Journal: JCI Insight

Article Title: Cardiac hypertrophy and arrhythmia in mice induced by a mutation in ryanodine receptor 2

doi: 10.1172/jci.insight.126544

Figure Lengend Snippet: (A) Linear representation of a single ryanodine receptor 2 (RyR2) subunit, indicating the regions where CPVT mutations are clustered (CPVT-I through -IV). The 3 SPRY domains are indicated and color-coded in all panels. (B) Amino acid sequence of a region of SPRY2 RyR2 from 5 relevant species. Residues corresponding to the human 1107 and 1124 are highlighted. Accession numbers and other species and isoforms are indicated in Supplemental Figure 2. (C) Electron density map of RyR2 from the literature (20). Subunits are delimited by black lines. (D) Modeled interface between the 3 SPRY domains of RyR2. The positions of the 2 HCM-associated mutations, A1107 and P1124, are indicated. (E and F) Superimposed crystal structures of the WT and P1124L SPRY2 domains obtained from the literature (19) and this work, respectively. Asterisk indicates positions of residue 1124 within the β2-β3 linker. Blue arrows indicate movement of 4 residues between WT and P1124L structures. Red dashed lines indicate salt bridges.

Article Snippet: Nonspecific binding was determined in the presence of 20 μM unlabeled ryanodine (2153770, MP Biomedicals).

Techniques: Sequencing

Journal: JCI Insight

Article Title: Cardiac hypertrophy and arrhythmia in mice induced by a mutation in ryanodine receptor 2

doi: 10.1172/jci.insight.126544

Figure Lengend Snippet: (A) Ca2+-dependent [3H]ryanodine binding curve corrected for ryanodine receptor 2 (RyR2) expression. The mouse Ryr2 cDNA was transiently transfected into HEK293 cells. Cells were lysed 48 hours after transfection. Recombinant RyR2 was then incubated with [3H]ryanodine in the presence of increasing concentrations of [Ca2+] to activate the channel. [3H]ryanodine was determined by liquid scintillation. (B) Maximum binding (BMax) calculated from the curves in A using Hill’s equation. (C) Ca2+-dependent [3H]ryanodine binding curves (same as in A) normalized to 10 μM [Ca2+]. (D) EC50 for [Ca2+]-dependent [3H]ryanodine binding (A–D, n = 7 curves from at least 4 independent transfections. *P < 0.05, **P < 0.01, rank sum test). (E) Representative single RyR2 channel recordings from cardiac microsomes prepared WT and homozygous (Homo) P1124L hearts. Channels were fused into artificial planar lipid bilayers. Single channel current and open probability (PO) were recorded at nominally free [Ca2+] in a 300/50 mM Cs+ gradient. (F) Overlapped histograms of normalized current calculated from 2 representative channels. (G) Average PO of WT and P1124L channels. (H) Average single channel current calculated from recordings in a 300/50 mM Cs+ gradient (G and H, n = 3 WT, 5–6 P1124L channels. *P < 0.05, 2-tailed t test).

Article Snippet: Nonspecific binding was determined in the presence of 20 μM unlabeled ryanodine (2153770, MP Biomedicals).

Techniques: Binding Assay, Expressing, Transfection, Recombinant, Incubation

Journal: JCI Insight

Article Title: Cardiac hypertrophy and arrhythmia in mice induced by a mutation in ryanodine receptor 2

doi: 10.1172/jci.insight.126544

Figure Lengend Snippet: (A) Representative Western blots of ryanodine receptor 2 (RyR2) and calmodulin (CaM) expression in heart homogenates from mice under at 3–6 months of age and over 1 year of age. Band intensities were normalized to the GAPDH signal from the same gel. A separate loading control (GAPDH) is shown for proteins run in different gels. (B) Quantification of RyR2 expression (1-way ANOVA [3–6 months] or ANOVA on ranks [1 year]). (C) Quantification of CaM expression (n = 10 WT, 8 [3–6 months] and 9 [1 year] Het, 10 Homo hearts for each age group. *P < 0.05, ANOVA on ranks).

Article Snippet: Nonspecific binding was determined in the presence of 20 μM unlabeled ryanodine (2153770, MP Biomedicals).

Techniques: Western Blot, Expressing

Journal: Cell reports

Article Title: Electrical signals in the ER are cell type and stimulus specific with extreme spatial compartmentalization in neurons

doi: 10.1016/j.celrep.2022.111943

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Activity was manipulated via bath application of the following: 2-Methyl-6-(phenylethynyl)pyridine hydrochloride (MPEP; 1 μM; Tocris Bioscience, Bristol, UK), 7-(Hydroxyimino)cyclopropa[b]chromen-1a-carboxylate ethyl ester (CPCCOEt; 20 μM; Tocris Bioscience, Bristol, UK), Iberiotoxin (20 μM; Tocris Bioscience, Bristol, UK), KCl (40 mM; Sigma-Aldrich, St. Louis, MO), Paxilline (1 μM; Tocris Bioscience, Bristol, UK), Penitrem-A (650 nM; Tocris Bioscience, Bristol, UK), Picrotoxin (PTX; 50 μM; Tocris Bioscience, Bristol, UK), Ryanodine (Ry; 10 μM; Tocris Bioscience, Bristol, UK), sodium tetrodotoxin citrate (TTX; 1 μM; Tocris Bioscience, Bristol, UK), Thapsigargin (thaps; 10 μM; Thermo Fisher Scientific, Waltham, MA), as indicated.

Techniques: Virus, Recombinant, Staining, Plasmid Preparation, Software

Journal: Microcirculation (New York, N.Y. : 1994)

Article Title: Aging alters spontaneous and neurotransmitter-mediated Ca 2+ signaling in smooth muscle cells of mouse mesenteric arteries

doi: 10.1111/micc.12607

Figure Lengend Snippet: Transcript levels for ryanodine receptor isoforms in mesenteric artery smooth muscle cells from young and old mice. Data are from 22 with permission.

Article Snippet: Thus prepared, slides were incubated 60 min in primary antibody [Alamone Labs, 1:250] for RyR1 (Cat. #ARR-001), RyR2 (Cat. #ARR-002), or RyR3 (Cat. #ARR-003).

Techniques:

Journal: Microcirculation (New York, N.Y. : 1994)

Article Title: Aging alters spontaneous and neurotransmitter-mediated Ca 2+ signaling in smooth muscle cells of mouse mesenteric arteries

doi: 10.1111/micc.12607

Figure Lengend Snippet: Representative immunofluorescence images depicting green fluorescence for RyR1 (top rows), RyR2 (center rows) and RyR3 (bottom rows) in 3 separate SMCs from MAs of (A) young and (B) old mice, (C) SMCs incubated with respective blocking peptides, and (D) SMC with primary omitted. ToPro nuclear stain (blue) is included in all images. Scale bars = 20 μm and apply to all panels.

Article Snippet: Thus prepared, slides were incubated 60 min in primary antibody [Alamone Labs, 1:250] for RyR1 (Cat. #ARR-001), RyR2 (Cat. #ARR-002), or RyR3 (Cat. #ARR-003).

Techniques: Immunofluorescence, Fluorescence, Incubation, Blocking Assay, Staining