rabbit anti cav1 3  (Alomone Labs)


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    Structured Review

    Alomone Labs rabbit anti cav1 3
    RS1 augments the current density of <t>Cav1.3-LTCC</t> in HEK cells. (A) Cells transfected with Cav1.3 subunit without other LTCC auxiliary subunits (Cav1.3 + EGFP) do not have functional LTCCs. Co-transfection with Cav1.3 and RS1 do not elicit LTCC currents carried by Ba 2+ ( I Ba ). (B) Cells transfected with Cav1.3 and β2 (Cav1.3 + β2 + EGFP), or Cav1.3, β2, and α2δ1 (Cav1.3 + β2 + α2δ1 + EGFP) display functional Cav1.3-LTCC currents. RS1 significantly enhances Cav1.3-LTCC when co-transfected with functional Cav1.3-LTCC (Cav1.3 + β2 + RS1, or Cav1.3 + β2 + α2δ1 + RS1). (C) The maximal current densities ( I Ba ) elicited at −20 mV are (in pA/pF): −9.22 ± 1.20 for EGFP (Cav1.3 + β2 + EGFP), −16.51 ± 1.27 for RS1 (Cav1.3 + β2 + RS1), −12.58 ± 1.40 for α2δ1 + EGFP (Cav1.3 + β2 + α2δ1 + EGFP), and −18.06 ± 1.13 for α2δ1 + RS1 (Cav1.3 + β2 + α2δ1 + RS1). *Indicates a significant difference between the groups (* p
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    Images

    1) Product Images from "Retinoschisin Facilitates the Function of L-Type Voltage-Gated Calcium Channels"

    Article Title: Retinoschisin Facilitates the Function of L-Type Voltage-Gated Calcium Channels

    Journal: Frontiers in Cellular Neuroscience

    doi: 10.3389/fncel.2017.00232

    RS1 augments the current density of Cav1.3-LTCC in HEK cells. (A) Cells transfected with Cav1.3 subunit without other LTCC auxiliary subunits (Cav1.3 + EGFP) do not have functional LTCCs. Co-transfection with Cav1.3 and RS1 do not elicit LTCC currents carried by Ba 2+ ( I Ba ). (B) Cells transfected with Cav1.3 and β2 (Cav1.3 + β2 + EGFP), or Cav1.3, β2, and α2δ1 (Cav1.3 + β2 + α2δ1 + EGFP) display functional Cav1.3-LTCC currents. RS1 significantly enhances Cav1.3-LTCC when co-transfected with functional Cav1.3-LTCC (Cav1.3 + β2 + RS1, or Cav1.3 + β2 + α2δ1 + RS1). (C) The maximal current densities ( I Ba ) elicited at −20 mV are (in pA/pF): −9.22 ± 1.20 for EGFP (Cav1.3 + β2 + EGFP), −16.51 ± 1.27 for RS1 (Cav1.3 + β2 + RS1), −12.58 ± 1.40 for α2δ1 + EGFP (Cav1.3 + β2 + α2δ1 + EGFP), and −18.06 ± 1.13 for α2δ1 + RS1 (Cav1.3 + β2 + α2δ1 + RS1). *Indicates a significant difference between the groups (* p
    Figure Legend Snippet: RS1 augments the current density of Cav1.3-LTCC in HEK cells. (A) Cells transfected with Cav1.3 subunit without other LTCC auxiliary subunits (Cav1.3 + EGFP) do not have functional LTCCs. Co-transfection with Cav1.3 and RS1 do not elicit LTCC currents carried by Ba 2+ ( I Ba ). (B) Cells transfected with Cav1.3 and β2 (Cav1.3 + β2 + EGFP), or Cav1.3, β2, and α2δ1 (Cav1.3 + β2 + α2δ1 + EGFP) display functional Cav1.3-LTCC currents. RS1 significantly enhances Cav1.3-LTCC when co-transfected with functional Cav1.3-LTCC (Cav1.3 + β2 + RS1, or Cav1.3 + β2 + α2δ1 + RS1). (C) The maximal current densities ( I Ba ) elicited at −20 mV are (in pA/pF): −9.22 ± 1.20 for EGFP (Cav1.3 + β2 + EGFP), −16.51 ± 1.27 for RS1 (Cav1.3 + β2 + RS1), −12.58 ± 1.40 for α2δ1 + EGFP (Cav1.3 + β2 + α2δ1 + EGFP), and −18.06 ± 1.13 for α2δ1 + RS1 (Cav1.3 + β2 + α2δ1 + RS1). *Indicates a significant difference between the groups (* p

    Techniques Used: Transfection, Functional Assay, Cotransfection

    Deletion of RS1 decreases the protein expression of Cav1.3 and Cav1.4. (A) The upper panel (a1-c5) contains retinal sections of wild type (WT), and the lower panel (d1-f5) contains retinal sections of RS1 −/− . (a1-a2) and (d1-d2) are the immunostaining for RS1. (b1-b5) and (e1-e5) are the double immunostaining for Cav1.3 and Ribeye; (c1-c5) and (f1-f5) are the double immunostaining for Cav1.4 and Ribeye. The scale bar represents 50 μm. (B) The same immunostained retinal sections are shown at a higher magnification (40×). The upper panel contains retinal sections from WT (a1-a5) and RS1 −/− (b1-b5) that were double-stained for Cav1.3 and Ribeye. Images in (a1-a5) and (b1-b5) include retinal layers of IS, ONL, OPL and INL. The lower panel contains retinal sections from WT (c1-c5) and RS1 −/− (d1-d5) that were double-stained for Cav1.4 and Ribeye. Images in (c1-c5) and (d1-d5) include retinal layers of ONL, OPL and INL. The scale bar represents 50 μm. 4′ s ,6-diamidino-2-phenylindole (DAPI) stains the nuclei. BF, bright field; IS, photoreceptor inner segments; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer.
    Figure Legend Snippet: Deletion of RS1 decreases the protein expression of Cav1.3 and Cav1.4. (A) The upper panel (a1-c5) contains retinal sections of wild type (WT), and the lower panel (d1-f5) contains retinal sections of RS1 −/− . (a1-a2) and (d1-d2) are the immunostaining for RS1. (b1-b5) and (e1-e5) are the double immunostaining for Cav1.3 and Ribeye; (c1-c5) and (f1-f5) are the double immunostaining for Cav1.4 and Ribeye. The scale bar represents 50 μm. (B) The same immunostained retinal sections are shown at a higher magnification (40×). The upper panel contains retinal sections from WT (a1-a5) and RS1 −/− (b1-b5) that were double-stained for Cav1.3 and Ribeye. Images in (a1-a5) and (b1-b5) include retinal layers of IS, ONL, OPL and INL. The lower panel contains retinal sections from WT (c1-c5) and RS1 −/− (d1-d5) that were double-stained for Cav1.4 and Ribeye. Images in (c1-c5) and (d1-d5) include retinal layers of ONL, OPL and INL. The scale bar represents 50 μm. 4′ s ,6-diamidino-2-phenylindole (DAPI) stains the nuclei. BF, bright field; IS, photoreceptor inner segments; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer.

    Techniques Used: Expressing, Immunostaining, Double Immunostaining, Staining

    Deletion of Cav1.3 decreases RS1 distribution in the retina. (A) Images were taken at a lower magnification (20×) of WT (a1-a3, b1-b3) and Cav1.3 −/− (c1-c3, d1-d3). Retinal sections were immunostained for Cav1.3 (a1-a3, c1-c3) and RS1 (b1-b3, d1-d3). The scale bar represents 50 μm. (B) Images were taken at a higher magnification (40×) of WT (a1-a3, c1-c3) and Cav1.3 −/− (b1-b3, d1-d3). Retinal sections were immunostained for RS1. The scale bar represents 50 μm. DAPI stains the nuclei. BF, bright field; IS, photoreceptor inner segments; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer.
    Figure Legend Snippet: Deletion of Cav1.3 decreases RS1 distribution in the retina. (A) Images were taken at a lower magnification (20×) of WT (a1-a3, b1-b3) and Cav1.3 −/− (c1-c3, d1-d3). Retinal sections were immunostained for Cav1.3 (a1-a3, c1-c3) and RS1 (b1-b3, d1-d3). The scale bar represents 50 μm. (B) Images were taken at a higher magnification (40×) of WT (a1-a3, c1-c3) and Cav1.3 −/− (b1-b3, d1-d3). Retinal sections were immunostained for RS1. The scale bar represents 50 μm. DAPI stains the nuclei. BF, bright field; IS, photoreceptor inner segments; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer.

    Techniques Used:

    There is a physical interaction between retinoschisin (RS1) and L-type voltage-gated calcium channel (LTCC)α1 subunits. (A) Anti-RS1 antibody (RS1 Ab) is able to co-immunoprecipitate Cav1.3 from the porcine retina. (B) RS1 Ab is able to co-immunoprecipitate Cav1.4 from the porcine retina. (C) The whole cell lysates as loading control for (A,B) . (D) Mammalian two-hybrid (luciferase reporter) assays show that hRS1 is able to interact with the first 500 amino acids from the N-terminus of Cav1.4 (hCav1.4-N) including the first motif (I). Cells co-transfected with hRS1 and hCav1.4-N (hRS1 + hCav1.4-N) have significantly higher luciferase activities than the other two control groups ( n = 6 for each group, * p
    Figure Legend Snippet: There is a physical interaction between retinoschisin (RS1) and L-type voltage-gated calcium channel (LTCC)α1 subunits. (A) Anti-RS1 antibody (RS1 Ab) is able to co-immunoprecipitate Cav1.3 from the porcine retina. (B) RS1 Ab is able to co-immunoprecipitate Cav1.4 from the porcine retina. (C) The whole cell lysates as loading control for (A,B) . (D) Mammalian two-hybrid (luciferase reporter) assays show that hRS1 is able to interact with the first 500 amino acids from the N-terminus of Cav1.4 (hCav1.4-N) including the first motif (I). Cells co-transfected with hRS1 and hCav1.4-N (hRS1 + hCav1.4-N) have significantly higher luciferase activities than the other two control groups ( n = 6 for each group, * p

    Techniques Used: Luciferase, Transfection

    2) Product Images from "Cell-type-specific tuning of Cav1.3 Ca2+-channels by a C-terminal automodulatory domain"

    Article Title: Cell-type-specific tuning of Cav1.3 Ca2+-channels by a C-terminal automodulatory domain

    Journal: Frontiers in Cellular Neuroscience

    doi: 10.3389/fncel.2015.00309

    Western blot analysis of α1-subunits in WT and mutant mouse brain homogenates. (A) Proteins (100 μg/lane) were separated on 5% SDS page and immunostained with polyclonal antibody Cav1.3 α1 CT . The Cav1.3 α1 subunit was specifically detected as a 231 kDa protein at expression levels indistinguishable between WT and homozygous mutants (HA) (see Results). KO, Cav1.3 -/- negative control. (B) Same separation as in A (100 μg/lane) but detection with anti-Cav1.3α1 NT . The migration of recombinant mCav1.3 L (L) and mCav1.343 S (43S) on the same gel (not shown) and their calculated molecular mass are indicated by arrows (left). Migration of molecular mass standards as well as the brain long and short α1-subunit species are also indicated (right). An unspecific ∼120 kDa band served as loading control. One representative experiment of at least three independent experiments is shown for all panels. KO, Cav1.3 -/- ; HA, Cav1.3DCRD HA/HA , WT, wild-type littermate.
    Figure Legend Snippet: Western blot analysis of α1-subunits in WT and mutant mouse brain homogenates. (A) Proteins (100 μg/lane) were separated on 5% SDS page and immunostained with polyclonal antibody Cav1.3 α1 CT . The Cav1.3 α1 subunit was specifically detected as a 231 kDa protein at expression levels indistinguishable between WT and homozygous mutants (HA) (see Results). KO, Cav1.3 -/- negative control. (B) Same separation as in A (100 μg/lane) but detection with anti-Cav1.3α1 NT . The migration of recombinant mCav1.3 L (L) and mCav1.343 S (43S) on the same gel (not shown) and their calculated molecular mass are indicated by arrows (left). Migration of molecular mass standards as well as the brain long and short α1-subunit species are also indicated (right). An unspecific ∼120 kDa band served as loading control. One representative experiment of at least three independent experiments is shown for all panels. KO, Cav1.3 -/- ; HA, Cav1.3DCRD HA/HA , WT, wild-type littermate.

    Techniques Used: Western Blot, Mutagenesis, SDS Page, Expressing, Negative Control, Migration, Recombinant

    Cav1.3 α1 transcripts containing exons 43 S and 43 L in mouse IHCs and OHCs, at P6 and P22 using nested PCR. Fragments containing 43S (403 bp) or 43L (557 bp) were amplified using nested PCR (see Materials and Methods) with primers specific for exon 42 (forward) and 45 (reverse) of mouse Cav1.3. S1–S14 represent samples from independent preparations. For each cell type and developmental stage at least three independent experiments were performed. Whole brain (WB) and heart (WH) served as positive controls, H 2 O (no template) as negative control. Specificity of PCR products was confirmed by sequencing. When two independent PCR reactions with three different RNA samples of each cell type were performed, the number of successful detections for each transcript was as follows: detection of 43L: 6 (out of six experiments) in IHC and OHC preparations of all developmental stages; detection of 43S: 4 (6) in IHC P06 and IHC P22, 6 (6) in OHC P06 and 5 (6) in OHC P22. Bp, basepair markers.
    Figure Legend Snippet: Cav1.3 α1 transcripts containing exons 43 S and 43 L in mouse IHCs and OHCs, at P6 and P22 using nested PCR. Fragments containing 43S (403 bp) or 43L (557 bp) were amplified using nested PCR (see Materials and Methods) with primers specific for exon 42 (forward) and 45 (reverse) of mouse Cav1.3. S1–S14 represent samples from independent preparations. For each cell type and developmental stage at least three independent experiments were performed. Whole brain (WB) and heart (WH) served as positive controls, H 2 O (no template) as negative control. Specificity of PCR products was confirmed by sequencing. When two independent PCR reactions with three different RNA samples of each cell type were performed, the number of successful detections for each transcript was as follows: detection of 43L: 6 (out of six experiments) in IHC and OHC preparations of all developmental stages; detection of 43S: 4 (6) in IHC P06 and IHC P22, 6 (6) in OHC P06 and 5 (6) in OHC P22. Bp, basepair markers.

