evidence [ , , ], it is likely that four independently functioning Ca V 1.1 channels (tetrads) are associated with one RyR giving four sets of four VSDs, requiring a total of 16 VSDs. However, recent fluorometric experiments [ , ] suggest some features and reconsiderations for this model: not all VSDs within Ca V 1.1 contribute equally to gate Cav1.1 pore opening and not all VSDs contribute equally to gate RyR1 Ca 2+ release. How many VSDs per tetrad and which of the four VSDs of Ca V 1.1 are needed for RyR1-mediated Ca 2+ release is unknown.

    Article Snippet: Further refinement of the structures of the Ca V 1.1 and RyR1, ideally in their native membranes with cryo-EM and single-particle image reconstruction, is needed to identify the key determinants for Ca V 1.1-RyR1 electromechanical coupling.

    Techniques: Activation Assay

    Functional site-directed fluorometry of Ca V 1.1 S4 signals evaluated in Xenopus Oocytes and muscle fibers. A) Ionic current recording from cut-open oocytes (black) with 2 mM Ba 2+ in the external and superimposed fluorometric signal from each VSDs. Note the overlap of the fluorometric and ionic signal for VSD-I. Mean voltage dependence of the fluorometric signal for each VSD from cut open oocytes voltage clamp in presence of 2 mM Ba 2+ and fitted with a Boltzmann function. Note the differences in voltage dependence and slope of each VSDs fluorometric signal. C) Normalized fluorometric signal recorded from muscle fibers in response to self-propagated action potential by field stimulation and its comparison with optically measured membrane voltage (AP, yellow), action potential-evoked charge movement (Q, gray), Ca 2+ transient (Ca 2+ , black), and estimated SR Ca 2+ release flux (Rel, olive). D) Overlay of normalized fluorometric signals presented in C and kinetics quantification. Note the differences in kinetics for fluorometric signals from different VSDs. Time to peak, rise time, and time to 50% are faster for VSD-II. Panels A and B, and C, reproduced with permission from Refs. . and , respectively. Panel D, unpublished analysis from Ref.

    Journal: Channels

    Article Title: Advances in Ca V 1.1 gating: New insights into permeation and voltage-sensing mechanisms

    doi: 10.1080/19336950.2023.2167569

    Figure Lengend Snippet: Functional site-directed fluorometry of Ca V 1.1 S4 signals evaluated in Xenopus Oocytes and muscle fibers. A) Ionic current recording from cut-open oocytes (black) with 2 mM Ba 2+ in the external and superimposed fluorometric signal from each VSDs. Note the overlap of the fluorometric and ionic signal for VSD-I. Mean voltage dependence of the fluorometric signal for each VSD from cut open oocytes voltage clamp in presence of 2 mM Ba 2+ and fitted with a Boltzmann function. Note the differences in voltage dependence and slope of each VSDs fluorometric signal. C) Normalized fluorometric signal recorded from muscle fibers in response to self-propagated action potential by field stimulation and its comparison with optically measured membrane voltage (AP, yellow), action potential-evoked charge movement (Q, gray), Ca 2+ transient (Ca 2+ , black), and estimated SR Ca 2+ release flux (Rel, olive). D) Overlay of normalized fluorometric signals presented in C and kinetics quantification. Note the differences in kinetics for fluorometric signals from different VSDs. Time to peak, rise time, and time to 50% are faster for VSD-II. Panels A and B, and C, reproduced with permission from Refs. . and , respectively. Panel D, unpublished analysis from Ref.

    Article Snippet: Further refinement of the structures of the Ca V 1.1 and RyR1, ideally in their native membranes with cryo-EM and single-particle image reconstruction, is needed to identify the key determinants for Ca V 1.1-RyR1 electromechanical coupling.

    Techniques: Functional Assay

    Journal: Channels

    Article Title: Advances in Ca V 1.1 gating: New insights into permeation and voltage-sensing mechanisms

    doi: 10.1080/19336950.2023.2167569

    Figure Lengend Snippet: Comparison of Ca V 1.1 fluorometric signals obtained using cut-open voltage clamp in Xenopus oocytes or field stimulation in mouse muscle fibers.

    Article Snippet: Further refinement of the structures of the Ca V 1.1 and RyR1, ideally in their native membranes with cryo-EM and single-particle image reconstruction, is needed to identify the key determinants for Ca V 1.1-RyR1 electromechanical coupling.

    Techniques: Microscopy, Construct, Labeling, Expressing, Injection

    Tertiary and quaternary structure of CaM variants in complex with RyR1/2 peptides. (A) Cartoon representation of CaM in three different conformational states, namely apo (PDB: 1DMO ), Ca 2+ -bound (PDB: 1CLL ) and complexed with the RyR2 peptide used in this study (PDB: 6Y4O ). The N-terminal domain is colored in yellow, the linker region in grey, while the C-terminal domain in green. The RyR2 peptide is represented in magenta. Ca 2+ ions are represented as red spheres while the side chains of N97 and Q135 are represented in sticks with C atoms colored according to the structural region, O atoms in red and N atoms in blue. (B) Pairwise sequence alignment of the Calmodulin Binding Domain-2 of human RyR1 (Uniprot entry P21817) and RyR2 (Uniprot entry Q92736). The sequence relative to the two RyR peptides employed in this study is highlighted in yellow, the residues that are not identical in such region are represented in bold and colored in red. (C) Near-UV CD spectra (250–320 nm) of 50 μM CaM were collected in the presence of 500 µM EGTA (black dashed line) and after sequential additions of 1 mM Ca 2+ (black solid line, 500 µM free Ca 2+ ) and 100 μM RyR peptides (blue solid line for RyR1, red solid line for RyR2). The spectrum of sole buffer was considered as blank and subtracted; each curve represents the average of five accumulations. Temperature was set at 25°C and signal was normalized to protein concentration.

    Journal: Frontiers in Molecular Biosciences

    Article Title: Calmodulin variants associated with congenital arrhythmia impair selectivity for ryanodine receptors

    doi: 10.3389/fmolb.2022.1100992

    Figure Lengend Snippet: Tertiary and quaternary structure of CaM variants in complex with RyR1/2 peptides. (A) Cartoon representation of CaM in three different conformational states, namely apo (PDB: 1DMO ), Ca 2+ -bound (PDB: 1CLL ) and complexed with the RyR2 peptide used in this study (PDB: 6Y4O ). The N-terminal domain is colored in yellow, the linker region in grey, while the C-terminal domain in green. The RyR2 peptide is represented in magenta. Ca 2+ ions are represented as red spheres while the side chains of N97 and Q135 are represented in sticks with C atoms colored according to the structural region, O atoms in red and N atoms in blue. (B) Pairwise sequence alignment of the Calmodulin Binding Domain-2 of human RyR1 (Uniprot entry P21817) and RyR2 (Uniprot entry Q92736). The sequence relative to the two RyR peptides employed in this study is highlighted in yellow, the residues that are not identical in such region are represented in bold and colored in red. (C) Near-UV CD spectra (250–320 nm) of 50 μM CaM were collected in the presence of 500 µM EGTA (black dashed line) and after sequential additions of 1 mM Ca 2+ (black solid line, 500 µM free Ca 2+ ) and 100 μM RyR peptides (blue solid line for RyR1, red solid line for RyR2). The spectrum of sole buffer was considered as blank and subtracted; each curve represents the average of five accumulations. Temperature was set at 25°C and signal was normalized to protein concentration.

    Article Snippet: Cryo-EM revealed a complex CaM-RyR2 recognition mechanism, in which apo- and Ca 2+ -bound CaM bind to distinct but overlapping RyR2 sites, and demonstrated that Ca 2+ -bound CaM is one of many possible regulators competing for RyR2 gating ( ).

    Techniques: Sequencing, Binding Assay, Protein Concentration

    Investigation of secondary structure of CaM variants and their RyR1/2 peptide complexes. Far-UV CD spectra (200–250 nm) of 10 μM CaM variants alone (top panels, black), and incubated with 20 μM RyR1 (center panels, blue), or 20 μM RyR2 (bottom panels, red), were collected in the presence of 300 µM EGTA (dashed lines) and after the addition of 600 µM Ca 2+ (300 µM free Ca 2+ , solid lines). The spectrum of sole buffer was considered as blank and subtracted; each curve represents the average of five accumulations. Temperature was set at 25°C.

    Journal: Frontiers in Molecular Biosciences

    Article Title: Calmodulin variants associated with congenital arrhythmia impair selectivity for ryanodine receptors

    doi: 10.3389/fmolb.2022.1100992

    Figure Lengend Snippet: Investigation of secondary structure of CaM variants and their RyR1/2 peptide complexes. Far-UV CD spectra (200–250 nm) of 10 μM CaM variants alone (top panels, black), and incubated with 20 μM RyR1 (center panels, blue), or 20 μM RyR2 (bottom panels, red), were collected in the presence of 300 µM EGTA (dashed lines) and after the addition of 600 µM Ca 2+ (300 µM free Ca 2+ , solid lines). The spectrum of sole buffer was considered as blank and subtracted; each curve represents the average of five accumulations. Temperature was set at 25°C.

    Article Snippet: Cryo-EM revealed a complex CaM-RyR2 recognition mechanism, in which apo- and Ca 2+ -bound CaM bind to distinct but overlapping RyR2 sites, and demonstrated that Ca 2+ -bound CaM is one of many possible regulators competing for RyR2 gating ( ).