    Techniques Used: Nested PCR, Amplification, Western Blot, Negative Control, Polymerase Chain Reaction, Sequencing, Immunohistochemistry

    Absence of smaller C-terminally–derived Cav1.3 α1 fragments in WT and Cav1.3DCRD HA/HA brain preparations. (A) Mouse brain homogenate (100 μg of protein/lane) prepared from WT or Cav1.3DCRD HA/HA (HA) mice were separated on 4–15% gradient SDS-PAGE and immunostained with anti-HA antibody. The blot was overexposed to also visualize less abundant smaller fragments. in separate experiments α1- associated HA-immunoreactivity could be detected with only 10% (10 μg/lane) of the protein amount used ( n = 3) demonstrating the sensitivity of the assay. (B) Mouse brain membranes (100 μg of protein/lane) were analyzed as in (A). (C) Mouse brain membranes (100 μg of protein/lane) from WT or Cav1.3 -/- (KO) mice were blotted as in (B) and stained with anti-Cav1.3α1 CT antibodies. To some WT samples (33 μg/lane) a 45 kDa recombinant C-terminal control peptide was added (arrow, amounts indicated) before separation to demonstrate successful transfer and sensitive detection as a positive control for sensitivity.
    Figure Legend Snippet: Absence of smaller C-terminally–derived Cav1.3 α1 fragments in WT and Cav1.3DCRD HA/HA brain preparations. (A) Mouse brain homogenate (100 μg of protein/lane) prepared from WT or Cav1.3DCRD HA/HA (HA) mice were separated on 4–15% gradient SDS-PAGE and immunostained with anti-HA antibody. The blot was overexposed to also visualize less abundant smaller fragments. in separate experiments α1- associated HA-immunoreactivity could be detected with only 10% (10 μg/lane) of the protein amount used ( n = 3) demonstrating the sensitivity of the assay. (B) Mouse brain membranes (100 μg of protein/lane) were analyzed as in (A). (C) Mouse brain membranes (100 μg of protein/lane) from WT or Cav1.3 -/- (KO) mice were blotted as in (B) and stained with anti-Cav1.3α1 CT antibodies. To some WT samples (33 μg/lane) a 45 kDa recombinant C-terminal control peptide was added (arrow, amounts indicated) before separation to demonstrate successful transfer and sensitive detection as a positive control for sensitivity.

    Techniques Used: Derivative Assay, Mouse Assay, SDS Page, Staining, Recombinant, Positive Control

    Comparison of the protein localization of anti-HA-labeled Cav1.3 with immunolabeled Ca v 1.3, Ca v β2 and CtBP2/RIBEYE in IHCs. (A–D) Whole-mount preparations of apical turns of the organ of Corti from adult Ca v 1.3DCRD HA/HA (A,C,D) and WT (B) mice were co-immunolabeled with anti-HA and anti-Ca v 1.3 ( A, B , 11 weeks), anti-HA and anti-Ca v β2 ( C , P28), or anti-HA and anti-CtBP2/RIBEYE antibodies ( D , P37). Every image shows the basolateral poles of two adjacent IHCs the nuclei of which are indicated by asterisks in the rightmost column, respectively. HA staining (A1,C1,D1) largely overlapped with Ca v 1.3 (A2) , Ca v ß2 (C2) and CtBP2 (D2) staining at the basal poles of IHCs as evident upon merging corresponding images (A3,C3,D3) . In the WT, no specific HA-labeling (B1) was present at the position of Ca v 1.3 labeling (B2,B3) . The weak ‘cloudy’ green anti-HA staining was present in all specimen investigated and therefore considered unspecific. Cell nuclei of IHCs were counterstained with DAPI (blue). 1 of 3 ( A , age: 2–3 months), 1 of 4 ( B , age: P25 – 3 month), 1 of 5 ( C , P25–P31) and 1 of 5 ( D , P28–P37) independent experiments is illustrated, respectively. Scale bars: 5 μm.
    Figure Legend Snippet: Comparison of the protein localization of anti-HA-labeled Cav1.3 with immunolabeled Ca v 1.3, Ca v β2 and CtBP2/RIBEYE in IHCs. (A–D) Whole-mount preparations of apical turns of the organ of Corti from adult Ca v 1.3DCRD HA/HA (A,C,D) and WT (B) mice were co-immunolabeled with anti-HA and anti-Ca v 1.3 ( A, B , 11 weeks), anti-HA and anti-Ca v β2 ( C , P28), or anti-HA and anti-CtBP2/RIBEYE antibodies ( D , P37). Every image shows the basolateral poles of two adjacent IHCs the nuclei of which are indicated by asterisks in the rightmost column, respectively. HA staining (A1,C1,D1) largely overlapped with Ca v 1.3 (A2) , Ca v ß2 (C2) and CtBP2 (D2) staining at the basal poles of IHCs as evident upon merging corresponding images (A3,C3,D3) . In the WT, no specific HA-labeling (B1) was present at the position of Ca v 1.3 labeling (B2,B3) . The weak ‘cloudy’ green anti-HA staining was present in all specimen investigated and therefore considered unspecific. Cell nuclei of IHCs were counterstained with DAPI (blue). 1 of 3 ( A , age: 2–3 months), 1 of 4 ( B , age: P25 – 3 month), 1 of 5 ( C , P25–P31) and 1 of 5 ( D , P28–P37) independent experiments is illustrated, respectively. Scale bars: 5 μm.

    Techniques Used: Labeling, Immunolabeling, Mouse Assay, Staining

    Activation and inactivation properties of I Ca through mCav1.3 L and mCav1.3 L -HA channels. (A) α1-subunits were heterologously expressed in tsA-201 cells together with β 3 and α 2 δ 1 (at least three independent transfections). Whole-cell patch-clamp current–voltage relationship obtained by depolarizations from a Vh of -80 mV to the indicated test potentials in cells transfected with mouse wild-type (WT) Cav1.3 (mCav1.3 L , black) and mCav1.3 L -HA (blue). All data were junction potential – corrected. (B) Percent I Ca inactivation (15 mM Ca 2+ ) during a test pulse from -80 mV to the V max . ∗∗∗ p
    Figure Legend Snippet: Activation and inactivation properties of I Ca through mCav1.3 L and mCav1.3 L -HA channels. (A) α1-subunits were heterologously expressed in tsA-201 cells together with β 3 and α 2 δ 1 (at least three independent transfections). Whole-cell patch-clamp current–voltage relationship obtained by depolarizations from a Vh of -80 mV to the indicated test potentials in cells transfected with mouse wild-type (WT) Cav1.3 (mCav1.3 L , black) and mCav1.3 L -HA (blue). All data were junction potential – corrected. (B) Percent I Ca inactivation (15 mM Ca 2+ ) during a test pulse from -80 mV to the V max . ∗∗∗ p

    Techniques Used: Activation Assay, Transfection, Patch Clamp

    3) Product Images from "Thyroid hormone is required for pruning, functioning and long-term maintenance of afferent inner hair cell synapses"

    Article Title: Thyroid hormone is required for pruning, functioning and long-term maintenance of afferent inner hair cell synapses

    Journal: The European journal of neuroscience

    doi: 10.1111/ejn.13081

    A cartoon representing normal and hypothyroid adult IHC synapses. (a) WT IHC. (b) Hypothyroid IHC. (c) Magnification of the box in b. A hypothyroid IHC is characterized by an excess of afferent synapses caused by the abnormal retention of afferent neurite branches and terminals, calcium channels being more widely distributed around the hair cell, and higher glutamate buildup at the synaptic cleft, owing to lower GLAST levels (b and c). In comparison, a normal IHC shows fewer but well-organized afferent terminals and a more clustered pattern of CaV1.3 calcium channel expression at the basal region of the hair cell. a, afferent fibre; e, efferent fibre; r, ribbon synapse.
    Figure Legend Snippet: A cartoon representing normal and hypothyroid adult IHC synapses. (a) WT IHC. (b) Hypothyroid IHC. (c) Magnification of the box in b. A hypothyroid IHC is characterized by an excess of afferent synapses caused by the abnormal retention of afferent neurite branches and terminals, calcium channels being more widely distributed around the hair cell, and higher glutamate buildup at the synaptic cleft, owing to lower GLAST levels (b and c). In comparison, a normal IHC shows fewer but well-organized afferent terminals and a more clustered pattern of CaV1.3 calcium channel expression at the basal region of the hair cell. a, afferent fibre; e, efferent fibre; r, ribbon synapse.

    Techniques Used: Immunohistochemistry, Expressing

    Abnormal CaV1.3 puncta clustering at the synapses of Pit1 dw IHCs. (a and b) Projection of confocal sections obtained from the mid-turn of cochlear whole mounts stained with the afferent presynaptic marker RIBEYE (green) and the calcium channel CaV1.3 (red) at P7 in WT mice (a) and Pit1 dw mice (b). (c–f) The same markers at P14 for WT (c) and Pit1 dw mice (d), and at P24 for WT (e) and Pit1 dw mice (f). (g–i) Box plots of the quantification of RIBEYE (g), CaV1.3 (h) and RIBEYE–CaV1.3 (i) puncta from the mid-turn of the cochlea in WT and Pit1 dw mice. significant comparisons ( P
    Figure Legend Snippet: Abnormal CaV1.3 puncta clustering at the synapses of Pit1 dw IHCs. (a and b) Projection of confocal sections obtained from the mid-turn of cochlear whole mounts stained with the afferent presynaptic marker RIBEYE (green) and the calcium channel CaV1.3 (red) at P7 in WT mice (a) and Pit1 dw mice (b). (c–f) The same markers at P14 for WT (c) and Pit1 dw mice (d), and at P24 for WT (e) and Pit1 dw mice (f). (g–i) Box plots of the quantification of RIBEYE (g), CaV1.3 (h) and RIBEYE–CaV1.3 (i) puncta from the mid-turn of the cochlea in WT and Pit1 dw mice. significant comparisons ( P

    Techniques Used: Staining, Marker, Mouse Assay

    4) Product Images from "Different roles attributed to Cav1 channel subtypes in spontaneous action potential firing and fine tuning of exocytosis in mouse chromaffin cells"

    Article Title: Different roles attributed to Cav1 channel subtypes in spontaneous action potential firing and fine tuning of exocytosis in mouse chromaffin cells

    Journal: Journal of Neurochemistry

    doi: 10.1111/j.1471-4159.2010.07089.x

    Cav1 channel deletion compensated by the increased expression of other Cav channel types. Pharmacological dissection of Ca 2+ channels in mouse chromaffin cells from WT and Cav1.3 −/− cells. (a and b) Time course of the Ca 2+ charge density obtained after sequentially and cumulatively adding the different Ca 2+ channel blockers, in WT and Cav1.3 −/− cells, respectively: 3 μM nifedipine was used to block Cav1 channels, 1 μM ω‐CTX‐GVIA to block Cav2.2 channels, 3 μM ω‐CTX‐MVIIC to block Cav2.1 channels, and 200 μM Cd 2+ to block the residual Ca 2+ current. (c and d) Original traces of the Ca 2+ currents recorded at the stationary stage using each Ca 2+ channel blocker (corresponding to points a–f in panels 3a and b, where a : control, b : after 3 μM nifedipine perfusion, c : after 3 μM nifedipine and 1 μM ω‐CTX‐GVIA perfusion and d : after 3 μM nifedipine, ω‐CTX‐GVIA and 3 μM ω‐CTX‐MVIIC perfusion). (e) Ca 2+ charge density of the different Ca 2+ channel types for WT (black columns) and Cav1.3 −/− cells (white columns), respectively. (f) Total Ca 2+ charge obtained under control conditions for WT (black column) and Cav1.3 −/− cells (white column). (g) Sizes of chromaffin cells obtained from WT (black column) and Cav1.3 −/− mice (white column). Experiments were performed on nine paired cultures of WT ( n = 18 cells) and Cav1.3 −/− cells ( n = 17 cells), using 1–2 mice of each strain. Bars represent means ± SEM. ** p
    Figure Legend Snippet: Cav1 channel deletion compensated by the increased expression of other Cav channel types. Pharmacological dissection of Ca 2+ channels in mouse chromaffin cells from WT and Cav1.3 −/− cells. (a and b) Time course of the Ca 2+ charge density obtained after sequentially and cumulatively adding the different Ca 2+ channel blockers, in WT and Cav1.3 −/− cells, respectively: 3 μM nifedipine was used to block Cav1 channels, 1 μM ω‐CTX‐GVIA to block Cav2.2 channels, 3 μM ω‐CTX‐MVIIC to block Cav2.1 channels, and 200 μM Cd 2+ to block the residual Ca 2+ current. (c and d) Original traces of the Ca 2+ currents recorded at the stationary stage using each Ca 2+ channel blocker (corresponding to points a–f in panels 3a and b, where a : control, b : after 3 μM nifedipine perfusion, c : after 3 μM nifedipine and 1 μM ω‐CTX‐GVIA perfusion and d : after 3 μM nifedipine, ω‐CTX‐GVIA and 3 μM ω‐CTX‐MVIIC perfusion). (e) Ca 2+ charge density of the different Ca 2+ channel types for WT (black columns) and Cav1.3 −/− cells (white columns), respectively. (f) Total Ca 2+ charge obtained under control conditions for WT (black column) and Cav1.3 −/− cells (white column). (g) Sizes of chromaffin cells obtained from WT (black column) and Cav1.3 −/− mice (white column). Experiments were performed on nine paired cultures of WT ( n = 18 cells) and Cav1.3 −/− cells ( n = 17 cells), using 1–2 mice of each strain. Bars represent means ± SEM. ** p

    Techniques Used: Expressing, Dissection, Blocking Assay, Mouse Assay

    Contribution of Cav1 channel subtypes to pacemaking activity, and shaping of action potential waveform. (a–b) Recordings of the spontaneous firing of action potentials performed in the current clamp configuration in WT (a) or Cav1.3 −/− cells (b) under control conditions, and after perfusion with 300 nM nifedipine and 3 μM nifedipine. (c–d) Mean action potential obtained by averaging the action potentials recorded over 10 s under control conditions or after 300 nM and 3 μM nifedipine application to WT ( n = 6) (c) or Cav1.3 −/− cells ( n = 4) (d). Data were obtained in two paired cultures of WT and Cav1.3 −/− cells, using 1–2 mice of each strain.
    Figure Legend Snippet: Contribution of Cav1 channel subtypes to pacemaking activity, and shaping of action potential waveform. (a–b) Recordings of the spontaneous firing of action potentials performed in the current clamp configuration in WT (a) or Cav1.3 −/− cells (b) under control conditions, and after perfusion with 300 nM nifedipine and 3 μM nifedipine. (c–d) Mean action potential obtained by averaging the action potentials recorded over 10 s under control conditions or after 300 nM and 3 μM nifedipine application to WT ( n = 6) (c) or Cav1.3 −/− cells ( n = 4) (d). Data were obtained in two paired cultures of WT and Cav1.3 −/− cells, using 1–2 mice of each strain.