    Techniques: Incubation

    CaM affinity for RyR1 and RyR2 peptides assessed by fluorescence spectroscopy. (A) One micromolar RyR1 (left column) or RyR2 (right column) peptide was incubated with increasing amounts of WT (top row), N97I (middle row) and Q135P (bottom row) CaM in the presence of 100 µM Ca 2+ . Data are reported as a function of the peptide fraction bound to CaM (see Materials and Methods for details). Curves report the mean ± std of each point obtained in three technical replicas. Representative fitting to one-site saturation ligand binding curve is superposed to each titration set. (B) Scatter plot of replicates reporting the K D values calculated from fitting procedures in each individual dataset using a one-site binding model. Stars represent t -test statistical significance: * p -value ≤ 0.05, ** p -value ≤ 0.01.

    Journal: Frontiers in Molecular Biosciences

    Article Title: Calmodulin variants associated with congenital arrhythmia impair selectivity for ryanodine receptors

    doi: 10.3389/fmolb.2022.1100992

    Figure Lengend Snippet: CaM affinity for RyR1 and RyR2 peptides assessed by fluorescence spectroscopy. (A) One micromolar RyR1 (left column) or RyR2 (right column) peptide was incubated with increasing amounts of WT (top row), N97I (middle row) and Q135P (bottom row) CaM in the presence of 100 µM Ca 2+ . Data are reported as a function of the peptide fraction bound to CaM (see Materials and Methods for details). Curves report the mean ± std of each point obtained in three technical replicas. Representative fitting to one-site saturation ligand binding curve is superposed to each titration set. (B) Scatter plot of replicates reporting the K D values calculated from fitting procedures in each individual dataset using a one-site binding model. Stars represent t -test statistical significance: * p -value ≤ 0.05, ** p -value ≤ 0.01.

    Article Snippet: Cryo-EM revealed a complex CaM-RyR2 recognition mechanism, in which apo- and Ca 2+ -bound CaM bind to distinct but overlapping RyR2 sites, and demonstrated that Ca 2+ -bound CaM is one of many possible regulators competing for RyR2 gating ( ).

    Techniques: Fluorescence, Spectroscopy, Incubation, Ligand Binding Assay, Titration, Binding Assay

    Apparent affinities of CaM-RyR1/2 complexes assessed by fluorescence spectroscopy.

    Journal: Frontiers in Molecular Biosciences

    Article Title: Calmodulin variants associated with congenital arrhythmia impair selectivity for ryanodine receptors

    doi: 10.3389/fmolb.2022.1100992

    Figure Lengend Snippet: Apparent affinities of CaM-RyR1/2 complexes assessed by fluorescence spectroscopy.

    Article Snippet: Cryo-EM revealed a complex CaM-RyR2 recognition mechanism, in which apo- and Ca 2+ -bound CaM bind to distinct but overlapping RyR2 sites, and demonstrated that Ca 2+ -bound CaM is one of many possible regulators competing for RyR2 gating ( ).

    Techniques: Fluorescence, Spectroscopy

    Thermodynamics of CaM-RyR1/2 peptide interaction assessed by isothermal titration calorimetry. (A) Examples of ITC titration curves obtained for each CaM variant upon interaction with RyR1 or RyR2 peptides. Measurements were performed at 25°C using 20 mM Tris pH 7.5, 150 mM KCl, 5 mM Ca 2+ as working buffer and setting stirring at 750 rpm. Each titration consisted in thirty 1-µL injections of 125 µM RyR1 or RyR2 (into the titrant syringe) with 10 µM CaM variants. (B) Scatter plot of replicates summarizing the K D values calculated from the fitting using a one-site binding model (see Materials and Methods). Stars represent the p -values: * p ≤ 0.05, *** p ≤ 0.001. Data for WT CaM titration with RyR2 are from .

    Journal: Frontiers in Molecular Biosciences

    Article Title: Calmodulin variants associated with congenital arrhythmia impair selectivity for ryanodine receptors

    doi: 10.3389/fmolb.2022.1100992

    Figure Lengend Snippet: Thermodynamics of CaM-RyR1/2 peptide interaction assessed by isothermal titration calorimetry. (A) Examples of ITC titration curves obtained for each CaM variant upon interaction with RyR1 or RyR2 peptides. Measurements were performed at 25°C using 20 mM Tris pH 7.5, 150 mM KCl, 5 mM Ca 2+ as working buffer and setting stirring at 750 rpm. Each titration consisted in thirty 1-µL injections of 125 µM RyR1 or RyR2 (into the titrant syringe) with 10 µM CaM variants. (B) Scatter plot of replicates summarizing the K D values calculated from the fitting using a one-site binding model (see Materials and Methods). Stars represent the p -values: * p ≤ 0.05, *** p ≤ 0.001. Data for WT CaM titration with RyR2 are from .

    Article Snippet: Cryo-EM revealed a complex CaM-RyR2 recognition mechanism, in which apo- and Ca 2+ -bound CaM bind to distinct but overlapping RyR2 sites, and demonstrated that Ca 2+ -bound CaM is one of many possible regulators competing for RyR2 gating ( ).

    Techniques: Isothermal Titration Calorimetry, Titration, Variant Assay, Binding Assay

    Thermodynamics of CaM-RyR1/2 peptide association assessed by isothermal titration calorimetry.

    Journal: Frontiers in Molecular Biosciences

    Article Title: Calmodulin variants associated with congenital arrhythmia impair selectivity for ryanodine receptors

    doi: 10.3389/fmolb.2022.1100992

    Figure Lengend Snippet: Thermodynamics of CaM-RyR1/2 peptide association assessed by isothermal titration calorimetry.

    Article Snippet: Cryo-EM revealed a complex CaM-RyR2 recognition mechanism, in which apo- and Ca 2+ -bound CaM bind to distinct but overlapping RyR2 sites, and demonstrated that Ca 2+ -bound CaM is one of many possible regulators competing for RyR2 gating ( ).

    Techniques: Titration

    Kinetics of CaM-RyR1/2 peptide interaction investigated by surface plasmon resonance. (A) Sensorgrams collected by flowing different amounts of RyR1 and RyR2 (125 nM—2 µM) on immobilized His-CaM variants using 20 mM Tris pH 7.5, 150 mM KCl, 0.005% Tween 20, 5 mM Ca 2+ , 100 µM DTT as a running buffer. Association and dissociation phases were followed for 60 s and 300 s, respectively. Experimental curves (black solid lines) are shown together with theoretical curves (red or blue solid lines) according to a 1:1 Langmuir binding model; fitting for association and dissociation phases led to the rate constants ( k on and k off ) reported in each panel (mean ± s.e.m. of 8-20 independent binding curves). (B) Scatter plot of replicates and statistical analysis comparing rate constants for WT and each pathogenic CaM variant. Stars represent p -values: * p ≤ 0.05, ** p ≤ 0.01, **** p ≤ 0.0001.

    Journal: Frontiers in Molecular Biosciences

    Article Title: Calmodulin variants associated with congenital arrhythmia impair selectivity for ryanodine receptors

    doi: 10.3389/fmolb.2022.1100992

    Figure Lengend Snippet: Kinetics of CaM-RyR1/2 peptide interaction investigated by surface plasmon resonance. (A) Sensorgrams collected by flowing different amounts of RyR1 and RyR2 (125 nM—2 µM) on immobilized His-CaM variants using 20 mM Tris pH 7.5, 150 mM KCl, 0.005% Tween 20, 5 mM Ca 2+ , 100 µM DTT as a running buffer. Association and dissociation phases were followed for 60 s and 300 s, respectively. Experimental curves (black solid lines) are shown together with theoretical curves (red or blue solid lines) according to a 1:1 Langmuir binding model; fitting for association and dissociation phases led to the rate constants ( k on and k off ) reported in each panel (mean ± s.e.m. of 8-20 independent binding curves). (B) Scatter plot of replicates and statistical analysis comparing rate constants for WT and each pathogenic CaM variant. Stars represent p -values: * p ≤ 0.05, ** p ≤ 0.01, **** p ≤ 0.0001.

    Article Snippet: Cryo-EM revealed a complex CaM-RyR2 recognition mechanism, in which apo- and Ca 2+ -bound CaM bind to distinct but overlapping RyR2 sites, and demonstrated that Ca 2+ -bound CaM is one of many possible regulators competing for RyR2 gating ( ).

    Techniques: SPR Assay, Binding Assay, Variant Assay

    Backbone flexibility of CaM-RyR1/2 complexes. Cα-Root-Mean Square Fluctuation (RMSF) of CaM (top panels) and RyR1 or RyR2 peptides (bottom panels) calculated over 1.2 μs MD simulations of the respective complex (WT: black, N97I: blue, and Q135P: red).

    Journal: Frontiers in Molecular Biosciences

    Article Title: Calmodulin variants associated with congenital arrhythmia impair selectivity for ryanodine receptors

    doi: 10.3389/fmolb.2022.1100992

    Figure Lengend Snippet: Backbone flexibility of CaM-RyR1/2 complexes. Cα-Root-Mean Square Fluctuation (RMSF) of CaM (top panels) and RyR1 or RyR2 peptides (bottom panels) calculated over 1.2 μs MD simulations of the respective complex (WT: black, N97I: blue, and Q135P: red).

    Article Snippet: Cryo-EM revealed a complex CaM-RyR2 recognition mechanism, in which apo- and Ca 2+ -bound CaM bind to distinct but overlapping RyR2 sites, and demonstrated that Ca 2+ -bound CaM is one of many possible regulators competing for RyR2 gating ( ).

    Techniques:

    Effects of CaM variants on the connectivity of hub residues of CaM (top panels) and RyR1/2 peptides (bottom panels) . Hubs were defined as residues with degree ≥6 in the Protein Structure Network (PSN) of at least one variant. ∆Degree is calculated as the difference in hub degree between the variant (N97I: black, Q135P: red) and the WT.

    Journal: Frontiers in Molecular Biosciences

    Article Title: Calmodulin variants associated with congenital arrhythmia impair selectivity for ryanodine receptors

    doi: 10.3389/fmolb.2022.1100992

    Figure Lengend Snippet: Effects of CaM variants on the connectivity of hub residues of CaM (top panels) and RyR1/2 peptides (bottom panels) . Hubs were defined as residues with degree ≥6 in the Protein Structure Network (PSN) of at least one variant. ∆Degree is calculated as the difference in hub degree between the variant (N97I: black, Q135P: red) and the WT.

    Article Snippet: Cryo-EM revealed a complex CaM-RyR2 recognition mechanism, in which apo- and Ca 2+ -bound CaM bind to distinct but overlapping RyR2 sites, and demonstrated that Ca 2+ -bound CaM is one of many possible regulators competing for RyR2 gating ( ).

    Techniques: Variant Assay

    Robustness of intramolecular communication between EF-hands in CaM-RyR1/2 complexes. (A) Intramolecular communication among EF-hands in the CaM-RyR1 (left) and CaM-RyR2 (right) complexes. Communication robustness between EF-hands (EF1 to EF4, represented by their bidentate Glu residues) in CaM WT (black), N97I (red), and Q135P (green) variants in complex with RyR peptides. (B) Effects of specific RyR peptides on the intramolecular communication among EF-hands in CaM-RyR complexes. Communication robustness between EF-hands (EF1 to EF4, represented by their bidentate Glu residues) in CaM variants in complex with RyR1 (black) and RyR2 (red) peptides.

    Journal: Frontiers in Molecular Biosciences

    Article Title: Calmodulin variants associated with congenital arrhythmia impair selectivity for ryanodine receptors

    doi: 10.3389/fmolb.2022.1100992

    Figure Lengend Snippet: Robustness of intramolecular communication between EF-hands in CaM-RyR1/2 complexes. (A) Intramolecular communication among EF-hands in the CaM-RyR1 (left) and CaM-RyR2 (right) complexes. Communication robustness between EF-hands (EF1 to EF4, represented by their bidentate Glu residues) in CaM WT (black), N97I (red), and Q135P (green) variants in complex with RyR peptides. (B) Effects of specific RyR peptides on the intramolecular communication among EF-hands in CaM-RyR complexes. Communication robustness between EF-hands (EF1 to EF4, represented by their bidentate Glu residues) in CaM variants in complex with RyR1 (black) and RyR2 (red) peptides.

    Article Snippet: Cryo-EM revealed a complex CaM-RyR2 recognition mechanism, in which apo- and Ca 2+ -bound CaM bind to distinct but overlapping RyR2 sites, and demonstrated that Ca 2+ -bound CaM is one of many possible regulators competing for RyR2 gating ( ).

    Techniques:

    Tertiary and quaternary structure of CaM variants in complex with RyR1/2 peptides. (A) Cartoon representation of CaM in three different conformational states, namely apo (PDB: 1DMO ), Ca 2+ -bound (PDB: 1CLL ) and complexed with the RyR2 peptide used in this study (PDB: 6Y4O ). The N-terminal domain is colored in yellow, the linker region in grey, while the C-terminal domain in green. The RyR2 peptide is represented in magenta. Ca 2+ ions are represented as red spheres while the side chains of N97 and Q135 are represented in sticks with C atoms colored according to the structural region, O atoms in red and N atoms in blue. (B) Pairwise sequence alignment of the Calmodulin Binding Domain-2 of human RyR1 (Uniprot entry P21817) and RyR2 (Uniprot entry Q92736). The sequence relative to the two RyR peptides employed in this study is highlighted in yellow, the residues that are not identical in such region are represented in bold and colored in red. (C) Near-UV CD spectra (250–320 nm) of 50 μM CaM were collected in the presence of 500 µM EGTA (black dashed line) and after sequential additions of 1 mM Ca 2+ (black solid line, 500 µM free Ca 2+ ) and 100 μM RyR peptides (blue solid line for RyR1, red solid line for RyR2). The spectrum of sole buffer was considered as blank and subtracted; each curve represents the average of five accumulations. Temperature was set at 25°C and signal was normalized to protein concentration.

    Journal: Frontiers in Molecular Biosciences

    Article Title: Calmodulin variants associated with congenital arrhythmia impair selectivity for ryanodine receptors

    doi: 10.3389/fmolb.2022.1100992

    Figure Lengend Snippet: Tertiary and quaternary structure of CaM variants in complex with RyR1/2 peptides. (A) Cartoon representation of CaM in three different conformational states, namely apo (PDB: 1DMO ), Ca 2+ -bound (PDB: 1CLL ) and complexed with the RyR2 peptide used in this study (PDB: 6Y4O ). The N-terminal domain is colored in yellow, the linker region in grey, while the C-terminal domain in green. The RyR2 peptide is represented in magenta. Ca 2+ ions are represented as red spheres while the side chains of N97 and Q135 are represented in sticks with C atoms colored according to the structural region, O atoms in red and N atoms in blue. (B) Pairwise sequence alignment of the Calmodulin Binding Domain-2 of human RyR1 (Uniprot entry P21817) and RyR2 (Uniprot entry Q92736). The sequence relative to the two RyR peptides employed in this study is highlighted in yellow, the residues that are not identical in such region are represented in bold and colored in red. (C) Near-UV CD spectra (250–320 nm) of 50 μM CaM were collected in the presence of 500 µM EGTA (black dashed line) and after sequential additions of 1 mM Ca 2+ (black solid line, 500 µM free Ca 2+ ) and 100 μM RyR peptides (blue solid line for RyR1, red solid line for RyR2). The spectrum of sole buffer was considered as blank and subtracted; each curve represents the average of five accumulations. Temperature was set at 25°C and signal was normalized to protein concentration.

    Article Snippet: Cryo-EM revealed a complex CaM-RyR2 recognition mechanism, in which apo- and Ca 2+ -bound CaM bind to distinct but overlapping RyR2 sites, and demonstrated that Ca 2+ -bound CaM is one of many possible regulators competing for RyR2 gating ( ).

    Techniques: Sequencing, Binding Assay, Protein Concentration

    Investigation of secondary structure of CaM variants and their RyR1/2 peptide complexes. Far-UV CD spectra (200–250 nm) of 10 μM CaM variants alone (top panels, black), and incubated with 20 μM RyR1 (center panels, blue), or 20 μM RyR2 (bottom panels, red), were collected in the presence of 300 µM EGTA (dashed lines) and after the addition of 600 µM Ca 2+ (300 µM free Ca 2+ , solid lines). The spectrum of sole buffer was considered as blank and subtracted; each curve represents the average of five accumulations. Temperature was set at 25°C.

    Journal: Frontiers in Molecular Biosciences

    Article Title: Calmodulin variants associated with congenital arrhythmia impair selectivity for ryanodine receptors

    doi: 10.3389/fmolb.2022.1100992

    Figure Lengend Snippet: Investigation of secondary structure of CaM variants and their RyR1/2 peptide complexes. Far-UV CD spectra (200–250 nm) of 10 μM CaM variants alone (top panels, black), and incubated with 20 μM RyR1 (center panels, blue), or 20 μM RyR2 (bottom panels, red), were collected in the presence of 300 µM EGTA (dashed lines) and after the addition of 600 µM Ca 2+ (300 µM free Ca 2+ , solid lines). The spectrum of sole buffer was considered as blank and subtracted; each curve represents the average of five accumulations. Temperature was set at 25°C.

    Article Snippet: Cryo-EM revealed a complex CaM-RyR2 recognition mechanism, in which apo- and Ca 2+ -bound CaM bind to distinct but overlapping RyR2 sites, and demonstrated that Ca 2+ -bound CaM is one of many possible regulators competing for RyR2 gating ( ).

    Techniques: Incubation

    CaM affinity for RyR1 and RyR2 peptides assessed by fluorescence spectroscopy. (A) One micromolar RyR1 (left column) or RyR2 (right column) peptide was incubated with increasing amounts of WT (top row), N97I (middle row) and Q135P (bottom row) CaM in the presence of 100 µM Ca 2+ . Data are reported as a function of the peptide fraction bound to CaM (see Materials and Methods for details). Curves report the mean ± std of each point obtained in three technical replicas. Representative fitting to one-site saturation ligand binding curve is superposed to each titration set. (B) Scatter plot of replicates reporting the K D values calculated from fitting procedures in each individual dataset using a one-site binding model. Stars represent t -test statistical significance: * p -value ≤ 0.05, ** p -value ≤ 0.01.

    Journal: Frontiers in Molecular Biosciences

    Article Title: Calmodulin variants associated with congenital arrhythmia impair selectivity for ryanodine receptors

    doi: 10.3389/fmolb.2022.1100992

    Figure Lengend Snippet: CaM affinity for RyR1 and RyR2 peptides assessed by fluorescence spectroscopy. (A) One micromolar RyR1 (left column) or RyR2 (right column) peptide was incubated with increasing amounts of WT (top row), N97I (middle row) and Q135P (bottom row) CaM in the presence of 100 µM Ca 2+ . Data are reported as a function of the peptide fraction bound to CaM (see Materials and Methods for details). Curves report the mean ± std of each point obtained in three technical replicas. Representative fitting to one-site saturation ligand binding curve is superposed to each titration set. (B) Scatter plot of replicates reporting the K D values calculated from fitting procedures in each individual dataset using a one-site binding model. Stars represent t -test statistical significance: * p -value ≤ 0.05, ** p -value ≤ 0.01.