    Techniques Used: Activity Assay, Mouse Assay

    Contribution of Cav1 channel subtypes to pacemaking activity. (a–b) Phase‐plane plot obtained by plotting dV/dt versus the voltage stimulus in WT and Cav1.3 −/− , respectively. Arrows indicate the points at which dV/dt increased from the initial baseline (threshold potential). Estimated threshold potentials were −27 mV and −22 mV for WT and Cav1.3 −/− cells, respectively; (c–d) action potential clamp experiments were performed by applying the mean voltage stimulus obtained under control conditions in Fig. 8(g and h) every 30 s. Starting from ‘Solution 3’ (see Material and Methods ), different blockers were sequentially added to that solution: 2 μM TTX (Solution 3 + TTX), 45 mM TEA (Solution 3 + TTX + TEA), 3 μM nifedipine (Solution 3 + TTX + TEA + Nife) and 200 μM CdCl 2 (Solution 3 + TTX + TEA + Nife + Cd). Perfusion with each solution was continued for at least 2 min so that the currents reached the steady‐state. Number of cell: 22 WT cells, 25 Cav1.3 −/− cells. Data were obtained in four paired cultures of WT and Cav1.3 −/− cells, using two mice of each strain. (e–f) Ion currents were calculated from the recordings of panels (c) and (d), for WT and Cav1.3 −/− cells, respectively. To obtain the Na + current, the current obtained after perfusion with ‘Solution 3 + TTX’ was subtracted from the ‘Solution 3’ current. The K + current was calculated as the difference in the current yielded under ‘Solution 3 + TTX + TEA’ minus ‘Solution 3 + TTX’. The Cav1 current was obtained as the difference between ‘Solution 3 + TTX + TEA’ and ‘Solution 3 + TTX + TEA + Nife’ and the total Ca 2+ current as the difference between ‘Solution 3 + TTX + TEA’ and ‘Solution 3 + TTX + TEA + Cd’. The voltage stimulus, obtained from Fig. 8(g and h) , control conditions, was superimposed on the ion currents. Data were obtained in 4 paired cultures of WT and Cav1.3 −/− cells, using two mice of each strain.
    Figure Legend Snippet: Contribution of Cav1 channel subtypes to pacemaking activity. (a–b) Phase‐plane plot obtained by plotting dV/dt versus the voltage stimulus in WT and Cav1.3 −/− , respectively. Arrows indicate the points at which dV/dt increased from the initial baseline (threshold potential). Estimated threshold potentials were −27 mV and −22 mV for WT and Cav1.3 −/− cells, respectively; (c–d) action potential clamp experiments were performed by applying the mean voltage stimulus obtained under control conditions in Fig. 8(g and h) every 30 s. Starting from ‘Solution 3’ (see Material and Methods ), different blockers were sequentially added to that solution: 2 μM TTX (Solution 3 + TTX), 45 mM TEA (Solution 3 + TTX + TEA), 3 μM nifedipine (Solution 3 + TTX + TEA + Nife) and 200 μM CdCl 2 (Solution 3 + TTX + TEA + Nife + Cd). Perfusion with each solution was continued for at least 2 min so that the currents reached the steady‐state. Number of cell: 22 WT cells, 25 Cav1.3 −/− cells. Data were obtained in four paired cultures of WT and Cav1.3 −/− cells, using two mice of each strain. (e–f) Ion currents were calculated from the recordings of panels (c) and (d), for WT and Cav1.3 −/− cells, respectively. To obtain the Na + current, the current obtained after perfusion with ‘Solution 3 + TTX’ was subtracted from the ‘Solution 3’ current. The K + current was calculated as the difference in the current yielded under ‘Solution 3 + TTX + TEA’ minus ‘Solution 3 + TTX’. The Cav1 current was obtained as the difference between ‘Solution 3 + TTX + TEA’ and ‘Solution 3 + TTX + TEA + Nife’ and the total Ca 2+ current as the difference between ‘Solution 3 + TTX + TEA’ and ‘Solution 3 + TTX + TEA + Cd’. The voltage stimulus, obtained from Fig. 8(g and h) , control conditions, was superimposed on the ion currents. Data were obtained in 4 paired cultures of WT and Cav1.3 −/− cells, using two mice of each strain.

    Techniques Used: Activity Assay, Mouse Assay

    Cav1 channel subtypes expressed in mouse chromaffin cells. Sensitivity of Cav1 channel subtypes to DHPs. Square‐step depolarizing pulses of 50 ms duration were applied every 30 s to the peak current voltage. (a, c and e) Ca 2+ charge density blockade obtained after perfusion with 300 nM (black columns) and 3 μM (white columns) nifedipine (Nife) in WT, Cav1.3 −/− and Cav1.2DHP −/− cells, respectively. A large fraction of Cav1.3 −/− cells ( n = 21) did not respond to 300 nM nifedipine. (b, d and f) Original Ca 2+ current traces under control conditions or after perfusion with 300 nM or 3 μM nifedipine in WT, Cav1.3 −/− and Cav1.2DHP −/− cells, respectively. Experiments were performed on seven paired cultures of WT and Cav1.3 −/− cells and five paired cultures of WT and Cav1.2DHP −/− cells, using two mice from each strain. Numbers of cells indicated in parentheses. Bars represent means ± SEM. *** p
    Figure Legend Snippet: Cav1 channel subtypes expressed in mouse chromaffin cells. Sensitivity of Cav1 channel subtypes to DHPs. Square‐step depolarizing pulses of 50 ms duration were applied every 30 s to the peak current voltage. (a, c and e) Ca 2+ charge density blockade obtained after perfusion with 300 nM (black columns) and 3 μM (white columns) nifedipine (Nife) in WT, Cav1.3 −/− and Cav1.2DHP −/− cells, respectively. A large fraction of Cav1.3 −/− cells ( n = 21) did not respond to 300 nM nifedipine. (b, d and f) Original Ca 2+ current traces under control conditions or after perfusion with 300 nM or 3 μM nifedipine in WT, Cav1.3 −/− and Cav1.2DHP −/− cells, respectively. Experiments were performed on seven paired cultures of WT and Cav1.3 −/− cells and five paired cultures of WT and Cav1.2DHP −/− cells, using two mice from each strain. Numbers of cells indicated in parentheses. Bars represent means ± SEM. *** p

    Techniques Used: Mouse Assay

    Voltage dependent activation of Ca 2+ channels. (a) I – V curves obtained under control conditions in WT and Cav1.3 −/− cells ( n = 8–12). 200 ms square‐step depolarizing pulses at increasing potentials (voltage increments of 10 mV), from −50 mV to 80 mV, were applied every 1 min. Data were obtained from four paired cultures of WT and Cav1.3 −/− cells, using 1–2 mice from each strain and normalized as the percentage of current in control conditions at 10 mV, plotted as the mean ± SEM. * p
    Figure Legend Snippet: Voltage dependent activation of Ca 2+ channels. (a) I – V curves obtained under control conditions in WT and Cav1.3 −/− cells ( n = 8–12). 200 ms square‐step depolarizing pulses at increasing potentials (voltage increments of 10 mV), from −50 mV to 80 mV, were applied every 1 min. Data were obtained from four paired cultures of WT and Cav1.3 −/− cells, using 1–2 mice from each strain and normalized as the percentage of current in control conditions at 10 mV, plotted as the mean ± SEM. * p

    Techniques Used: Activation Assay, Mouse Assay

    Cav1 channel deletion compensated by the increased expression of other Ca 2+ channel types. Contribution of Cav channels to the exocytosis of neurotransmitters in mouse chromaffin cells from WT and Cav1.3 −/− mice. (a and b) C m traces recorded simultaneously to the Ca 2+ currents of Fig. 2(c and d) in WT and Cav1.3 −/− cells, respectively. (c) Percentage of total secretion attributed to each Ca 2+ channel type in WT (black columns) and Cav1.3 −/− cells (white columns). (d) Total secretion attained under control conditions for WT (black column) and Cav1.3 −/− cells (white column). Experiments were performed on seven paired cultures of WT ( n = 15 cells) and Cav1.3 −/− cells ( n = 11 cells), using 1–2 mice from each strain. Bars represent means ± SEM. * p
    Figure Legend Snippet: Cav1 channel deletion compensated by the increased expression of other Ca 2+ channel types. Contribution of Cav channels to the exocytosis of neurotransmitters in mouse chromaffin cells from WT and Cav1.3 −/− mice. (a and b) C m traces recorded simultaneously to the Ca 2+ currents of Fig. 2(c and d) in WT and Cav1.3 −/− cells, respectively. (c) Percentage of total secretion attributed to each Ca 2+ channel type in WT (black columns) and Cav1.3 −/− cells (white columns). (d) Total secretion attained under control conditions for WT (black column) and Cav1.3 −/− cells (white column). Experiments were performed on seven paired cultures of WT ( n = 15 cells) and Cav1.3 −/− cells ( n = 11 cells), using 1–2 mice from each strain. Bars represent means ± SEM. * p

    Techniques Used: Expressing, Mouse Assay

    Contribution of Cav1 channel subtypes to the Ca 2+ charge and exocytosis of chromaffin vesicles. Ca 2+ charge density (a) and the corresponding exocytosis (b) versus the voltage in WT and Cav1.3 −/− cells. Data were obtained in the same experiments as in Fig. 3 ( n = 8–12, from four paired cultures of WT and Cav1.3 −/− cells, using 1–2 mice of each strain) and normalized as the percentage of charge density in control conditions at 10 mV, plotted as the mean ± SEM. (c) Original traces of Ca 2+ current density and the corresponding exocytosis elicited at −20 mV, −10 mV and 0 mV in WT and Cav1.3 −/− cells. * p
    Figure Legend Snippet: Contribution of Cav1 channel subtypes to the Ca 2+ charge and exocytosis of chromaffin vesicles. Ca 2+ charge density (a) and the corresponding exocytosis (b) versus the voltage in WT and Cav1.3 −/− cells. Data were obtained in the same experiments as in Fig. 3 ( n = 8–12, from four paired cultures of WT and Cav1.3 −/− cells, using 1–2 mice of each strain) and normalized as the percentage of charge density in control conditions at 10 mV, plotted as the mean ± SEM. (c) Original traces of Ca 2+ current density and the corresponding exocytosis elicited at −20 mV, −10 mV and 0 mV in WT and Cav1.3 −/− cells. * p

    Techniques Used: Mouse Assay

    Kinetics of the Cav1 channel subtypes. One‐second square‐step depolarizing pulses were applied at −10 mV every 5 min. (a) Inactivation kinetics. Left, Ca 2+ current remaining at the end of a 1‐s pulse expressed as a percentage of the peak current ( I 1000 / I peak ) in WT (black column) and Cav1.3 −/− cells (grey column); middle, percentage of cells whose inactivation kinetics could be well fitted to a single (τ inact single , black columns) or to a double (τ inact double , grey columns) exponential function in WT and Cav1.3 −/− ; right, the average τ inact single yielded by the single exponential fitting, and τ inact double , which exhibited two components, a fast component (τ inact fast ) and a slow component (τ inact slow ), were plotted for WT and Cav1.3 −/− cells (black and grey columns, respectively). (b) Original traces of the Cav1 channel currents recorded in WT and Cav1.3 −/− cells were averaged, superimposed and scaled to the peak WT Cav1 channel current. Number of cells indicated in parentheses. Data were obtained in three paired cultures of WT and Cav1.3 −/− cells, using two mice of each strain.
    Figure Legend Snippet: Kinetics of the Cav1 channel subtypes. One‐second square‐step depolarizing pulses were applied at −10 mV every 5 min. (a) Inactivation kinetics. Left, Ca 2+ current remaining at the end of a 1‐s pulse expressed as a percentage of the peak current ( I 1000 / I peak ) in WT (black column) and Cav1.3 −/− cells (grey column); middle, percentage of cells whose inactivation kinetics could be well fitted to a single (τ inact single , black columns) or to a double (τ inact double , grey columns) exponential function in WT and Cav1.3 −/− ; right, the average τ inact single yielded by the single exponential fitting, and τ inact double , which exhibited two components, a fast component (τ inact fast ) and a slow component (τ inact slow ), were plotted for WT and Cav1.3 −/− cells (black and grey columns, respectively). (b) Original traces of the Cav1 channel currents recorded in WT and Cav1.3 −/− cells were averaged, superimposed and scaled to the peak WT Cav1 channel current. Number of cells indicated in parentheses. Data were obtained in three paired cultures of WT and Cav1.3 −/− cells, using two mice of each strain.

    Techniques Used: Mouse Assay

    Cav1 channel subtypes expressed in mouse chromaffin cells. Immunocytochemical characterization of Cav1 channel subtypes. (a–b) Confocal images of isolated mouse chromaffin cells from WT (a) or Cav1.3 −/− mice (b) labeled with antibodies against Cav1.1, Cav1.2, Cav1.3 and Cav1.4 channels (dilution 1 : 200) and the corresponding secondary antibody (dilution 1 : 200) Alexa Fluor excited at a wavelength of 594 nm (dilution 1 : 200). Experiments were performed on four paired cultures of WT and Cav1.3 −/− cells. Calibration bar: 75 microns.
    Figure Legend Snippet: Cav1 channel subtypes expressed in mouse chromaffin cells. Immunocytochemical characterization of Cav1 channel subtypes. (a–b) Confocal images of isolated mouse chromaffin cells from WT (a) or Cav1.3 −/− mice (b) labeled with antibodies against Cav1.1, Cav1.2, Cav1.3 and Cav1.4 channels (dilution 1 : 200) and the corresponding secondary antibody (dilution 1 : 200) Alexa Fluor excited at a wavelength of 594 nm (dilution 1 : 200). Experiments were performed on four paired cultures of WT and Cav1.3 −/− cells. Calibration bar: 75 microns.

    Techniques Used: Isolation, Mouse Assay, Labeling

    Contribution of Cav1 channel subtypes to pacemaking activity. (a–b) In a different set of experiments performed under the current‐clamp configuration, the spontaneous oscillatory activity resistant to TTX, obtained in half the cells treated with this toxin, was reversibly abolished by 300 nM nifedipine in WT (a) or Cav1.3 −/− cells (b). In the other half of the cells, reversible blockade of spontaneous action potentials by 2 μM TTX in WT (c) or Cav1.3 −/− (d) cells was achieved. Data were obtained in two paired cultures of WT and Cav1.3 −/− cells using two mice of each strain.
    Figure Legend Snippet: Contribution of Cav1 channel subtypes to pacemaking activity. (a–b) In a different set of experiments performed under the current‐clamp configuration, the spontaneous oscillatory activity resistant to TTX, obtained in half the cells treated with this toxin, was reversibly abolished by 300 nM nifedipine in WT (a) or Cav1.3 −/− cells (b). In the other half of the cells, reversible blockade of spontaneous action potentials by 2 μM TTX in WT (c) or Cav1.3 −/− (d) cells was achieved. Data were obtained in two paired cultures of WT and Cav1.3 −/− cells using two mice of each strain.

    Techniques Used: Activity Assay, Mouse Assay

    Coupling of Cav1 channel subtypes to BK channels. (a) Upper section: double‐pulse protocol used to recruit BK channels. This included a 400 ms test pulse ( V t ) to 140 mV or above that potential (trace 1), followed by a 10‐ms pre‐pulse applied at 0 mV before V t (trace 2). The Ca 2+ dependent K + currents activated using this protocol were BK channels. Lower section: original K + current traces recorded using the above protocol under control conditions and after perfusion with different K + channel blockers, added sequentially and cumulatively: first, 200 nM apamin, 100 nM charibdotoxin (ChTx), and finally 45 mM TEA. Pulses were applied every 2 min. Numbers of cells are indicated in parentheses. (b) The K + charge density was averaged and normalized for each condition with respect to the current in the absence of a pre‐pulse. (c–d) Effects of 3 μM nifedipine on BK channel currents in WT (c) and Cav1.3 −/− cells (d). Number of cells: 14 WT cells, 11 Cav1.3 −/− cells. Data were obtained in four paired cultures of WT and Cav1.3 −/− cells, using two mice of each strain.
    Figure Legend Snippet: Coupling of Cav1 channel subtypes to BK channels. (a) Upper section: double‐pulse protocol used to recruit BK channels. This included a 400 ms test pulse ( V t ) to 140 mV or above that potential (trace 1), followed by a 10‐ms pre‐pulse applied at 0 mV before V t (trace 2). The Ca 2+ dependent K + currents activated using this protocol were BK channels. Lower section: original K + current traces recorded using the above protocol under control conditions and after perfusion with different K + channel blockers, added sequentially and cumulatively: first, 200 nM apamin, 100 nM charibdotoxin (ChTx), and finally 45 mM TEA. Pulses were applied every 2 min. Numbers of cells are indicated in parentheses. (b) The K + charge density was averaged and normalized for each condition with respect to the current in the absence of a pre‐pulse. (c–d) Effects of 3 μM nifedipine on BK channel currents in WT (c) and Cav1.3 −/− cells (d). Number of cells: 14 WT cells, 11 Cav1.3 −/− cells. Data were obtained in four paired cultures of WT and Cav1.3 −/− cells, using two mice of each strain.

    Techniques Used: Mouse Assay

    5) Product Images from "Biophysical classification of a CACNA1D de novo mutation as a high-risk mutation for a severe neurodevelopmental disorder"

    Article Title: Biophysical classification of a CACNA1D de novo mutation as a high-risk mutation for a severe neurodevelopmental disorder

    Journal: Molecular Autism

    doi: 10.1186/s13229-019-0310-4

    Mutation S652L increases intracellular Ca 2+ during simulated action potential firing. a Upper left: Shape of single action potential waveform (APW) mimicked by the following voltage steps: HP: − 80 mV, − 80 to − 60 mV for 2.5 ms, − 60 to + 20 mV in 1 ms, + 20 to − 70 mV in 1.5 ms, − 70 to − 60 mV in 5 ms, − 60 mV for 90 ms. The corresponding I Ca of WT L and S652L L are shown below. Right: Representative current responses of WT L and S652L L during 30 s of stimulation with APW-like stimuli at a frequency of 10 Hz. b Peak I Ca of S652L L Cav1.3 channels decayed faster than WT L during stimulation. Statistics: unpaired student´s t-test ([mean ± SEM]; WT L , 14.94 ± 2.19, n = 20; S652L L , 30.94 ± 2.85***, n = 21; *** p
    Figure Legend Snippet: Mutation S652L increases intracellular Ca 2+ during simulated action potential firing. a Upper left: Shape of single action potential waveform (APW) mimicked by the following voltage steps: HP: − 80 mV, − 80 to − 60 mV for 2.5 ms, − 60 to + 20 mV in 1 ms, + 20 to − 70 mV in 1.5 ms, − 70 to − 60 mV in 5 ms, − 60 mV for 90 ms. The corresponding I Ca of WT L and S652L L are shown below. Right: Representative current responses of WT L and S652L L during 30 s of stimulation with APW-like stimuli at a frequency of 10 Hz. b Peak I Ca of S652L L Cav1.3 channels decayed faster than WT L during stimulation. Statistics: unpaired student´s t-test ([mean ± SEM]; WT L , 14.94 ± 2.19, n = 20; S652L L , 30.94 ± 2.85***, n = 21; *** p

    Techniques Used: Mutagenesis, Mass Spectrometry

    Molecular modeling of Cav1.3 WT α 1 -subunits, mutations S652L and S652W. Top: Top view and side view of the Cav1.3 α 1 -subunit structure. The region involving the inter-domain interactions (IIS4-S5–IS4-IS5) affected by the mutation is highlighted (left). Bottom: a WT inter-domain interaction of S652 in repeat II and S256 in the S4-S5 linker in repeat I. b Weaker hydrophobic interactions of the mutated residue L652 with the hydrophobic cloud in the S4-S5 linker of repeat I. c Stabilizing effect of the W652 mutation; the tryptophan residue can form an intra-domain hydrogen bond with the backbone of K648 and due to its aromatic character an inter-domain pi-H interaction with S256.
    Figure Legend Snippet: Molecular modeling of Cav1.3 WT α 1 -subunits, mutations S652L and S652W. Top: Top view and side view of the Cav1.3 α 1 -subunit structure. The region involving the inter-domain interactions (IIS4-S5–IS4-IS5) affected by the mutation is highlighted (left). Bottom: a WT inter-domain interaction of S652 in repeat II and S256 in the S4-S5 linker in repeat I. b Weaker hydrophobic interactions of the mutated residue L652 with the hydrophobic cloud in the S4-S5 linker of repeat I. c Stabilizing effect of the W652 mutation; the tryptophan residue can form an intra-domain hydrogen bond with the backbone of K648 and due to its aromatic character an inter-domain pi-H interaction with S256.

    Techniques Used: Mutagenesis

    Mutation S652L induces severe gating changes. a , b Current-voltage relationship ( I Ca ; mean ± SEM) of WT and mutant C-terminal long (WT L , S652L L , A) and short (WT S , S652L S , B) Cav1.3 splice variants recorded in parallel on the same day using 50-ms depolarizing square pulses to various test potentials from a holding potential (HP) of -89 mV. Inset: Representative I Ca traces upon depolarization to the potential of maximal inward current ( V max ). Statistics: two-way ANOVA followed by Bonferroni post hoc test, * p
    Figure Legend Snippet: Mutation S652L induces severe gating changes. a , b Current-voltage relationship ( I Ca ; mean ± SEM) of WT and mutant C-terminal long (WT L , S652L L , A) and short (WT S , S652L S , B) Cav1.3 splice variants recorded in parallel on the same day using 50-ms depolarizing square pulses to various test potentials from a holding potential (HP) of -89 mV. Inset: Representative I Ca traces upon depolarization to the potential of maximal inward current ( V max ). Statistics: two-way ANOVA followed by Bonferroni post hoc test, * p

    Techniques Used: Mutagenesis, Mass Spectrometry

    6) Product Images from "RBP2 stabilizes slow Cav1.3 Ca2+ channel inactivation properties of cochlear inner hair cells"

    Article Title: RBP2 stabilizes slow Cav1.3 Ca2+ channel inactivation properties of cochlear inner hair cells

    Journal: Pflugers Archiv

    doi: 10.1007/s00424-019-02338-4

    Modulation of Cav1.3 L /α2δ1/β2a Ba 2+ currents (15 mM) by co-expression of RIM2α and/or RBP2. Color code: control (black), plus RIM2α (blue), plus RBP2 (green), and plus RIM2α/RBP2 (red). Experimental conditions and statistical analysis are as described in Fig. 8
    Figure Legend Snippet: Modulation of Cav1.3 L /α2δ1/β2a Ba 2+ currents (15 mM) by co-expression of RIM2α and/or RBP2. Color code: control (black), plus RIM2α (blue), plus RBP2 (green), and plus RIM2α/RBP2 (red). Experimental conditions and statistical analysis are as described in Fig. 8

    Techniques Used: Expressing

    Modulation of Cav1.3 42A /α2δ1/β3 Ba 2+ currents (15 mM) by co-expression of RIM2α and/or RBP2. Color code: control (black), plus RIM2α (blue), plus RBP2 (green), and plus RIM2α/RBP2 (red). Experimental conditions and statistical analysis are as described in Fig. 8
    Figure Legend Snippet: Modulation of Cav1.3 42A /α2δ1/β3 Ba 2+ currents (15 mM) by co-expression of RIM2α and/or RBP2. Color code: control (black), plus RIM2α (blue), plus RBP2 (green), and plus RIM2α/RBP2 (red). Experimental conditions and statistical analysis are as described in Fig. 8

    Techniques Used: Expressing

    Modulation of Cav1.3 L /α2δ1/β3 Ba 2+ currents (15 mM) by co-expression of RIM2α and/or RBP2. a Schematic illustration of measured LTCC complexes, from left to right: control (Cav1.3 L /α2δ1/β3); plus RIM2α; plus RBP2; plus RIM2α/RBP2. Data in panels b and c are shown for each recording condition. Color code: control (black), plus RIM2α (blue), plus RBP2 (green), and plus RIM2α/RBP2 (red). b I Ba inactivation time course during a 5-s long depolarization to the V max ( y -axis labels as in the left panel). Traces were normalized to the I Ba peak and are shown as mean ± SEM for the indicated number of recordings. c Voltage dependence of I Ba steady-state activation and inactivation ( y -axis label as in the left panel). For parameters and statistics, see panel d and Table 1 . d Statistics of two activation parameters ( V 0.5,act and activation threshold) are shown. e Bar graphs showing the remaining I Ba after 250, 500, 1000, or 5000 ms. Data shown as mean ± SEM. Statistical significance was determined using one-way ANOVA with Bonferroni’s multiple comparison post hoc test as indicated in the graph: versus control (Cav1.3 without RIM2α and/or RBP2): *** p
    Figure Legend Snippet: Modulation of Cav1.3 L /α2δ1/β3 Ba 2+ currents (15 mM) by co-expression of RIM2α and/or RBP2. a Schematic illustration of measured LTCC complexes, from left to right: control (Cav1.3 L /α2δ1/β3); plus RIM2α; plus RBP2; plus RIM2α/RBP2. Data in panels b and c are shown for each recording condition. Color code: control (black), plus RIM2α (blue), plus RBP2 (green), and plus RIM2α/RBP2 (red). b I Ba inactivation time course during a 5-s long depolarization to the V max ( y -axis labels as in the left panel). Traces were normalized to the I Ba peak and are shown as mean ± SEM for the indicated number of recordings. c Voltage dependence of I Ba steady-state activation and inactivation ( y -axis label as in the left panel). For parameters and statistics, see panel d and Table 1 . d Statistics of two activation parameters ( V 0.5,act and activation threshold) are shown. e Bar graphs showing the remaining I Ba after 250, 500, 1000, or 5000 ms. Data shown as mean ± SEM. Statistical significance was determined using one-way ANOVA with Bonferroni’s multiple comparison post hoc test as indicated in the graph: versus control (Cav1.3 without RIM2α and/or RBP2): *** p

    Techniques Used: Expressing, Activation Assay

    Comparison of RIM2α/RBP2-stabilized Cav1.3 L I Ba inactivation (β3 and β2a; tsA-201 cells) with I Ba VDI measured in IHCs. Mean I Ba (15 mM) traces of Cav1.3 L /α2δ1 with β3 (black), β3/RIM2α/RBP2 (dark red), or β2a/RIM2α/RBP2 (red) during the first 2 s of a depolarization to V max . For comparison, we recorded I Ba in mature mouse IHCs measured as recently described [ 53 ] in mature mouse IHCs (mean I Ba trace from 5 individual recordings; gray; 10 mM Ba 2+ ). Circles indicate the remaining Ba 2+ current at the indicated time points recorded from IHCs taken from previously published papers: [ 33 ] (dark blue; 10 mM Ba 2+ , mouse P20); [ 42 ] (turquoise; 5 mM Ba 2+ , mouse P40–70); [ 9 ] (purple; 5 mM Ba 2+ , mouse 2–4 weeks); [ 24 ] (yellow; 5 mM Ba 2+ , gerbil P50); [ 34 ] (green; 20 mM Ba 2+ , chicken 1–21 days). Turquoise and purple circles are overlapping and are therefore shown together as half-filled circle
    Figure Legend Snippet: Comparison of RIM2α/RBP2-stabilized Cav1.3 L I Ba inactivation (β3 and β2a; tsA-201 cells) with I Ba VDI measured in IHCs. Mean I Ba (15 mM) traces of Cav1.3 L /α2δ1 with β3 (black), β3/RIM2α/RBP2 (dark red), or β2a/RIM2α/RBP2 (red) during the first 2 s of a depolarization to V max . For comparison, we recorded I Ba in mature mouse IHCs measured as recently described [ 53 ] in mature mouse IHCs (mean I Ba trace from 5 individual recordings; gray; 10 mM Ba 2+ ). Circles indicate the remaining Ba 2+ current at the indicated time points recorded from IHCs taken from previously published papers: [ 33 ] (dark blue; 10 mM Ba 2+ , mouse P20); [ 42 ] (turquoise; 5 mM Ba 2+ , mouse P40–70); [ 9 ] (purple; 5 mM Ba 2+ , mouse 2–4 weeks); [ 24 ] (yellow; 5 mM Ba 2+ , gerbil P50); [ 34 ] (green; 20 mM Ba 2+ , chicken 1–21 days). Turquoise and purple circles are overlapping and are therefore shown together as half-filled circle