    Article Snippet: Cryo-EM revealed a complex CaM-RyR2 recognition mechanism, in which apo- and Ca 2+ -bound CaM bind to distinct but overlapping RyR2 sites, and demonstrated that Ca 2+ -bound CaM is one of many possible regulators competing for RyR2 gating ( ).

    Techniques: Fluorescence, Spectroscopy, Incubation, Ligand Binding Assay, Titration, Binding Assay

    Apparent affinities of CaM-RyR1/2 complexes assessed by fluorescence spectroscopy.

    Journal: Frontiers in Molecular Biosciences

    Article Title: Calmodulin variants associated with congenital arrhythmia impair selectivity for ryanodine receptors

    doi: 10.3389/fmolb.2022.1100992

    Figure Lengend Snippet: Apparent affinities of CaM-RyR1/2 complexes assessed by fluorescence spectroscopy.

    Article Snippet: Cryo-EM revealed a complex CaM-RyR2 recognition mechanism, in which apo- and Ca 2+ -bound CaM bind to distinct but overlapping RyR2 sites, and demonstrated that Ca 2+ -bound CaM is one of many possible regulators competing for RyR2 gating ( ).

    Techniques: Fluorescence, Spectroscopy

    Thermodynamics of CaM-RyR1/2 peptide interaction assessed by isothermal titration calorimetry. (A) Examples of ITC titration curves obtained for each CaM variant upon interaction with RyR1 or RyR2 peptides. Measurements were performed at 25°C using 20 mM Tris pH 7.5, 150 mM KCl, 5 mM Ca 2+ as working buffer and setting stirring at 750 rpm. Each titration consisted in thirty 1-µL injections of 125 µM RyR1 or RyR2 (into the titrant syringe) with 10 µM CaM variants. (B) Scatter plot of replicates summarizing the K D values calculated from the fitting using a one-site binding model (see Materials and Methods). Stars represent the p -values: * p ≤ 0.05, *** p ≤ 0.001. Data for WT CaM titration with RyR2 are from .

    Journal: Frontiers in Molecular Biosciences

    Article Title: Calmodulin variants associated with congenital arrhythmia impair selectivity for ryanodine receptors

    doi: 10.3389/fmolb.2022.1100992

    Figure Lengend Snippet: Thermodynamics of CaM-RyR1/2 peptide interaction assessed by isothermal titration calorimetry. (A) Examples of ITC titration curves obtained for each CaM variant upon interaction with RyR1 or RyR2 peptides. Measurements were performed at 25°C using 20 mM Tris pH 7.5, 150 mM KCl, 5 mM Ca 2+ as working buffer and setting stirring at 750 rpm. Each titration consisted in thirty 1-µL injections of 125 µM RyR1 or RyR2 (into the titrant syringe) with 10 µM CaM variants. (B) Scatter plot of replicates summarizing the K D values calculated from the fitting using a one-site binding model (see Materials and Methods). Stars represent the p -values: * p ≤ 0.05, *** p ≤ 0.001. Data for WT CaM titration with RyR2 are from .

    Article Snippet: Cryo-EM revealed a complex CaM-RyR2 recognition mechanism, in which apo- and Ca 2+ -bound CaM bind to distinct but overlapping RyR2 sites, and demonstrated that Ca 2+ -bound CaM is one of many possible regulators competing for RyR2 gating ( ).

    Techniques: Isothermal Titration Calorimetry, Titration, Variant Assay, Binding Assay

    Thermodynamics of CaM-RyR1/2 peptide association assessed by isothermal titration calorimetry.

    Journal: Frontiers in Molecular Biosciences

    Article Title: Calmodulin variants associated with congenital arrhythmia impair selectivity for ryanodine receptors

    doi: 10.3389/fmolb.2022.1100992

    Figure Lengend Snippet: Thermodynamics of CaM-RyR1/2 peptide association assessed by isothermal titration calorimetry.

    Article Snippet: Cryo-EM revealed a complex CaM-RyR2 recognition mechanism, in which apo- and Ca 2+ -bound CaM bind to distinct but overlapping RyR2 sites, and demonstrated that Ca 2+ -bound CaM is one of many possible regulators competing for RyR2 gating ( ).

    Techniques: Titration

    Kinetics of CaM-RyR1/2 peptide interaction investigated by surface plasmon resonance. (A) Sensorgrams collected by flowing different amounts of RyR1 and RyR2 (125 nM—2 µM) on immobilized His-CaM variants using 20 mM Tris pH 7.5, 150 mM KCl, 0.005% Tween 20, 5 mM Ca 2+ , 100 µM DTT as a running buffer. Association and dissociation phases were followed for 60 s and 300 s, respectively. Experimental curves (black solid lines) are shown together with theoretical curves (red or blue solid lines) according to a 1:1 Langmuir binding model; fitting for association and dissociation phases led to the rate constants ( k on and k off ) reported in each panel (mean ± s.e.m. of 8-20 independent binding curves). (B) Scatter plot of replicates and statistical analysis comparing rate constants for WT and each pathogenic CaM variant. Stars represent p -values: * p ≤ 0.05, ** p ≤ 0.01, **** p ≤ 0.0001.

    Journal: Frontiers in Molecular Biosciences

    Article Title: Calmodulin variants associated with congenital arrhythmia impair selectivity for ryanodine receptors

    doi: 10.3389/fmolb.2022.1100992

    Figure Lengend Snippet: Kinetics of CaM-RyR1/2 peptide interaction investigated by surface plasmon resonance. (A) Sensorgrams collected by flowing different amounts of RyR1 and RyR2 (125 nM—2 µM) on immobilized His-CaM variants using 20 mM Tris pH 7.5, 150 mM KCl, 0.005% Tween 20, 5 mM Ca 2+ , 100 µM DTT as a running buffer. Association and dissociation phases were followed for 60 s and 300 s, respectively. Experimental curves (black solid lines) are shown together with theoretical curves (red or blue solid lines) according to a 1:1 Langmuir binding model; fitting for association and dissociation phases led to the rate constants ( k on and k off ) reported in each panel (mean ± s.e.m. of 8-20 independent binding curves). (B) Scatter plot of replicates and statistical analysis comparing rate constants for WT and each pathogenic CaM variant. Stars represent p -values: * p ≤ 0.05, ** p ≤ 0.01, **** p ≤ 0.0001.

    Article Snippet: Cryo-EM revealed a complex CaM-RyR2 recognition mechanism, in which apo- and Ca 2+ -bound CaM bind to distinct but overlapping RyR2 sites, and demonstrated that Ca 2+ -bound CaM is one of many possible regulators competing for RyR2 gating ( ).

    Techniques: SPR Assay, Binding Assay, Variant Assay

    Backbone flexibility of CaM-RyR1/2 complexes. Cα-Root-Mean Square Fluctuation (RMSF) of CaM (top panels) and RyR1 or RyR2 peptides (bottom panels) calculated over 1.2 μs MD simulations of the respective complex (WT: black, N97I: blue, and Q135P: red).

    Journal: Frontiers in Molecular Biosciences

    Article Title: Calmodulin variants associated with congenital arrhythmia impair selectivity for ryanodine receptors

    doi: 10.3389/fmolb.2022.1100992

    Figure Lengend Snippet: Backbone flexibility of CaM-RyR1/2 complexes. Cα-Root-Mean Square Fluctuation (RMSF) of CaM (top panels) and RyR1 or RyR2 peptides (bottom panels) calculated over 1.2 μs MD simulations of the respective complex (WT: black, N97I: blue, and Q135P: red).

    Article Snippet: Cryo-EM revealed a complex CaM-RyR2 recognition mechanism, in which apo- and Ca 2+ -bound CaM bind to distinct but overlapping RyR2 sites, and demonstrated that Ca 2+ -bound CaM is one of many possible regulators competing for RyR2 gating ( ).

    Techniques:

    Effects of CaM variants on the connectivity of hub residues of CaM (top panels) and RyR1/2 peptides (bottom panels) . Hubs were defined as residues with degree ≥6 in the Protein Structure Network (PSN) of at least one variant. ∆Degree is calculated as the difference in hub degree between the variant (N97I: black, Q135P: red) and the WT.

    Journal: Frontiers in Molecular Biosciences

    Article Title: Calmodulin variants associated with congenital arrhythmia impair selectivity for ryanodine receptors

    doi: 10.3389/fmolb.2022.1100992

    Figure Lengend Snippet: Effects of CaM variants on the connectivity of hub residues of CaM (top panels) and RyR1/2 peptides (bottom panels) . Hubs were defined as residues with degree ≥6 in the Protein Structure Network (PSN) of at least one variant. ∆Degree is calculated as the difference in hub degree between the variant (N97I: black, Q135P: red) and the WT.

    Article Snippet: Cryo-EM revealed a complex CaM-RyR2 recognition mechanism, in which apo- and Ca 2+ -bound CaM bind to distinct but overlapping RyR2 sites, and demonstrated that Ca 2+ -bound CaM is one of many possible regulators competing for RyR2 gating ( ).

    Techniques: Variant Assay

    Robustness of intramolecular communication between EF-hands in CaM-RyR1/2 complexes. (A) Intramolecular communication among EF-hands in the CaM-RyR1 (left) and CaM-RyR2 (right) complexes. Communication robustness between EF-hands (EF1 to EF4, represented by their bidentate Glu residues) in CaM WT (black), N97I (red), and Q135P (green) variants in complex with RyR peptides. (B) Effects of specific RyR peptides on the intramolecular communication among EF-hands in CaM-RyR complexes. Communication robustness between EF-hands (EF1 to EF4, represented by their bidentate Glu residues) in CaM variants in complex with RyR1 (black) and RyR2 (red) peptides.

    Journal: Frontiers in Molecular Biosciences

    Article Title: Calmodulin variants associated with congenital arrhythmia impair selectivity for ryanodine receptors

    doi: 10.3389/fmolb.2022.1100992

    Figure Lengend Snippet: Robustness of intramolecular communication between EF-hands in CaM-RyR1/2 complexes. (A) Intramolecular communication among EF-hands in the CaM-RyR1 (left) and CaM-RyR2 (right) complexes. Communication robustness between EF-hands (EF1 to EF4, represented by their bidentate Glu residues) in CaM WT (black), N97I (red), and Q135P (green) variants in complex with RyR peptides. (B) Effects of specific RyR peptides on the intramolecular communication among EF-hands in CaM-RyR complexes. Communication robustness between EF-hands (EF1 to EF4, represented by their bidentate Glu residues) in CaM variants in complex with RyR1 (black) and RyR2 (red) peptides.

    Article Snippet: Cryo-EM revealed a complex CaM-RyR2 recognition mechanism, in which apo- and Ca 2+ -bound CaM bind to distinct but overlapping RyR2 sites, and demonstrated that Ca 2+ -bound CaM is one of many possible regulators competing for RyR2 gating ( ).

    Techniques:

    Tertiary and quaternary structure of CaM variants in complex with RyR1/2 peptides. (A) Cartoon representation of CaM in three different conformational states, namely apo (PDB: 1DMO ), Ca 2+ -bound (PDB: 1CLL ) and complexed with the RyR2 peptide used in this study (PDB: 6Y4O ). The N-terminal domain is colored in yellow, the linker region in grey, while the C-terminal domain in green. The RyR2 peptide is represented in magenta. Ca 2+ ions are represented as red spheres while the side chains of N97 and Q135 are represented in sticks with C atoms colored according to the structural region, O atoms in red and N atoms in blue. (B) Pairwise sequence alignment of the Calmodulin Binding Domain-2 of human RyR1 (Uniprot entry P21817) and RyR2 (Uniprot entry Q92736). The sequence relative to the two RyR peptides employed in this study is highlighted in yellow, the residues that are not identical in such region are represented in bold and colored in red. (C) Near-UV CD spectra (250–320 nm) of 50 μM CaM were collected in the presence of 500 µM EGTA (black dashed line) and after sequential additions of 1 mM Ca 2+ (black solid line, 500 µM free Ca 2+ ) and 100 μM RyR peptides (blue solid line for RyR1, red solid line for RyR2). The spectrum of sole buffer was considered as blank and subtracted; each curve represents the average of five accumulations. Temperature was set at 25°C and signal was normalized to protein concentration.

    Journal: Frontiers in Molecular Biosciences

    Article Title: Calmodulin variants associated with congenital arrhythmia impair selectivity for ryanodine receptors

    doi: 10.3389/fmolb.2022.1100992

    Figure Lengend Snippet: Tertiary and quaternary structure of CaM variants in complex with RyR1/2 peptides. (A) Cartoon representation of CaM in three different conformational states, namely apo (PDB: 1DMO ), Ca 2+ -bound (PDB: 1CLL ) and complexed with the RyR2 peptide used in this study (PDB: 6Y4O ). The N-terminal domain is colored in yellow, the linker region in grey, while the C-terminal domain in green. The RyR2 peptide is represented in magenta. Ca 2+ ions are represented as red spheres while the side chains of N97 and Q135 are represented in sticks with C atoms colored according to the structural region, O atoms in red and N atoms in blue. (B) Pairwise sequence alignment of the Calmodulin Binding Domain-2 of human RyR1 (Uniprot entry P21817) and RyR2 (Uniprot entry Q92736). The sequence relative to the two RyR peptides employed in this study is highlighted in yellow, the residues that are not identical in such region are represented in bold and colored in red. (C) Near-UV CD spectra (250–320 nm) of 50 μM CaM were collected in the presence of 500 µM EGTA (black dashed line) and after sequential additions of 1 mM Ca 2+ (black solid line, 500 µM free Ca 2+ ) and 100 μM RyR peptides (blue solid line for RyR1, red solid line for RyR2). The spectrum of sole buffer was considered as blank and subtracted; each curve represents the average of five accumulations. Temperature was set at 25°C and signal was normalized to protein concentration.

    Article Snippet: Unprecedented insight into the functional properties of RyR2 and its regulation by CaM has come from cryogenic-electron microscopy (cryo-EM) ( ; ; ), which shed light on the three previously identified CaM-binding domains (CaMBD) shared by RyR receptors, which could interact individually or in groups with CaM lobes to regulate channel function ( ).

    Techniques: Sequencing, Binding Assay, Protein Concentration

    Investigation of secondary structure of CaM variants and their RyR1/2 peptide complexes. Far-UV CD spectra (200–250 nm) of 10 μM CaM variants alone (top panels, black), and incubated with 20 μM RyR1 (center panels, blue), or 20 μM RyR2 (bottom panels, red), were collected in the presence of 300 µM EGTA (dashed lines) and after the addition of 600 µM Ca 2+ (300 µM free Ca 2+ , solid lines). The spectrum of sole buffer was considered as blank and subtracted; each curve represents the average of five accumulations. Temperature was set at 25°C.

    Journal: Frontiers in Molecular Biosciences

    Article Title: Calmodulin variants associated with congenital arrhythmia impair selectivity for ryanodine receptors

    doi: 10.3389/fmolb.2022.1100992

    Figure Lengend Snippet: Investigation of secondary structure of CaM variants and their RyR1/2 peptide complexes. Far-UV CD spectra (200–250 nm) of 10 μM CaM variants alone (top panels, black), and incubated with 20 μM RyR1 (center panels, blue), or 20 μM RyR2 (bottom panels, red), were collected in the presence of 300 µM EGTA (dashed lines) and after the addition of 600 µM Ca 2+ (300 µM free Ca 2+ , solid lines). The spectrum of sole buffer was considered as blank and subtracted; each curve represents the average of five accumulations. Temperature was set at 25°C.

    Article Snippet: Unprecedented insight into the functional properties of RyR2 and its regulation by CaM has come from cryogenic-electron microscopy (cryo-EM) ( ; ; ), which shed light on the three previously identified CaM-binding domains (CaMBD) shared by RyR receptors, which could interact individually or in groups with CaM lobes to regulate channel function ( ).

    Techniques: Incubation

    CaM affinity for RyR1 and RyR2 peptides assessed by fluorescence spectroscopy. (A) One micromolar RyR1 (left column) or RyR2 (right column) peptide was incubated with increasing amounts of WT (top row), N97I (middle row) and Q135P (bottom row) CaM in the presence of 100 µM Ca 2+ . Data are reported as a function of the peptide fraction bound to CaM (see Materials and Methods for details). Curves report the mean ± std of each point obtained in three technical replicas. Representative fitting to one-site saturation ligand binding curve is superposed to each titration set. (B) Scatter plot of replicates reporting the K D values calculated from fitting procedures in each individual dataset using a one-site binding model. Stars represent t -test statistical significance: * p -value ≤ 0.05, ** p -value ≤ 0.01.

    Journal: Frontiers in Molecular Biosciences

    Article Title: Calmodulin variants associated with congenital arrhythmia impair selectivity for ryanodine receptors

    doi: 10.3389/fmolb.2022.1100992

    Figure Lengend Snippet: CaM affinity for RyR1 and RyR2 peptides assessed by fluorescence spectroscopy. (A) One micromolar RyR1 (left column) or RyR2 (right column) peptide was incubated with increasing amounts of WT (top row), N97I (middle row) and Q135P (bottom row) CaM in the presence of 100 µM Ca 2+ . Data are reported as a function of the peptide fraction bound to CaM (see Materials and Methods for details). Curves report the mean ± std of each point obtained in three technical replicas. Representative fitting to one-site saturation ligand binding curve is superposed to each titration set. (B) Scatter plot of replicates reporting the K D values calculated from fitting procedures in each individual dataset using a one-site binding model. Stars represent t -test statistical significance: * p -value ≤ 0.05, ** p -value ≤ 0.01.

    Article Snippet: Unprecedented insight into the functional properties of RyR2 and its regulation by CaM has come from cryogenic-electron microscopy (cryo-EM) ( ; ; ), which shed light on the three previously identified CaM-binding domains (CaMBD) shared by RyR receptors, which could interact individually or in groups with CaM lobes to regulate channel function ( ).

    Techniques: Fluorescence, Spectroscopy, Incubation, Ligand Binding Assay, Titration, Binding Assay

    Apparent affinities of CaM-RyR1/2 complexes assessed by fluorescence spectroscopy.

    Journal: Frontiers in Molecular Biosciences

    Article Title: Calmodulin variants associated with congenital arrhythmia impair selectivity for ryanodine receptors

    doi: 10.3389/fmolb.2022.1100992

    Figure Lengend Snippet: Apparent affinities of CaM-RyR1/2 complexes assessed by fluorescence spectroscopy.

    Article Snippet: Unprecedented insight into the functional properties of RyR2 and its regulation by CaM has come from cryogenic-electron microscopy (cryo-EM) ( ; ; ), which shed light on the three previously identified CaM-binding domains (CaMBD) shared by RyR receptors, which could interact individually or in groups with CaM lobes to regulate channel function ( ).