    Techniques Used:

    Modulation of Cav1.3 L /α2δ1/β2e Ba 2+ currents (15 mM) by co-expression of RIM2α and/or RBP2. Color code: control (black), plus RIM2α (blue), plus RBP2 (green), and plus RIM2α/RBP2 (red). Experimental conditions and statistical analysis are as described in Fig. 8
    Figure Legend Snippet: Modulation of Cav1.3 L /α2δ1/β2e Ba 2+ currents (15 mM) by co-expression of RIM2α and/or RBP2. Color code: control (black), plus RIM2α (blue), plus RBP2 (green), and plus RIM2α/RBP2 (red). Experimental conditions and statistical analysis are as described in Fig. 8

    Techniques Used: Expressing

    Modulation of VDI by β3 and different β2 subunit splic e variants (15 mM Ba 2+ ). a , b Left panels: mean (± SEM) I Ba traces for Cav1.3 L /α2δ1 ( a ) or Cav1.3 42A /α2δ1 ( b ) co-expressed with either β3 (black/gray), β2a (red), or β2e (purple). The number of individual recordings is indicated in parentheses. VDI was quantified using 15 mM Ba 2+ as charge carrier and calculated as residual I Ba at the indicated predefined time points (bar graphs). Statistical significance was determined using one-way ANOVA with Bonferroni post hoc test ( a ) or unpaired Student’s t test ( b ): *** p
    Figure Legend Snippet: Modulation of VDI by β3 and different β2 subunit splic e variants (15 mM Ba 2+ ). a , b Left panels: mean (± SEM) I Ba traces for Cav1.3 L /α2δ1 ( a ) or Cav1.3 42A /α2δ1 ( b ) co-expressed with either β3 (black/gray), β2a (red), or β2e (purple). The number of individual recordings is indicated in parentheses. VDI was quantified using 15 mM Ba 2+ as charge carrier and calculated as residual I Ba at the indicated predefined time points (bar graphs). Statistical significance was determined using one-way ANOVA with Bonferroni post hoc test ( a ) or unpaired Student’s t test ( b ): *** p

    Techniques Used:

    Interaction of RBP2 with Cav1.3 channels. RIMs and RBPs are multidomain proteins [ 41 , 46 ]. All RIM isoforms (RIM1α and 1β; RIM2α, 2β, and 2γ; RIM3γ and RIM4γ) bind via their C 2 B domain to the auxiliary β subunit of the Ca 2+ channel complex. Disruption of the SH3 or GK domain in the β subunit prevents the interaction with RIM [ 28 ]. All three RBP isoforms contain three SH3 domains and two (RBP3) or three (RBP1 and 2) FN3 domains [ 41 ]. The second SH3 domain of RBP binds to the proline-rich region (PXXP) present only in RIMα or β isoforms, located between the two C 2 domains. The other SH3 domains, marked by “x,” in turn can interact with a proline-rich region (PXXP) localized in the full-length Cav1.3 C terminus [ 23 ]. Note that incorporation of alternative exons 42A and 43S leads to short C-terminal splice variants (Cav1.3 42A or Cav1.3 43S , respectively; C-terminal ends indicated by orange dots) lacking the PXXP interaction site. AID, α-interaction domain; FN3, fibronectin 3 domain; GK, guanylate-kinase like domain; PXXP, proline-rich region; SH3, SRC homology 3 domain; Zn 2+ , zinc finger domain. Note that RIM may also interact via its C 2 B domain with the C terminus of Cav1.3, but the interaction site is unknown [ 49 ]
    Figure Legend Snippet: Interaction of RBP2 with Cav1.3 channels. RIMs and RBPs are multidomain proteins [ 41 , 46 ]. All RIM isoforms (RIM1α and 1β; RIM2α, 2β, and 2γ; RIM3γ and RIM4γ) bind via their C 2 B domain to the auxiliary β subunit of the Ca 2+ channel complex. Disruption of the SH3 or GK domain in the β subunit prevents the interaction with RIM [ 28 ]. All three RBP isoforms contain three SH3 domains and two (RBP3) or three (RBP1 and 2) FN3 domains [ 41 ]. The second SH3 domain of RBP binds to the proline-rich region (PXXP) present only in RIMα or β isoforms, located between the two C 2 domains. The other SH3 domains, marked by “x,” in turn can interact with a proline-rich region (PXXP) localized in the full-length Cav1.3 C terminus [ 23 ]. Note that incorporation of alternative exons 42A and 43S leads to short C-terminal splice variants (Cav1.3 42A or Cav1.3 43S , respectively; C-terminal ends indicated by orange dots) lacking the PXXP interaction site. AID, α-interaction domain; FN3, fibronectin 3 domain; GK, guanylate-kinase like domain; PXXP, proline-rich region; SH3, SRC homology 3 domain; Zn 2+ , zinc finger domain. Note that RIM may also interact via its C 2 B domain with the C terminus of Cav1.3, but the interaction site is unknown [ 49 ]

    Techniques Used:

    RIM, RBP, and Cav1.3 α1 subunit expression in IHCs. Control experiments in IHC preparations revealed the expected transcripts of long (containing exon 43) and short C-terminal splice variants (containing exon 43S) of Cav1.3 α1 subunits (top left). RIM2α was reliably detected in IHCs (4 out of 4 independent preparations) before (P6) and after hearing onset (after P12) (top right). RBP1 was the only isoform, which could not be detected in IHCs at any tested developmental stage (cDNA preparations from 5 different mice at different postnatal days, not shown). RBP2 transcripts (bottom left) were found only in 1 out of 5 different samples before hearing onset but were consistently detected in mature IHCs (8 out of 9 separate preparations). RBP3 transcripts (bottom right) were identified before as well as after hearing onset (6 out of 6 and 8 out of 10 independent samples, respectively). Brain samples from adult mice and reactions without template (“ctrl”) were used as positive and negative controls, respectively. Representative PCRs from > 3 independent experiments are shown
    Figure Legend Snippet: RIM, RBP, and Cav1.3 α1 subunit expression in IHCs. Control experiments in IHC preparations revealed the expected transcripts of long (containing exon 43) and short C-terminal splice variants (containing exon 43S) of Cav1.3 α1 subunits (top left). RIM2α was reliably detected in IHCs (4 out of 4 independent preparations) before (P6) and after hearing onset (after P12) (top right). RBP1 was the only isoform, which could not be detected in IHCs at any tested developmental stage (cDNA preparations from 5 different mice at different postnatal days, not shown). RBP2 transcripts (bottom left) were found only in 1 out of 5 different samples before hearing onset but were consistently detected in mature IHCs (8 out of 9 separate preparations). RBP3 transcripts (bottom right) were identified before as well as after hearing onset (6 out of 6 and 8 out of 10 independent samples, respectively). Brain samples from adult mice and reactions without template (“ctrl”) were used as positive and negative controls, respectively. Representative PCRs from > 3 independent experiments are shown

    Techniques Used: Expressing, Immunohistochemistry, Mouse Assay

    Modulation of Cav1.3 42A /α2δ1/β2a Ba 2+ currents (15 mM) by co-expression of RIM2α and/or RBP2. Color code: control (black), plus RIM2α (blue), plus RBP2 (green), and plus RIM2α/RBP2 (red). Experimental conditions and statistical analysis are as described in Fig. 8
    Figure Legend Snippet: Modulation of Cav1.3 42A /α2δ1/β2a Ba 2+ currents (15 mM) by co-expression of RIM2α and/or RBP2. Color code: control (black), plus RIM2α (blue), plus RBP2 (green), and plus RIM2α/RBP2 (red). Experimental conditions and statistical analysis are as described in Fig. 8

    Techniques Used: Expressing

    RBP2 interaction with Cav1.3 C-terminal splice variants. a Schematic representation of the Cav1.3 C-terminal GST-fusion proteins: GST-Cav1.3 42 C-term (GST-42), GST-Cav1.3 42A C-term (GST-42A), and GST-Cav1.3 43S C-term (GST-43S) including the binding position for the anti-Cav1.3α1 2022–2138 antibody (anti-42) in the full-length C terminus. Numbers indicate the amino acid position in the Cav1.3 protein (GenBank™ accession number NM_000720). b GST pull-down of whole-cell extracts prepared from HEK293 cells transfected with HA-RBP2 with the indicated Cav1.3 C termini coupled to GST; 1 of 4 similar experiments is illustrated. Bound HA-RBP2 was visualized by western blotting using anti-HA. Anti-GAPDH staining served as a negative control. Input—0.5, 0.25, and 0.1% of the lysate. GST, GST-RIIβ, and GST-max p14 were control peptides not binding to HA-RBP2. Migration of molecular mass markers is indicated. c Left: Ponceau staining of GST-fusion proteins. Arrows indicate the migration of the full-length construct. Despite the partial degradation of GST-fusion proteins GST-42 and GST-42A, we observed selective protein–protein interactions between GST-42 and RBP2. Right: Immunoblot from panel b was stripped and the presence of GST-Cav1.3 42 C-term was verified by immunoblotting using anti-Cav1.3α1 2022–2138 antibody directed against an epitope present only in the long C-terminal splice variant as illustrated in panel a . d Confirmation of HA-RBP2 interaction with the long Cav1.3 C terminus by co-immunoprecipitation of HA-rRBP2 expressed in tsA-201 cells with YFP-tagged long Cav1.3 C terminus (YFP-Cav1.3 42 C-term; YFP-42). Top: Verification of the presence of YFP-Cav1.3 42 C-term by immunoblotting using an YFP antibody. Bottom: Specific immunoprecipitation of RBP2 by Cav1.3 42 C-term (detection by anti-RBP2-1318). Input control—1 and 0.5% of the lysate. Mock: untransfected control
    Figure Legend Snippet: RBP2 interaction with Cav1.3 C-terminal splice variants. a Schematic representation of the Cav1.3 C-terminal GST-fusion proteins: GST-Cav1.3 42 C-term (GST-42), GST-Cav1.3 42A C-term (GST-42A), and GST-Cav1.3 43S C-term (GST-43S) including the binding position for the anti-Cav1.3α1 2022–2138 antibody (anti-42) in the full-length C terminus. Numbers indicate the amino acid position in the Cav1.3 protein (GenBank™ accession number NM_000720). b GST pull-down of whole-cell extracts prepared from HEK293 cells transfected with HA-RBP2 with the indicated Cav1.3 C termini coupled to GST; 1 of 4 similar experiments is illustrated. Bound HA-RBP2 was visualized by western blotting using anti-HA. Anti-GAPDH staining served as a negative control. Input—0.5, 0.25, and 0.1% of the lysate. GST, GST-RIIβ, and GST-max p14 were control peptides not binding to HA-RBP2. Migration of molecular mass markers is indicated. c Left: Ponceau staining of GST-fusion proteins. Arrows indicate the migration of the full-length construct. Despite the partial degradation of GST-fusion proteins GST-42 and GST-42A, we observed selective protein–protein interactions between GST-42 and RBP2. Right: Immunoblot from panel b was stripped and the presence of GST-Cav1.3 42 C-term was verified by immunoblotting using anti-Cav1.3α1 2022–2138 antibody directed against an epitope present only in the long C-terminal splice variant as illustrated in panel a . d Confirmation of HA-RBP2 interaction with the long Cav1.3 C terminus by co-immunoprecipitation of HA-rRBP2 expressed in tsA-201 cells with YFP-tagged long Cav1.3 C terminus (YFP-Cav1.3 42 C-term; YFP-42). Top: Verification of the presence of YFP-Cav1.3 42 C-term by immunoblotting using an YFP antibody. Bottom: Specific immunoprecipitation of RBP2 by Cav1.3 42 C-term (detection by anti-RBP2-1318). Input control—1 and 0.5% of the lysate. Mock: untransfected control

    Techniques Used: Binding Assay, Transfection, Western Blot, Staining, Negative Control, Migration, Construct, Variant Assay, Immunoprecipitation

    RBP2 co-localization with Cav1.3 at ribbon synapses in mouse IHCs. a – h Maximum intensity projection (MIP) of confocal stacks of whole-mount organs of Corti with stretches of 7–8 IHCs. a – d IHCs from the apical cochlear turn of a 4-week-old NMRI mouse co-immunolabeled for Cav1.3 and RBP2 demonstrate that almost every Cav1.3 cluster co-localized with RBP2 at the basolateral pole of the IHCs ( a ), which is shown in more detail in the enlargements of the box in a ( b – d ). e – h IHCs from the apical cochlear turn of a 4-week-old NMRI mouse co-immunolabeled for the ribbon synapse marker CtBP2 and RBP2 show that almost every ribbon co-localized with RBP2 at the basolateral pole ( e ), which is shown in more detail in the enlargements of the box in e ( f – h ). Nuclei stained in blue with DAPI are shown only in the merged images. The dotted lines in a and e outline the basolateral pole of one IHC in each specimen. Scale bars: a , e , 10 μm; d , h , 5 μm
    Figure Legend Snippet: RBP2 co-localization with Cav1.3 at ribbon synapses in mouse IHCs. a – h Maximum intensity projection (MIP) of confocal stacks of whole-mount organs of Corti with stretches of 7–8 IHCs. a – d IHCs from the apical cochlear turn of a 4-week-old NMRI mouse co-immunolabeled for Cav1.3 and RBP2 demonstrate that almost every Cav1.3 cluster co-localized with RBP2 at the basolateral pole of the IHCs ( a ), which is shown in more detail in the enlargements of the box in a ( b – d ). e – h IHCs from the apical cochlear turn of a 4-week-old NMRI mouse co-immunolabeled for the ribbon synapse marker CtBP2 and RBP2 show that almost every ribbon co-localized with RBP2 at the basolateral pole ( e ), which is shown in more detail in the enlargements of the box in e ( f – h ). Nuclei stained in blue with DAPI are shown only in the merged images. The dotted lines in a and e outline the basolateral pole of one IHC in each specimen. Scale bars: a , e , 10 μm; d , h , 5 μm