    Techniques: Fluorescence, Spectroscopy

    Thermodynamics of CaM-RyR1/2 peptide interaction assessed by isothermal titration calorimetry. (A) Examples of ITC titration curves obtained for each CaM variant upon interaction with RyR1 or RyR2 peptides. Measurements were performed at 25°C using 20 mM Tris pH 7.5, 150 mM KCl, 5 mM Ca 2+ as working buffer and setting stirring at 750 rpm. Each titration consisted in thirty 1-µL injections of 125 µM RyR1 or RyR2 (into the titrant syringe) with 10 µM CaM variants. (B) Scatter plot of replicates summarizing the K D values calculated from the fitting using a one-site binding model (see Materials and Methods). Stars represent the p -values: * p ≤ 0.05, *** p ≤ 0.001. Data for WT CaM titration with RyR2 are from .

    Journal: Frontiers in Molecular Biosciences

    Article Title: Calmodulin variants associated with congenital arrhythmia impair selectivity for ryanodine receptors

    doi: 10.3389/fmolb.2022.1100992

    Figure Lengend Snippet: Thermodynamics of CaM-RyR1/2 peptide interaction assessed by isothermal titration calorimetry. (A) Examples of ITC titration curves obtained for each CaM variant upon interaction with RyR1 or RyR2 peptides. Measurements were performed at 25°C using 20 mM Tris pH 7.5, 150 mM KCl, 5 mM Ca 2+ as working buffer and setting stirring at 750 rpm. Each titration consisted in thirty 1-µL injections of 125 µM RyR1 or RyR2 (into the titrant syringe) with 10 µM CaM variants. (B) Scatter plot of replicates summarizing the K D values calculated from the fitting using a one-site binding model (see Materials and Methods). Stars represent the p -values: * p ≤ 0.05, *** p ≤ 0.001. Data for WT CaM titration with RyR2 are from .

    Article Snippet: Unprecedented insight into the functional properties of RyR2 and its regulation by CaM has come from cryogenic-electron microscopy (cryo-EM) ( ; ; ), which shed light on the three previously identified CaM-binding domains (CaMBD) shared by RyR receptors, which could interact individually or in groups with CaM lobes to regulate channel function ( ).

    Techniques: Isothermal Titration Calorimetry, Titration, Variant Assay, Binding Assay

    Thermodynamics of CaM-RyR1/2 peptide association assessed by isothermal titration calorimetry.

    Journal: Frontiers in Molecular Biosciences

    Article Title: Calmodulin variants associated with congenital arrhythmia impair selectivity for ryanodine receptors

    doi: 10.3389/fmolb.2022.1100992

    Figure Lengend Snippet: Thermodynamics of CaM-RyR1/2 peptide association assessed by isothermal titration calorimetry.

    Article Snippet: Unprecedented insight into the functional properties of RyR2 and its regulation by CaM has come from cryogenic-electron microscopy (cryo-EM) ( ; ; ), which shed light on the three previously identified CaM-binding domains (CaMBD) shared by RyR receptors, which could interact individually or in groups with CaM lobes to regulate channel function ( ).

    Techniques: Titration

    Kinetics of CaM-RyR1/2 peptide interaction investigated by surface plasmon resonance. (A) Sensorgrams collected by flowing different amounts of RyR1 and RyR2 (125 nM—2 µM) on immobilized His-CaM variants using 20 mM Tris pH 7.5, 150 mM KCl, 0.005% Tween 20, 5 mM Ca 2+ , 100 µM DTT as a running buffer. Association and dissociation phases were followed for 60 s and 300 s, respectively. Experimental curves (black solid lines) are shown together with theoretical curves (red or blue solid lines) according to a 1:1 Langmuir binding model; fitting for association and dissociation phases led to the rate constants ( k on and k off ) reported in each panel (mean ± s.e.m. of 8-20 independent binding curves). (B) Scatter plot of replicates and statistical analysis comparing rate constants for WT and each pathogenic CaM variant. Stars represent p -values: * p ≤ 0.05, ** p ≤ 0.01, **** p ≤ 0.0001.

    Journal: Frontiers in Molecular Biosciences

    Article Title: Calmodulin variants associated with congenital arrhythmia impair selectivity for ryanodine receptors

    doi: 10.3389/fmolb.2022.1100992

    Figure Lengend Snippet: Kinetics of CaM-RyR1/2 peptide interaction investigated by surface plasmon resonance. (A) Sensorgrams collected by flowing different amounts of RyR1 and RyR2 (125 nM—2 µM) on immobilized His-CaM variants using 20 mM Tris pH 7.5, 150 mM KCl, 0.005% Tween 20, 5 mM Ca 2+ , 100 µM DTT as a running buffer. Association and dissociation phases were followed for 60 s and 300 s, respectively. Experimental curves (black solid lines) are shown together with theoretical curves (red or blue solid lines) according to a 1:1 Langmuir binding model; fitting for association and dissociation phases led to the rate constants ( k on and k off ) reported in each panel (mean ± s.e.m. of 8-20 independent binding curves). (B) Scatter plot of replicates and statistical analysis comparing rate constants for WT and each pathogenic CaM variant. Stars represent p -values: * p ≤ 0.05, ** p ≤ 0.01, **** p ≤ 0.0001.

    Article Snippet: Unprecedented insight into the functional properties of RyR2 and its regulation by CaM has come from cryogenic-electron microscopy (cryo-EM) ( ; ; ), which shed light on the three previously identified CaM-binding domains (CaMBD) shared by RyR receptors, which could interact individually or in groups with CaM lobes to regulate channel function ( ).

    Techniques: SPR Assay, Binding Assay, Variant Assay

    Backbone flexibility of CaM-RyR1/2 complexes. Cα-Root-Mean Square Fluctuation (RMSF) of CaM (top panels) and RyR1 or RyR2 peptides (bottom panels) calculated over 1.2 μs MD simulations of the respective complex (WT: black, N97I: blue, and Q135P: red).

    Journal: Frontiers in Molecular Biosciences

    Article Title: Calmodulin variants associated with congenital arrhythmia impair selectivity for ryanodine receptors

    doi: 10.3389/fmolb.2022.1100992

    Figure Lengend Snippet: Backbone flexibility of CaM-RyR1/2 complexes. Cα-Root-Mean Square Fluctuation (RMSF) of CaM (top panels) and RyR1 or RyR2 peptides (bottom panels) calculated over 1.2 μs MD simulations of the respective complex (WT: black, N97I: blue, and Q135P: red).

    Article Snippet: Unprecedented insight into the functional properties of RyR2 and its regulation by CaM has come from cryogenic-electron microscopy (cryo-EM) ( ; ; ), which shed light on the three previously identified CaM-binding domains (CaMBD) shared by RyR receptors, which could interact individually or in groups with CaM lobes to regulate channel function ( ).

    Techniques:

    Effects of CaM variants on the connectivity of hub residues of CaM (top panels) and RyR1/2 peptides (bottom panels) . Hubs were defined as residues with degree ≥6 in the Protein Structure Network (PSN) of at least one variant. ∆Degree is calculated as the difference in hub degree between the variant (N97I: black, Q135P: red) and the WT.

    Journal: Frontiers in Molecular Biosciences

    Article Title: Calmodulin variants associated with congenital arrhythmia impair selectivity for ryanodine receptors

    doi: 10.3389/fmolb.2022.1100992

    Figure Lengend Snippet: Effects of CaM variants on the connectivity of hub residues of CaM (top panels) and RyR1/2 peptides (bottom panels) . Hubs were defined as residues with degree ≥6 in the Protein Structure Network (PSN) of at least one variant. ∆Degree is calculated as the difference in hub degree between the variant (N97I: black, Q135P: red) and the WT.

    Article Snippet: Unprecedented insight into the functional properties of RyR2 and its regulation by CaM has come from cryogenic-electron microscopy (cryo-EM) ( ; ; ), which shed light on the three previously identified CaM-binding domains (CaMBD) shared by RyR receptors, which could interact individually or in groups with CaM lobes to regulate channel function ( ).

    Techniques: Variant Assay

    Robustness of intramolecular communication between EF-hands in CaM-RyR1/2 complexes. (A) Intramolecular communication among EF-hands in the CaM-RyR1 (left) and CaM-RyR2 (right) complexes. Communication robustness between EF-hands (EF1 to EF4, represented by their bidentate Glu residues) in CaM WT (black), N97I (red), and Q135P (green) variants in complex with RyR peptides. (B) Effects of specific RyR peptides on the intramolecular communication among EF-hands in CaM-RyR complexes. Communication robustness between EF-hands (EF1 to EF4, represented by their bidentate Glu residues) in CaM variants in complex with RyR1 (black) and RyR2 (red) peptides.

    Journal: Frontiers in Molecular Biosciences

    Article Title: Calmodulin variants associated with congenital arrhythmia impair selectivity for ryanodine receptors

    doi: 10.3389/fmolb.2022.1100992

    Figure Lengend Snippet: Robustness of intramolecular communication between EF-hands in CaM-RyR1/2 complexes. (A) Intramolecular communication among EF-hands in the CaM-RyR1 (left) and CaM-RyR2 (right) complexes. Communication robustness between EF-hands (EF1 to EF4, represented by their bidentate Glu residues) in CaM WT (black), N97I (red), and Q135P (green) variants in complex with RyR peptides. (B) Effects of specific RyR peptides on the intramolecular communication among EF-hands in CaM-RyR complexes. Communication robustness between EF-hands (EF1 to EF4, represented by their bidentate Glu residues) in CaM variants in complex with RyR1 (black) and RyR2 (red) peptides.

    Article Snippet: Unprecedented insight into the functional properties of RyR2 and its regulation by CaM has come from cryogenic-electron microscopy (cryo-EM) ( ; ; ), which shed light on the three previously identified CaM-binding domains (CaMBD) shared by RyR receptors, which could interact individually or in groups with CaM lobes to regulate channel function ( ).

    Techniques:

    Tertiary and quaternary structure of CaM variants in complex with RyR1/2 peptides. (A) Cartoon representation of CaM in three different conformational states, namely apo (PDB: 1DMO ), Ca 2+ -bound (PDB: 1CLL ) and complexed with the RyR2 peptide used in this study (PDB: 6Y4O ). The N-terminal domain is colored in yellow, the linker region in grey, while the C-terminal domain in green. The RyR2 peptide is represented in magenta. Ca 2+ ions are represented as red spheres while the side chains of N97 and Q135 are represented in sticks with C atoms colored according to the structural region, O atoms in red and N atoms in blue. (B) Pairwise sequence alignment of the Calmodulin Binding Domain-2 of human RyR1 (Uniprot entry P21817) and RyR2 (Uniprot entry Q92736). The sequence relative to the two RyR peptides employed in this study is highlighted in yellow, the residues that are not identical in such region are represented in bold and colored in red. (C) Near-UV CD spectra (250–320 nm) of 50 μM CaM were collected in the presence of 500 µM EGTA (black dashed line) and after sequential additions of 1 mM Ca 2+ (black solid line, 500 µM free Ca 2+ ) and 100 μM RyR peptides (blue solid line for RyR1, red solid line for RyR2). The spectrum of sole buffer was considered as blank and subtracted; each curve represents the average of five accumulations. Temperature was set at 25°C and signal was normalized to protein concentration.

    Journal: Frontiers in Molecular Biosciences

    Article Title: Calmodulin variants associated with congenital arrhythmia impair selectivity for ryanodine receptors

    doi: 10.3389/fmolb.2022.1100992

    Figure Lengend Snippet: Tertiary and quaternary structure of CaM variants in complex with RyR1/2 peptides. (A) Cartoon representation of CaM in three different conformational states, namely apo (PDB: 1DMO ), Ca 2+ -bound (PDB: 1CLL ) and complexed with the RyR2 peptide used in this study (PDB: 6Y4O ). The N-terminal domain is colored in yellow, the linker region in grey, while the C-terminal domain in green. The RyR2 peptide is represented in magenta. Ca 2+ ions are represented as red spheres while the side chains of N97 and Q135 are represented in sticks with C atoms colored according to the structural region, O atoms in red and N atoms in blue. (B) Pairwise sequence alignment of the Calmodulin Binding Domain-2 of human RyR1 (Uniprot entry P21817) and RyR2 (Uniprot entry Q92736). The sequence relative to the two RyR peptides employed in this study is highlighted in yellow, the residues that are not identical in such region are represented in bold and colored in red. (C) Near-UV CD spectra (250–320 nm) of 50 μM CaM were collected in the presence of 500 µM EGTA (black dashed line) and after sequential additions of 1 mM Ca 2+ (black solid line, 500 µM free Ca 2+ ) and 100 μM RyR peptides (blue solid line for RyR1, red solid line for RyR2). The spectrum of sole buffer was considered as blank and subtracted; each curve represents the average of five accumulations. Temperature was set at 25°C and signal was normalized to protein concentration.

    Article Snippet: Cryo-EM revealed a complex CaM-RyR2 recognition mechanism, in which apo- and Ca 2+ -bound CaM bind to distinct but overlapping RyR2 sites, and demonstrated that Ca 2+ -bound CaM is one of many possible regulators competing for RyR2 gating ( ).

    Techniques: Sequencing, Binding Assay, Protein Concentration

    Investigation of secondary structure of CaM variants and their RyR1/2 peptide complexes. Far-UV CD spectra (200–250 nm) of 10 μM CaM variants alone (top panels, black), and incubated with 20 μM RyR1 (center panels, blue), or 20 μM RyR2 (bottom panels, red), were collected in the presence of 300 µM EGTA (dashed lines) and after the addition of 600 µM Ca 2+ (300 µM free Ca 2+ , solid lines). The spectrum of sole buffer was considered as blank and subtracted; each curve represents the average of five accumulations. Temperature was set at 25°C.

    Journal: Frontiers in Molecular Biosciences

    Article Title: Calmodulin variants associated with congenital arrhythmia impair selectivity for ryanodine receptors

    doi: 10.3389/fmolb.2022.1100992

    Figure Lengend Snippet: Investigation of secondary structure of CaM variants and their RyR1/2 peptide complexes. Far-UV CD spectra (200–250 nm) of 10 μM CaM variants alone (top panels, black), and incubated with 20 μM RyR1 (center panels, blue), or 20 μM RyR2 (bottom panels, red), were collected in the presence of 300 µM EGTA (dashed lines) and after the addition of 600 µM Ca 2+ (300 µM free Ca 2+ , solid lines). The spectrum of sole buffer was considered as blank and subtracted; each curve represents the average of five accumulations. Temperature was set at 25°C.

    Article Snippet: Cryo-EM revealed a complex CaM-RyR2 recognition mechanism, in which apo- and Ca 2+ -bound CaM bind to distinct but overlapping RyR2 sites, and demonstrated that Ca 2+ -bound CaM is one of many possible regulators competing for RyR2 gating ( ).

    Techniques: Incubation

    CaM affinity for RyR1 and RyR2 peptides assessed by fluorescence spectroscopy. (A) One micromolar RyR1 (left column) or RyR2 (right column) peptide was incubated with increasing amounts of WT (top row), N97I (middle row) and Q135P (bottom row) CaM in the presence of 100 µM Ca 2+ . Data are reported as a function of the peptide fraction bound to CaM (see Materials and Methods for details). Curves report the mean ± std of each point obtained in three technical replicas. Representative fitting to one-site saturation ligand binding curve is superposed to each titration set. (B) Scatter plot of replicates reporting the K D values calculated from fitting procedures in each individual dataset using a one-site binding model. Stars represent t -test statistical significance: * p -value ≤ 0.05, ** p -value ≤ 0.01.

    Journal: Frontiers in Molecular Biosciences

    Article Title: Calmodulin variants associated with congenital arrhythmia impair selectivity for ryanodine receptors

    doi: 10.3389/fmolb.2022.1100992

    Figure Lengend Snippet: CaM affinity for RyR1 and RyR2 peptides assessed by fluorescence spectroscopy. (A) One micromolar RyR1 (left column) or RyR2 (right column) peptide was incubated with increasing amounts of WT (top row), N97I (middle row) and Q135P (bottom row) CaM in the presence of 100 µM Ca 2+ . Data are reported as a function of the peptide fraction bound to CaM (see Materials and Methods for details). Curves report the mean ± std of each point obtained in three technical replicas. Representative fitting to one-site saturation ligand binding curve is superposed to each titration set. (B) Scatter plot of replicates reporting the K D values calculated from fitting procedures in each individual dataset using a one-site binding model. Stars represent t -test statistical significance: * p -value ≤ 0.05, ** p -value ≤ 0.01.

    Article Snippet: Cryo-EM revealed a complex CaM-RyR2 recognition mechanism, in which apo- and Ca 2+ -bound CaM bind to distinct but overlapping RyR2 sites, and demonstrated that Ca 2+ -bound CaM is one of many possible regulators competing for RyR2 gating ( ).

    Techniques: Fluorescence, Spectroscopy, Incubation, Ligand Binding Assay, Titration, Binding Assay

    Apparent affinities of CaM-RyR1/2 complexes assessed by fluorescence spectroscopy.

    Journal: Frontiers in Molecular Biosciences

    Article Title: Calmodulin variants associated with congenital arrhythmia impair selectivity for ryanodine receptors

    doi: 10.3389/fmolb.2022.1100992

    Figure Lengend Snippet: Apparent affinities of CaM-RyR1/2 complexes assessed by fluorescence spectroscopy.

    Article Snippet: Cryo-EM revealed a complex CaM-RyR2 recognition mechanism, in which apo- and Ca 2+ -bound CaM bind to distinct but overlapping RyR2 sites, and demonstrated that Ca 2+ -bound CaM is one of many possible regulators competing for RyR2 gating ( ).

    Techniques: Fluorescence, Spectroscopy

    Thermodynamics of CaM-RyR1/2 peptide interaction assessed by isothermal titration calorimetry. (A) Examples of ITC titration curves obtained for each CaM variant upon interaction with RyR1 or RyR2 peptides. Measurements were performed at 25°C using 20 mM Tris pH 7.5, 150 mM KCl, 5 mM Ca 2+ as working buffer and setting stirring at 750 rpm. Each titration consisted in thirty 1-µL injections of 125 µM RyR1 or RyR2 (into the titrant syringe) with 10 µM CaM variants. (B) Scatter plot of replicates summarizing the K D values calculated from the fitting using a one-site binding model (see Materials and Methods). Stars represent the p -values: * p ≤ 0.05, *** p ≤ 0.001. Data for WT CaM titration with RyR2 are from .

    Journal: Frontiers in Molecular Biosciences

    Article Title: Calmodulin variants associated with congenital arrhythmia impair selectivity for ryanodine receptors

    doi: 10.3389/fmolb.2022.1100992

    Figure Lengend Snippet: Thermodynamics of CaM-RyR1/2 peptide interaction assessed by isothermal titration calorimetry. (A) Examples of ITC titration curves obtained for each CaM variant upon interaction with RyR1 or RyR2 peptides. Measurements were performed at 25°C using 20 mM Tris pH 7.5, 150 mM KCl, 5 mM Ca 2+ as working buffer and setting stirring at 750 rpm. Each titration consisted in thirty 1-µL injections of 125 µM RyR1 or RyR2 (into the titrant syringe) with 10 µM CaM variants. (B) Scatter plot of replicates summarizing the K D values calculated from the fitting using a one-site binding model (see Materials and Methods). Stars represent the p -values: * p ≤ 0.05, *** p ≤ 0.001. Data for WT CaM titration with RyR2 are from .