    Techniques Used: Immunolabeling, Marker, Staining, Immunohistochemistry

    7) Product Images from "Efficient stimulus-secretion coupling at ribbon synapses requires RIM-binding protein tethering of L-type Ca2+ channels"

    Article Title: Efficient stimulus-secretion coupling at ribbon synapses requires RIM-binding protein tethering of L-type Ca2+ channels

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.1702991114

    Deletion of RBPs reduces Ca 2+ currents in presynaptic rod bipolar cells forming ribbon synapses on postsynaptic AII amacrine cells. ( A ) Selective loss of presynaptic L-type Ca 2+ channels from ribbon synapses formed by rod bipolar cells. Representative confocal images of RBP WT ( Left ) and RBP DKO ( Right ) retina sections stained for PKCα (green, to identify rod bipolar neurons), VGlut1 (purple, to identify presynaptic terminals), and the L-type Ca 2+ -channel CaV1.3 (red). ( B ) Summary graphs of the intensity of PKCα fluorescent signals in WT(gray) and DKO (blue) rod bipolar cell terminals ( Left ), and terminal size (area, Right ) defined by PKC staining. Number of experiments (images/mice): RBP WT, 79/3; RBP DKO, 70/3. ( C ) Relative vGluT1 ( Left ) and CaV1.3 ( Right ) staining intensity normalized by PKCα signals. Number of experiments (images/mice): RBP WT, 79/3; RBP DKO, 70/3. ( D ) Experimental configuration for Ca 2+ -current recordings ( Left ) and representative experiment ( Right Top ) schematic of depolarization protocol; ( Right Bottom ) Sample traces for an RBP control (black) and an RBP DKO (blue) bipolar cell. ( E ) Deletion of RBP1,2 severely impairs presynaptic Ca 2+ -current density. Summary plot of the Ca 2+ -current charge transfer over 50 ms as a function of the membrane voltage in RBP1,2 DKO (blue) rod bipolar cells and corresponding littermate controls (gray). ( F and G ) Incremental contribution of RBP1 ( F ) and RBP2 ( G ) to the presynaptic Ca 2+ -channel density of ribbon synapses. Same as E , but for littermate control ( F and G , gray) and RBP1 KO ( F , orange) or RBP2 KO ( G , green) mice. ( H ) Summary graphs of the Ca 2+ -current charge transfer induced by a 50-ms depolarization to −20 mV in RBP1,2 DKO, RBP1 KO, or RBP2 KO mice, normalized to the controls analyzed in the same experiments. Number of experiments as in E – G . ( I ) Summary graphs of whole-cell capacitance ( Left ) and input resistance ( Right ) in the same rod bipolar cells used to measure whole-cell presynaptic Ca 2+ currents. Number of experiments as in E . All summary graphs are mean ± SD. Statistical analyses were performed by either Student's t test ( B , C , H , and I ) or by ANOVA followed by a Bonferroni post hoc test ( E – G ), comparing RBP DKO with RBP WT (* P
    Figure Legend Snippet: Deletion of RBPs reduces Ca 2+ currents in presynaptic rod bipolar cells forming ribbon synapses on postsynaptic AII amacrine cells. ( A ) Selective loss of presynaptic L-type Ca 2+ channels from ribbon synapses formed by rod bipolar cells. Representative confocal images of RBP WT ( Left ) and RBP DKO ( Right ) retina sections stained for PKCα (green, to identify rod bipolar neurons), VGlut1 (purple, to identify presynaptic terminals), and the L-type Ca 2+ -channel CaV1.3 (red). ( B ) Summary graphs of the intensity of PKCα fluorescent signals in WT(gray) and DKO (blue) rod bipolar cell terminals ( Left ), and terminal size (area, Right ) defined by PKC staining. Number of experiments (images/mice): RBP WT, 79/3; RBP DKO, 70/3. ( C ) Relative vGluT1 ( Left ) and CaV1.3 ( Right ) staining intensity normalized by PKCα signals. Number of experiments (images/mice): RBP WT, 79/3; RBP DKO, 70/3. ( D ) Experimental configuration for Ca 2+ -current recordings ( Left ) and representative experiment ( Right Top ) schematic of depolarization protocol; ( Right Bottom ) Sample traces for an RBP control (black) and an RBP DKO (blue) bipolar cell. ( E ) Deletion of RBP1,2 severely impairs presynaptic Ca 2+ -current density. Summary plot of the Ca 2+ -current charge transfer over 50 ms as a function of the membrane voltage in RBP1,2 DKO (blue) rod bipolar cells and corresponding littermate controls (gray). ( F and G ) Incremental contribution of RBP1 ( F ) and RBP2 ( G ) to the presynaptic Ca 2+ -channel density of ribbon synapses. Same as E , but for littermate control ( F and G , gray) and RBP1 KO ( F , orange) or RBP2 KO ( G , green) mice. ( H ) Summary graphs of the Ca 2+ -current charge transfer induced by a 50-ms depolarization to −20 mV in RBP1,2 DKO, RBP1 KO, or RBP2 KO mice, normalized to the controls analyzed in the same experiments. Number of experiments as in E – G . ( I ) Summary graphs of whole-cell capacitance ( Left ) and input resistance ( Right ) in the same rod bipolar cells used to measure whole-cell presynaptic Ca 2+ currents. Number of experiments as in E . All summary graphs are mean ± SD. Statistical analyses were performed by either Student's t test ( B , C , H , and I ) or by ANOVA followed by a Bonferroni post hoc test ( E – G ), comparing RBP DKO with RBP WT (* P

    Techniques Used: Staining, Mouse Assay, Mass Spectrometry

    8) Product Images from "Cell-type-specific tuning of Cav1.3 Ca2+-channels by a C-terminal automodulatory domain"

    Article Title: Cell-type-specific tuning of Cav1.3 Ca2+-channels by a C-terminal automodulatory domain

    Journal: Frontiers in Cellular Neuroscience

    doi: 10.3389/fncel.2015.00309

    Western blot analysis of α1-subunits in WT and mutant mouse brain homogenates. (A) Proteins (100 μg/lane) were separated on 5% SDS page and immunostained with polyclonal antibody Cav1.3 α1 CT . The Cav1.3 α1 subunit was specifically detected as a 231 kDa protein at expression levels indistinguishable between WT and homozygous mutants (HA) (see Results). KO, Cav1.3 -/- negative control. (B) Same separation as in A (100 μg/lane) but detection with anti-Cav1.3α1 NT . The migration of recombinant mCav1.3 L (L) and mCav1.343 S (43S) on the same gel (not shown) and their calculated molecular mass are indicated by arrows (left). Migration of molecular mass standards as well as the brain long and short α1-subunit species are also indicated (right). An unspecific ∼120 kDa band served as loading control. One representative experiment of at least three independent experiments is shown for all panels. KO, Cav1.3 -/- ; HA, Cav1.3DCRD HA/HA , WT, wild-type littermate.
    Figure Legend Snippet: Western blot analysis of α1-subunits in WT and mutant mouse brain homogenates. (A) Proteins (100 μg/lane) were separated on 5% SDS page and immunostained with polyclonal antibody Cav1.3 α1 CT . The Cav1.3 α1 subunit was specifically detected as a 231 kDa protein at expression levels indistinguishable between WT and homozygous mutants (HA) (see Results). KO, Cav1.3 -/- negative control. (B) Same separation as in A (100 μg/lane) but detection with anti-Cav1.3α1 NT . The migration of recombinant mCav1.3 L (L) and mCav1.343 S (43S) on the same gel (not shown) and their calculated molecular mass are indicated by arrows (left). Migration of molecular mass standards as well as the brain long and short α1-subunit species are also indicated (right). An unspecific ∼120 kDa band served as loading control. One representative experiment of at least three independent experiments is shown for all panels. KO, Cav1.3 -/- ; HA, Cav1.3DCRD HA/HA , WT, wild-type littermate.

    Techniques Used: Western Blot, Mutagenesis, SDS Page, Expressing, Negative Control, Migration, Recombinant

    Cav1.3 α1 transcripts containing exons 43 S and 43 L in mouse IHCs and OHCs, at P6 and P22 using nested PCR. Fragments containing 43S (403 bp) or 43L (557 bp) were amplified using nested PCR (see Materials and Methods) with primers specific for exon 42 (forward) and 45 (reverse) of mouse Cav1.3. S1–S14 represent samples from independent preparations. For each cell type and developmental stage at least three independent experiments were performed. Whole brain (WB) and heart (WH) served as positive controls, H 2 O (no template) as negative control. Specificity of PCR products was confirmed by sequencing. When two independent PCR reactions with three different RNA samples of each cell type were performed, the number of successful detections for each transcript was as follows: detection of 43L: 6 (out of six experiments) in IHC and OHC preparations of all developmental stages; detection of 43S: 4 (6) in IHC P06 and IHC P22, 6 (6) in OHC P06 and 5 (6) in OHC P22. Bp, basepair markers.
    Figure Legend Snippet: Cav1.3 α1 transcripts containing exons 43 S and 43 L in mouse IHCs and OHCs, at P6 and P22 using nested PCR. Fragments containing 43S (403 bp) or 43L (557 bp) were amplified using nested PCR (see Materials and Methods) with primers specific for exon 42 (forward) and 45 (reverse) of mouse Cav1.3. S1–S14 represent samples from independent preparations. For each cell type and developmental stage at least three independent experiments were performed. Whole brain (WB) and heart (WH) served as positive controls, H 2 O (no template) as negative control. Specificity of PCR products was confirmed by sequencing. When two independent PCR reactions with three different RNA samples of each cell type were performed, the number of successful detections for each transcript was as follows: detection of 43L: 6 (out of six experiments) in IHC and OHC preparations of all developmental stages; detection of 43S: 4 (6) in IHC P06 and IHC P22, 6 (6) in OHC P06 and 5 (6) in OHC P22. Bp, basepair markers.

    Techniques Used: Nested PCR, Amplification, Western Blot, Negative Control, Polymerase Chain Reaction, Sequencing, Immunohistochemistry

    Absence of smaller C-terminally–derived Cav1.3 α1 fragments in WT and Cav1.3DCRD HA/HA brain preparations. (A) Mouse brain homogenate (100 μg of protein/lane) prepared from WT or Cav1.3DCRD HA/HA (HA) mice were separated on 4–15% gradient SDS-PAGE and immunostained with anti-HA antibody. The blot was overexposed to also visualize less abundant smaller fragments. in separate experiments α1- associated HA-immunoreactivity could be detected with only 10% (10 μg/lane) of the protein amount used ( n = 3) demonstrating the sensitivity of the assay. (B) Mouse brain membranes (100 μg of protein/lane) were analyzed as in (A). (C) Mouse brain membranes (100 μg of protein/lane) from WT or Cav1.3 -/- (KO) mice were blotted as in (B) and stained with anti-Cav1.3α1 CT antibodies. To some WT samples (33 μg/lane) a 45 kDa recombinant C-terminal control peptide was added (arrow, amounts indicated) before separation to demonstrate successful transfer and sensitive detection as a positive control for sensitivity.
    Figure Legend Snippet: Absence of smaller C-terminally–derived Cav1.3 α1 fragments in WT and Cav1.3DCRD HA/HA brain preparations. (A) Mouse brain homogenate (100 μg of protein/lane) prepared from WT or Cav1.3DCRD HA/HA (HA) mice were separated on 4–15% gradient SDS-PAGE and immunostained with anti-HA antibody. The blot was overexposed to also visualize less abundant smaller fragments. in separate experiments α1- associated HA-immunoreactivity could be detected with only 10% (10 μg/lane) of the protein amount used ( n = 3) demonstrating the sensitivity of the assay. (B) Mouse brain membranes (100 μg of protein/lane) were analyzed as in (A). (C) Mouse brain membranes (100 μg of protein/lane) from WT or Cav1.3 -/- (KO) mice were blotted as in (B) and stained with anti-Cav1.3α1 CT antibodies. To some WT samples (33 μg/lane) a 45 kDa recombinant C-terminal control peptide was added (arrow, amounts indicated) before separation to demonstrate successful transfer and sensitive detection as a positive control for sensitivity.

    Techniques Used: Derivative Assay, Mouse Assay, SDS Page, Staining, Recombinant, Positive Control

    Comparison of the protein localization of anti-HA-labeled Cav1.3 with immunolabeled Ca v 1.3, Ca v β2 and CtBP2/RIBEYE in IHCs. (A–D) Whole-mount preparations of apical turns of the organ of Corti from adult Ca v 1.3DCRD HA/HA (A,C,D) and WT (B) mice were co-immunolabeled with anti-HA and anti-Ca v 1.3 ( A, B , 11 weeks), anti-HA and anti-Ca v β2 ( C , P28), or anti-HA and anti-CtBP2/RIBEYE antibodies ( D , P37). Every image shows the basolateral poles of two adjacent IHCs the nuclei of which are indicated by asterisks in the rightmost column, respectively. HA staining (A1,C1,D1) largely overlapped with Ca v 1.3 (A2) , Ca v ß2 (C2) and CtBP2 (D2) staining at the basal poles of IHCs as evident upon merging corresponding images (A3,C3,D3) . In the WT, no specific HA-labeling (B1) was present at the position of Ca v 1.3 labeling (B2,B3) . The weak ‘cloudy’ green anti-HA staining was present in all specimen investigated and therefore considered unspecific. Cell nuclei of IHCs were counterstained with DAPI (blue). 1 of 3 ( A , age: 2–3 months), 1 of 4 ( B , age: P25 – 3 month), 1 of 5 ( C , P25–P31) and 1 of 5 ( D , P28–P37) independent experiments is illustrated, respectively. Scale bars: 5 μm.
    Figure Legend Snippet: Comparison of the protein localization of anti-HA-labeled Cav1.3 with immunolabeled Ca v 1.3, Ca v β2 and CtBP2/RIBEYE in IHCs. (A–D) Whole-mount preparations of apical turns of the organ of Corti from adult Ca v 1.3DCRD HA/HA (A,C,D) and WT (B) mice were co-immunolabeled with anti-HA and anti-Ca v 1.3 ( A, B , 11 weeks), anti-HA and anti-Ca v β2 ( C , P28), or anti-HA and anti-CtBP2/RIBEYE antibodies ( D , P37). Every image shows the basolateral poles of two adjacent IHCs the nuclei of which are indicated by asterisks in the rightmost column, respectively. HA staining (A1,C1,D1) largely overlapped with Ca v 1.3 (A2) , Ca v ß2 (C2) and CtBP2 (D2) staining at the basal poles of IHCs as evident upon merging corresponding images (A3,C3,D3) . In the WT, no specific HA-labeling (B1) was present at the position of Ca v 1.3 labeling (B2,B3) . The weak ‘cloudy’ green anti-HA staining was present in all specimen investigated and therefore considered unspecific. Cell nuclei of IHCs were counterstained with DAPI (blue). 1 of 3 ( A , age: 2–3 months), 1 of 4 ( B , age: P25 – 3 month), 1 of 5 ( C , P25–P31) and 1 of 5 ( D , P28–P37) independent experiments is illustrated, respectively. Scale bars: 5 μm.

    Techniques Used: Labeling, Immunolabeling, Mouse Assay, Staining

    Activation and inactivation properties of I Ca through mCav1.3 L and mCav1.3 L -HA channels. (A) α1-subunits were heterologously expressed in tsA-201 cells together with β 3 and α 2 δ 1 (at least three independent transfections). Whole-cell patch-clamp current–voltage relationship obtained by depolarizations from a Vh of -80 mV to the indicated test potentials in cells transfected with mouse wild-type (WT) Cav1.3 (mCav1.3 L , black) and mCav1.3 L -HA (blue). All data were junction potential – corrected. (B) Percent I Ca inactivation (15 mM Ca 2+ ) during a test pulse from -80 mV to the V max . ∗∗∗ p
    Figure Legend Snippet: Activation and inactivation properties of I Ca through mCav1.3 L and mCav1.3 L -HA channels. (A) α1-subunits were heterologously expressed in tsA-201 cells together with β 3 and α 2 δ 1 (at least three independent transfections). Whole-cell patch-clamp current–voltage relationship obtained by depolarizations from a Vh of -80 mV to the indicated test potentials in cells transfected with mouse wild-type (WT) Cav1.3 (mCav1.3 L , black) and mCav1.3 L -HA (blue). All data were junction potential – corrected. (B) Percent I Ca inactivation (15 mM Ca 2+ ) during a test pulse from -80 mV to the V max . ∗∗∗ p

    Techniques Used: Activation Assay, Transfection, Patch Clamp

    9) Product Images from "A synaptic F-actin network controls otoferlin-dependent exocytosis in auditory inner hair cells"

    Article Title: A synaptic F-actin network controls otoferlin-dependent exocytosis in auditory inner hair cells

    Journal: eLife

    doi: 10.7554/eLife.10988

    Confocal imaging of the synaptic F-actin cages in IHCs. (A) Confocal images from averaged Z-stack projection (20 slices of 0.25 µm) of P13-IHCs labeled in blue with otoferlin-immuno-reactivity. Directly visualized with fluorescent-phalloidin (purple), F-actin intensively labelled the cuticular plate and the stereocilia but also in a punctated manner the synaptic basal pole of the IHCs. In this latter area, at higher magnification (averaged Z-stack projection of 8 slices of 0.25 µm), the synaptic F-actin forms a mesh of cages (see right panel where the blue channel of otoferlin is omitted; the cages are indicated by the white asterisks). At each border of the synaptic F-actin cages was generally attached one synaptic ribbon (red) and one associated Cav1.3 patch (green) as indicated in the lower left panel. The graph represents an example of fluorescent intensity profile through the white dashed line crossing the ribbon and the associated Cav1.3. (B) The graph indicates the Gaussian distribution of the larger axis (double white arrow head) of each F-actin cage. (C) A 45 min treatment with extracellular latrunculin-A disrupted the synaptic F-actin cages. The black holes at the base of the IHCs likely indicated swollen IHC active zones produced by the synaptic F-actin disorganization. At higher magnification (right panel), note also the disorganization of the Cav1.3 clusters (green) at the ribbons, as indicated by a larger distance in their respective fluorescent intensity profile distribution (bottom graph). (D) Comparative Gaussian distribution of the center mass distance between Cav1.3 and ribbon in controls (black, n = 71 active zones) and latrunculin-treated (orange, n = 102 active zones) IHCs. The inset histogram indicates the mean ± SEM distance in both conditions. *p
    Figure Legend Snippet: Confocal imaging of the synaptic F-actin cages in IHCs. (A) Confocal images from averaged Z-stack projection (20 slices of 0.25 µm) of P13-IHCs labeled in blue with otoferlin-immuno-reactivity. Directly visualized with fluorescent-phalloidin (purple), F-actin intensively labelled the cuticular plate and the stereocilia but also in a punctated manner the synaptic basal pole of the IHCs. In this latter area, at higher magnification (averaged Z-stack projection of 8 slices of 0.25 µm), the synaptic F-actin forms a mesh of cages (see right panel where the blue channel of otoferlin is omitted; the cages are indicated by the white asterisks). At each border of the synaptic F-actin cages was generally attached one synaptic ribbon (red) and one associated Cav1.3 patch (green) as indicated in the lower left panel. The graph represents an example of fluorescent intensity profile through the white dashed line crossing the ribbon and the associated Cav1.3. (B) The graph indicates the Gaussian distribution of the larger axis (double white arrow head) of each F-actin cage. (C) A 45 min treatment with extracellular latrunculin-A disrupted the synaptic F-actin cages. The black holes at the base of the IHCs likely indicated swollen IHC active zones produced by the synaptic F-actin disorganization. At higher magnification (right panel), note also the disorganization of the Cav1.3 clusters (green) at the ribbons, as indicated by a larger distance in their respective fluorescent intensity profile distribution (bottom graph). (D) Comparative Gaussian distribution of the center mass distance between Cav1.3 and ribbon in controls (black, n = 71 active zones) and latrunculin-treated (orange, n = 102 active zones) IHCs. The inset histogram indicates the mean ± SEM distance in both conditions. *p

    Techniques Used: Imaging, Labeling, Immunohistochemistry, Produced

    10) Product Images from "Exocytotic Machineries of Vestibular Type I and Cochlear Ribbon Synapses Display Similar Intrinsic Otoferlin-Dependent Ca2+ Sensitivity But a Different Coupling to Ca2+ Channels"

    Article Title: Exocytotic Machineries of Vestibular Type I and Cochlear Ribbon Synapses Display Similar Intrinsic Otoferlin-Dependent Ca2+ Sensitivity But a Different Coupling to Ca2+ Channels

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.0947-14.2014

    Colocalization between Cav1.3 channels and ribbons. A , B , 3D stack reconstruction of confocal images of utricle ( A , striolar region) and OC (apical region, B ). Organs (P14) were labeled for F-actin (pink), Cav1.3 (green), and CtBP2 (red ribbons). Afferent fibers were labeled with Anti-NF200 (blue). Asterisks indicate VHC-I surrounded by large calyceal nerve terminals. Scale bar, 6 μm. An example of a Cav1.3 channel cluster (green) and its ribbon (red) is shown for VHC-I ( A′ ) and IHCs ( B′ ). Scale bar, 0.25 μm. C , D , Colocalization between Cav1.3 and ribbon (CtBP2-ribeye) was assessed by plotting the fluorescence intensity profile from a line scan ( A′ , B′ , white dashed line). An example of fluorescence intensity profile is shown for VHC-I ( C ) and IHCs ( D ). E , Colocalization analysis using JACoP plugin in ImageJ. Gaussian fit of the occurrences of center mass distance measured between ribbon and Cav1.3 VHC-I (167.77 ± 4.53 nm, n = 83) and IHCs (308.34 ± 4.68 nm, n = 106) suggested a tighter organization of Cav1.3 channels and ribbons in VHC-I.
    Figure Legend Snippet: Colocalization between Cav1.3 channels and ribbons. A , B , 3D stack reconstruction of confocal images of utricle ( A , striolar region) and OC (apical region, B ). Organs (P14) were labeled for F-actin (pink), Cav1.3 (green), and CtBP2 (red ribbons). Afferent fibers were labeled with Anti-NF200 (blue). Asterisks indicate VHC-I surrounded by large calyceal nerve terminals. Scale bar, 6 μm. An example of a Cav1.3 channel cluster (green) and its ribbon (red) is shown for VHC-I ( A′ ) and IHCs ( B′ ). Scale bar, 0.25 μm. C , D , Colocalization between Cav1.3 and ribbon (CtBP2-ribeye) was assessed by plotting the fluorescence intensity profile from a line scan ( A′ , B′ , white dashed line). An example of fluorescence intensity profile is shown for VHC-I ( C ) and IHCs ( D ). E , Colocalization analysis using JACoP plugin in ImageJ. Gaussian fit of the occurrences of center mass distance measured between ribbon and Cav1.3 VHC-I (167.77 ± 4.53 nm, n = 83) and IHCs (308.34 ± 4.68 nm, n = 106) suggested a tighter organization of Cav1.3 channels and ribbons in VHC-I.

    Techniques Used: Labeling, Fluorescence

    Spatial distribution of Cav1.3 channels in VHC-I and IHCs. A , B , 3D Imaris reconstructions of confocal images from the central striolar region of an utricle and the low-frequency apical turn of an OC, respectively. Both organs were dissected from mice at P14. Hair cells were stained for F-actin (phalloidin, pink), otoferlin (anti-otoferlin, blue), ribbons (anti-CtBP2, red), and Cav1.3 (anti-α 1D, green). A′ , B′ , 3D-reconstructed images at higher magnification of some ribbons and their associated Cav1.3 cluster in vestibular ( A′ ; VHC-I) and cochlear hair cells ( B′ ; IHCs). It is to be noted that these images are 3D stack reconstructions. The distance between ribbons as well as their size are here distorted in 2D representation. C , Fluoresbrite YO carboxylate microspheres were taken as reference for the ribbon volume analysis (see Materials and Methods). The hatched gray bars represent the observed volume distribution of the beads (after correction for oblate and prolate image distortion) compared with their theoretical value (red). D , Fluoresbrite Yellow Green microspheres were taken as references for the spheroid surface analysis of Cav1.3 clusters. Hatched gray bars represent the observed surface distribution of the bead surfaces compared with their theoretical value (green). E , Volume distribution analysis of ribbons in VHC-I (light red, filled squares) and IHCs (dark red, filled circles) displayed no significant difference (VHC-I, 291 ribbons, 0.038 ± 0.001 μm 3 ; IHCs, 333 ribbons, 0.033 ± 0.001 μm 3 , p = 0.90). F , Surface distribution analysis of Cav1.3 clusters. In VHC-I, Cav1.3 channel clusters distributed in two equal populations of small (0.056 ± 0.001 μm 2 ) and large (0.209 ± 0.002 μm 2 ) clusters (green dashed line). In IHCs, the distribution of Cav1.3 channel clusters was fitted with a simple Gaussian (0.194 ± 0.002 μm 2 ; dark green). E′ , F′ , Insets, Synaptic ribbons at high magnifications in one VHC-I ( E′ ) and one IHC ( F′ ). Scale bar, 1 μm.
    Figure Legend Snippet: Spatial distribution of Cav1.3 channels in VHC-I and IHCs. A , B , 3D Imaris reconstructions of confocal images from the central striolar region of an utricle and the low-frequency apical turn of an OC, respectively. Both organs were dissected from mice at P14. Hair cells were stained for F-actin (phalloidin, pink), otoferlin (anti-otoferlin, blue), ribbons (anti-CtBP2, red), and Cav1.3 (anti-α 1D, green). A′ , B′ , 3D-reconstructed images at higher magnification of some ribbons and their associated Cav1.3 cluster in vestibular ( A′ ; VHC-I) and cochlear hair cells ( B′ ; IHCs). It is to be noted that these images are 3D stack reconstructions. The distance between ribbons as well as their size are here distorted in 2D representation. C , Fluoresbrite YO carboxylate microspheres were taken as reference for the ribbon volume analysis (see Materials and Methods). The hatched gray bars represent the observed volume distribution of the beads (after correction for oblate and prolate image distortion) compared with their theoretical value (red). D , Fluoresbrite Yellow Green microspheres were taken as references for the spheroid surface analysis of Cav1.3 clusters. Hatched gray bars represent the observed surface distribution of the bead surfaces compared with their theoretical value (green). E , Volume distribution analysis of ribbons in VHC-I (light red, filled squares) and IHCs (dark red, filled circles) displayed no significant difference (VHC-I, 291 ribbons, 0.038 ± 0.001 μm 3 ; IHCs, 333 ribbons, 0.033 ± 0.001 μm 3 , p = 0.90). F , Surface distribution analysis of Cav1.3 clusters. In VHC-I, Cav1.3 channel clusters distributed in two equal populations of small (0.056 ± 0.001 μm 2 ) and large (0.209 ± 0.002 μm 2 ) clusters (green dashed line). In IHCs, the distribution of Cav1.3 channel clusters was fitted with a simple Gaussian (0.194 ± 0.002 μm 2 ; dark green). E′ , F′ , Insets, Synaptic ribbons at high magnifications in one VHC-I ( E′ ) and one IHC ( F′ ). Scale bar, 1 μm.