    Article Snippet: Cryo-EM revealed a complex CaM-RyR2 recognition mechanism, in which apo- and Ca 2+ -bound CaM bind to distinct but overlapping RyR2 sites, and demonstrated that Ca 2+ -bound CaM is one of many possible regulators competing for RyR2 gating ( ).

    Techniques: Isothermal Titration Calorimetry, Titration, Variant Assay, Binding Assay

    Thermodynamics of CaM-RyR1/2 peptide association assessed by isothermal titration calorimetry.

    Journal: Frontiers in Molecular Biosciences

    Article Title: Calmodulin variants associated with congenital arrhythmia impair selectivity for ryanodine receptors

    doi: 10.3389/fmolb.2022.1100992

    Figure Lengend Snippet: Thermodynamics of CaM-RyR1/2 peptide association assessed by isothermal titration calorimetry.

    Article Snippet: Cryo-EM revealed a complex CaM-RyR2 recognition mechanism, in which apo- and Ca 2+ -bound CaM bind to distinct but overlapping RyR2 sites, and demonstrated that Ca 2+ -bound CaM is one of many possible regulators competing for RyR2 gating ( ).

    Techniques: Titration

    Kinetics of CaM-RyR1/2 peptide interaction investigated by surface plasmon resonance. (A) Sensorgrams collected by flowing different amounts of RyR1 and RyR2 (125 nM—2 µM) on immobilized His-CaM variants using 20 mM Tris pH 7.5, 150 mM KCl, 0.005% Tween 20, 5 mM Ca 2+ , 100 µM DTT as a running buffer. Association and dissociation phases were followed for 60 s and 300 s, respectively. Experimental curves (black solid lines) are shown together with theoretical curves (red or blue solid lines) according to a 1:1 Langmuir binding model; fitting for association and dissociation phases led to the rate constants ( k on and k off ) reported in each panel (mean ± s.e.m. of 8-20 independent binding curves). (B) Scatter plot of replicates and statistical analysis comparing rate constants for WT and each pathogenic CaM variant. Stars represent p -values: * p ≤ 0.05, ** p ≤ 0.01, **** p ≤ 0.0001.

    Journal: Frontiers in Molecular Biosciences

    Article Title: Calmodulin variants associated with congenital arrhythmia impair selectivity for ryanodine receptors

    doi: 10.3389/fmolb.2022.1100992

    Figure Lengend Snippet: Kinetics of CaM-RyR1/2 peptide interaction investigated by surface plasmon resonance. (A) Sensorgrams collected by flowing different amounts of RyR1 and RyR2 (125 nM—2 µM) on immobilized His-CaM variants using 20 mM Tris pH 7.5, 150 mM KCl, 0.005% Tween 20, 5 mM Ca 2+ , 100 µM DTT as a running buffer. Association and dissociation phases were followed for 60 s and 300 s, respectively. Experimental curves (black solid lines) are shown together with theoretical curves (red or blue solid lines) according to a 1:1 Langmuir binding model; fitting for association and dissociation phases led to the rate constants ( k on and k off ) reported in each panel (mean ± s.e.m. of 8-20 independent binding curves). (B) Scatter plot of replicates and statistical analysis comparing rate constants for WT and each pathogenic CaM variant. Stars represent p -values: * p ≤ 0.05, ** p ≤ 0.01, **** p ≤ 0.0001.

    Article Snippet: Cryo-EM revealed a complex CaM-RyR2 recognition mechanism, in which apo- and Ca 2+ -bound CaM bind to distinct but overlapping RyR2 sites, and demonstrated that Ca 2+ -bound CaM is one of many possible regulators competing for RyR2 gating ( ).

    Techniques: SPR Assay, Binding Assay, Variant Assay

    Backbone flexibility of CaM-RyR1/2 complexes. Cα-Root-Mean Square Fluctuation (RMSF) of CaM (top panels) and RyR1 or RyR2 peptides (bottom panels) calculated over 1.2 μs MD simulations of the respective complex (WT: black, N97I: blue, and Q135P: red).

    Journal: Frontiers in Molecular Biosciences

    Article Title: Calmodulin variants associated with congenital arrhythmia impair selectivity for ryanodine receptors

    doi: 10.3389/fmolb.2022.1100992

    Figure Lengend Snippet: Backbone flexibility of CaM-RyR1/2 complexes. Cα-Root-Mean Square Fluctuation (RMSF) of CaM (top panels) and RyR1 or RyR2 peptides (bottom panels) calculated over 1.2 μs MD simulations of the respective complex (WT: black, N97I: blue, and Q135P: red).

    Article Snippet: Cryo-EM revealed a complex CaM-RyR2 recognition mechanism, in which apo- and Ca 2+ -bound CaM bind to distinct but overlapping RyR2 sites, and demonstrated that Ca 2+ -bound CaM is one of many possible regulators competing for RyR2 gating ( ).

    Techniques:

    Effects of CaM variants on the connectivity of hub residues of CaM (top panels) and RyR1/2 peptides (bottom panels) . Hubs were defined as residues with degree ≥6 in the Protein Structure Network (PSN) of at least one variant. ∆Degree is calculated as the difference in hub degree between the variant (N97I: black, Q135P: red) and the WT.

    Journal: Frontiers in Molecular Biosciences

    Article Title: Calmodulin variants associated with congenital arrhythmia impair selectivity for ryanodine receptors

    doi: 10.3389/fmolb.2022.1100992

    Figure Lengend Snippet: Effects of CaM variants on the connectivity of hub residues of CaM (top panels) and RyR1/2 peptides (bottom panels) . Hubs were defined as residues with degree ≥6 in the Protein Structure Network (PSN) of at least one variant. ∆Degree is calculated as the difference in hub degree between the variant (N97I: black, Q135P: red) and the WT.

    Article Snippet: Cryo-EM revealed a complex CaM-RyR2 recognition mechanism, in which apo- and Ca 2+ -bound CaM bind to distinct but overlapping RyR2 sites, and demonstrated that Ca 2+ -bound CaM is one of many possible regulators competing for RyR2 gating ( ).

    Techniques: Variant Assay

    Robustness of intramolecular communication between EF-hands in CaM-RyR1/2 complexes. (A) Intramolecular communication among EF-hands in the CaM-RyR1 (left) and CaM-RyR2 (right) complexes. Communication robustness between EF-hands (EF1 to EF4, represented by their bidentate Glu residues) in CaM WT (black), N97I (red), and Q135P (green) variants in complex with RyR peptides. (B) Effects of specific RyR peptides on the intramolecular communication among EF-hands in CaM-RyR complexes. Communication robustness between EF-hands (EF1 to EF4, represented by their bidentate Glu residues) in CaM variants in complex with RyR1 (black) and RyR2 (red) peptides.

    Journal: Frontiers in Molecular Biosciences

    Article Title: Calmodulin variants associated with congenital arrhythmia impair selectivity for ryanodine receptors

    doi: 10.3389/fmolb.2022.1100992

    Figure Lengend Snippet: Robustness of intramolecular communication between EF-hands in CaM-RyR1/2 complexes. (A) Intramolecular communication among EF-hands in the CaM-RyR1 (left) and CaM-RyR2 (right) complexes. Communication robustness between EF-hands (EF1 to EF4, represented by their bidentate Glu residues) in CaM WT (black), N97I (red), and Q135P (green) variants in complex with RyR peptides. (B) Effects of specific RyR peptides on the intramolecular communication among EF-hands in CaM-RyR complexes. Communication robustness between EF-hands (EF1 to EF4, represented by their bidentate Glu residues) in CaM variants in complex with RyR1 (black) and RyR2 (red) peptides.

    Article Snippet: Cryo-EM revealed a complex CaM-RyR2 recognition mechanism, in which apo- and Ca 2+ -bound CaM bind to distinct but overlapping RyR2 sites, and demonstrated that Ca 2+ -bound CaM is one of many possible regulators competing for RyR2 gating ( ).

    Techniques:

    Comparative real-time PCR mRNA expression of 47 genes related to contractile function in the afferent arterioles from Notch3 −/− and wild-type littermates. Note that the expression of Cacna1h coding the α 1H subunit of the T-type Ca 2+ channel (Ca v 3.2) gene was strongly downregulated in Notch3 −/− ( p < 0.001 vs. wild-type). In contrast, Cacna1c coding the α 1C subunit of the L-type Ca 2+ channel was similarly expressed in the two strains (No. 26 on the list).

    Journal: International Journal of Molecular Sciences

    Article Title: Deletion of Notch3 Impairs Contractility of Renal Resistance Vessels Due to Deficient Ca 2+ Entry

    doi: 10.3390/ijms232416068

    Figure Lengend Snippet: Comparative real-time PCR mRNA expression of 47 genes related to contractile function in the afferent arterioles from Notch3 −/− and wild-type littermates. Note that the expression of Cacna1h coding the α 1H subunit of the T-type Ca 2+ channel (Ca v 3.2) gene was strongly downregulated in Notch3 −/− ( p < 0.001 vs. wild-type). In contrast, Cacna1c coding the α 1C subunit of the L-type Ca 2+ channel was similarly expressed in the two strains (No. 26 on the list).

    Article Snippet: 20 , ryanodine receptor 2 , ryr2 , Ryr2-Mm00465877_m1 , 1.13 ± 0.16 , 0.85 ± 0.1 , −0.42 , 0.14.

    Techniques: Real-time Polymerase Chain Reaction, Expressing