    Techniques Used: Mouse Assay, Staining, Immunohistochemistry

    Ca 2+ channel organization in VHC-I and IHCs by STED microscopy. A , B , Two examples of STED images of immunolabeled ribbon (anti-CtBP2, red) and Ca 2+ -channel (anti-Cav1.3 green) in P21 VHC-I ( A ) and P21 IHCs ( B ). Scale bars, 200 nm. C , D , Semiaxis (a, b) size distribution in VHC-I ( C ) and IHCs ( D ). Data points were fitted by a simple Gaussian. The long semi axis “a” and short semi-axis “b” are of similar values in VHC-I ( C ), whereas they show a nearly 2 times difference in IHCs ( D ). This suggested that the arrangement of the Ca 2+ channels clusters at the ribbon membrane forms an elongated stripe-like ellipsoide in IHCs and a nearly circular disk in VHC-I. C , D , Top, Schematic representation of the active zone below the ribbon in VHC-I and IHCs. Assuming that the Ca 2+ channels are uniformly distributed at the edge of the ellipse, an elongated ellipsoid distribution would give a larger distance for the interacting focus points of Ca 2+ influx in IHCs compared with VHC-I. “F” represents the distance between the center of the ellipse and each focus point. The model assumes that Ca 2+ channels are organized as individual nanodoamains for brief low-voltage stimulation (near threshold) and as microdomains with linear summation of Ca 2+ activity for long high-voltage stimulations. This summation of Ca 2+ influx would be more efficient in VHC-I than in IHCs. E , Similar mean surface distribution of Cav1.3 clusters in VHC-I and IHCs. F , Distribution and Gaussian fit of the distance between focus points and the center of the ellipse (“F”) in VHC-I (“F” = 54.4 ± 5.9 nm) and IHCs (“F” = 179.9 ± 0.9 nm).
    Figure Legend Snippet: Ca 2+ channel organization in VHC-I and IHCs by STED microscopy. A , B , Two examples of STED images of immunolabeled ribbon (anti-CtBP2, red) and Ca 2+ -channel (anti-Cav1.3 green) in P21 VHC-I ( A ) and P21 IHCs ( B ). Scale bars, 200 nm. C , D , Semiaxis (a, b) size distribution in VHC-I ( C ) and IHCs ( D ). Data points were fitted by a simple Gaussian. The long semi axis “a” and short semi-axis “b” are of similar values in VHC-I ( C ), whereas they show a nearly 2 times difference in IHCs ( D ). This suggested that the arrangement of the Ca 2+ channels clusters at the ribbon membrane forms an elongated stripe-like ellipsoide in IHCs and a nearly circular disk in VHC-I. C , D , Top, Schematic representation of the active zone below the ribbon in VHC-I and IHCs. Assuming that the Ca 2+ channels are uniformly distributed at the edge of the ellipse, an elongated ellipsoid distribution would give a larger distance for the interacting focus points of Ca 2+ influx in IHCs compared with VHC-I. “F” represents the distance between the center of the ellipse and each focus point. The model assumes that Ca 2+ channels are organized as individual nanodoamains for brief low-voltage stimulation (near threshold) and as microdomains with linear summation of Ca 2+ activity for long high-voltage stimulations. This summation of Ca 2+ influx would be more efficient in VHC-I than in IHCs. E , Similar mean surface distribution of Cav1.3 clusters in VHC-I and IHCs. F , Distribution and Gaussian fit of the distance between focus points and the center of the ellipse (“F”) in VHC-I (“F” = 54.4 ± 5.9 nm) and IHCs (“F” = 179.9 ± 0.9 nm).

    Techniques Used: Microscopy, Immunolabeling, Activity Assay

    11) Product Images from "TRPC7 regulates the electrophysiological functions of embryonic stem cell-derived cardiomyocytes"

    Article Title: TRPC7 regulates the electrophysiological functions of embryonic stem cell-derived cardiomyocytes

    Journal: Stem Cell Research & Therapy

    doi: 10.1186/s13287-021-02308-7

    Knockdown or overexpression of TRPC7 did not alter the expression of several important ion channels/pump in NRVMs. a – g Western blots showing the expression of a TRPC7, b HCN4, c Cav1.3, d IP3R1, e Cav3.1, f Cav3.2, g SERCA in NRVMs infected with different adenoviruses to knockdown or overexpress TRPC7. h – n Bar charts showing the quantification of each protein from a – g . To eliminate the loading bias, intensity of each target protein was normalized to that of its corresponding β-tubulin. TRPC7 was successfully knocked down or overexpressed in NRVMs but the change of TRPC7 expression did not alter the expression of HCN4, Cav1.3, IP3R1, Cav3.1, Cav3.2, and SERCA. Data were presented as mean ± SEM ( n = 4). * P
    Figure Legend Snippet: Knockdown or overexpression of TRPC7 did not alter the expression of several important ion channels/pump in NRVMs. a – g Western blots showing the expression of a TRPC7, b HCN4, c Cav1.3, d IP3R1, e Cav3.1, f Cav3.2, g SERCA in NRVMs infected with different adenoviruses to knockdown or overexpress TRPC7. h – n Bar charts showing the quantification of each protein from a – g . To eliminate the loading bias, intensity of each target protein was normalized to that of its corresponding β-tubulin. TRPC7 was successfully knocked down or overexpressed in NRVMs but the change of TRPC7 expression did not alter the expression of HCN4, Cav1.3, IP3R1, Cav3.1, Cav3.2, and SERCA. Data were presented as mean ± SEM ( n = 4). * P

    Techniques Used: Over Expression, Expressing, Western Blot, Infection

    12) Product Images from "L-Type Cav1.3 Calcium Channels Are Required for Beta-Adrenergic Triggered Automaticity in Dormant Mouse Sinoatrial Pacemaker Cells"

    Article Title: L-Type Cav1.3 Calcium Channels Are Required for Beta-Adrenergic Triggered Automaticity in Dormant Mouse Sinoatrial Pacemaker Cells

    Journal: Cells

    doi: 10.3390/cells11071114

    Dormant SANC express reduced I Cav1.3 and I f . Representative traces and I-V curves of I f (( A ), n = 10 dormant and n = 8 firing) and Nife-sensitive current ( I Cav1.3 ) (( B ), n = 12 dormant and n = 9 firing) in dormant (blue) and firing (red) SANC. ( C ) Nife-sensitive current ( I Cav1.3 ) and Nife-insensitive current ( I Cav1.2 ) densities at 0 mV in dormant and firing Ca v 1.2 DHP−/− SANC. Averaged SR Ca 2+ load ( D ), time constant of caffeine-induced Ca 2+ transient ( E ), number of LCRs ( F ) and diastolic Ca 2+ ( G ), in dormant (blue, n = 9) and firing (red, n = 15) SANC. ns: non-significant, * p
    Figure Legend Snippet: Dormant SANC express reduced I Cav1.3 and I f . Representative traces and I-V curves of I f (( A ), n = 10 dormant and n = 8 firing) and Nife-sensitive current ( I Cav1.3 ) (( B ), n = 12 dormant and n = 9 firing) in dormant (blue) and firing (red) SANC. ( C ) Nife-sensitive current ( I Cav1.3 ) and Nife-insensitive current ( I Cav1.2 ) densities at 0 mV in dormant and firing Ca v 1.2 DHP−/− SANC. Averaged SR Ca 2+ load ( D ), time constant of caffeine-induced Ca 2+ transient ( E ), number of LCRs ( F ) and diastolic Ca 2+ ( G ), in dormant (blue, n = 9) and firing (red, n = 15) SANC. ns: non-significant, * p

    Techniques Used:

    13) Product Images from "Down-regulation of Cav1.3 in auditory pathway promotes age-related hearing loss by enhancing calcium-mediated oxidative stress in male mice"

    Article Title: Down-regulation of Cav1.3 in auditory pathway promotes age-related hearing loss by enhancing calcium-mediated oxidative stress in male mice

    Journal: Aging (Albany NY)

    doi: 10.18632/aging.102203

    Age-related expression of Cav1.3 in auditory pathway. ( A ) The immunofluorescence of CaV1.3 in the auditory cortex (green, magnification, ×400). ( B ) the quantitative analysis of CaV1.3 expression in the auditory cortex. ( C ) the western-blotting analysis of CaV1.3 expression in auditory cortex (top), the bottom panel is the quantitative analysis. ( D ) the mRNA expression of CaV1.3 in auditory cortex, inferior colliculus and cochlear nucleus. ( E ) CaV1.3 expression in auditory cortex was analyzed by flow cytometry, the right panel is the quantitative analysis. ( F ) Cav1.3 expression in neurons of auditory cortex, the neuron cells were gated as NeuN+, the right panel is the quantitative analysis. Error bars represent mean ± s.d.; *P
    Figure Legend Snippet: Age-related expression of Cav1.3 in auditory pathway. ( A ) The immunofluorescence of CaV1.3 in the auditory cortex (green, magnification, ×400). ( B ) the quantitative analysis of CaV1.3 expression in the auditory cortex. ( C ) the western-blotting analysis of CaV1.3 expression in auditory cortex (top), the bottom panel is the quantitative analysis. ( D ) the mRNA expression of CaV1.3 in auditory cortex, inferior colliculus and cochlear nucleus. ( E ) CaV1.3 expression in auditory cortex was analyzed by flow cytometry, the right panel is the quantitative analysis. ( F ) Cav1.3 expression in neurons of auditory cortex, the neuron cells were gated as NeuN+, the right panel is the quantitative analysis. Error bars represent mean ± s.d.; *P

    Techniques Used: Expressing, Immunofluorescence, Western Blot, Flow Cytometry

    Age-related Cav1.3 expression in cochlea. ( A , B ) immunofluorescence of CaV1.3(green) and Myo7a (red) in the organ of Corti (left) and spiral ganglion (right) (magnification, ×400), nuclei was visualized by DAPI (blue). ( C ) the immunofluorescent staining for CaV1.3 (green) in the whole cochlear basilar membrane. ( D ) quantitative analysis of CaV1.3 expression in hair cells, spiral ganglion and cochlea basilar membrane.
    Figure Legend Snippet: Age-related Cav1.3 expression in cochlea. ( A , B ) immunofluorescence of CaV1.3(green) and Myo7a (red) in the organ of Corti (left) and spiral ganglion (right) (magnification, ×400), nuclei was visualized by DAPI (blue). ( C ) the immunofluorescent staining for CaV1.3 (green) in the whole cochlear basilar membrane. ( D ) quantitative analysis of CaV1.3 expression in hair cells, spiral ganglion and cochlea basilar membrane.

    Techniques Used: Expressing, Immunofluorescence, Staining

    Hair cells were vulnerable to ROS injury after Cav1.3 was knocked out. ( A ) the effect of CaV1.3 knock out in HEI-OC1 was analyzed by flow cytometry. ( B ) membrane potential (top) and non-linear capacitance (NLC) (bottom) studies in WT HEI-OC1 and CaV1.3 KO HEI-OC1 cells (n=5). ( C ) western-blotting analysis of CaV1.3 and p53 expression in control and senescence HEI-OC1 cells induced by D-galactose (D-Gal) or hydrogen peroxide (H 2 O 2 ), the bottom panel is the quantitative analysis. ( D ) β-Galactosidase staining (top) and C12FDG staining (bottom) of control and senescent HEI-OC1 cells induced by H 2 O 2 . ( E ) flow cytometry analysis of CaV1.3 in control and H 2 O 2 induced HEI-OC1 cells. ( F ) C12FDG staining (top) and β-Galactosidase staining (bottom) of NC (negative control) and KO (CaV1.3 knock out) HEI-OC1 cells after H 2 O 2 induction. ( G, H ) CFSE staining and red dot staining of NC and KO HEI-OC1 cells with or without H 2 O 2 induction. ( I ) LDH assay of NC and KO HEI-OC1 cells after H 2 O 2 induction (n=3). ( J ) caspase-3/7-AAD staining of NC and KO HEI-OC1 cells after H 2 O 2 induction (n=3). Error bars represent mean ± s.d.; *P
    Figure Legend Snippet: Hair cells were vulnerable to ROS injury after Cav1.3 was knocked out. ( A ) the effect of CaV1.3 knock out in HEI-OC1 was analyzed by flow cytometry. ( B ) membrane potential (top) and non-linear capacitance (NLC) (bottom) studies in WT HEI-OC1 and CaV1.3 KO HEI-OC1 cells (n=5). ( C ) western-blotting analysis of CaV1.3 and p53 expression in control and senescence HEI-OC1 cells induced by D-galactose (D-Gal) or hydrogen peroxide (H 2 O 2 ), the bottom panel is the quantitative analysis. ( D ) β-Galactosidase staining (top) and C12FDG staining (bottom) of control and senescent HEI-OC1 cells induced by H 2 O 2 . ( E ) flow cytometry analysis of CaV1.3 in control and H 2 O 2 induced HEI-OC1 cells. ( F ) C12FDG staining (top) and β-Galactosidase staining (bottom) of NC (negative control) and KO (CaV1.3 knock out) HEI-OC1 cells after H 2 O 2 induction. ( G, H ) CFSE staining and red dot staining of NC and KO HEI-OC1 cells with or without H 2 O 2 induction. ( I ) LDH assay of NC and KO HEI-OC1 cells after H 2 O 2 induction (n=3). ( J ) caspase-3/7-AAD staining of NC and KO HEI-OC1 cells after H 2 O 2 induction (n=3). Error bars represent mean ± s.d.; *P

    Techniques Used: Knock-Out, Flow Cytometry, Western Blot, Expressing, Staining, Negative Control, Lactate Dehydrogenase Assay

    Cav1.3 knock out decrease intra cellular calcium and subsequently result in reduction of complex I derived ROS inactivation. ( A ) intra-cellular ROS detection by flow cytometry (n=3). ( B – E ) the intra cellular calcium, intra cellular ROS, caspase-3/7-AAD staining and C12FDG staining of NC and KO HEI-OC1 cells with or without Ionmycin (n=3). ( F ) immunofluorescence of mitoSOX (red) and mitotrackor (green) in NC and KO HEI-OC1 cells, nuclei was visualized by DAPI (magnification, ×400, scal bar: 50μm), the right panels are the quantitative analysis of mitoSOX (top) and mitoTrackor (bottom). ( G ) the intra cellular ROS of KO HEI-OC1 cells with or without gradient Retenone (Ret) and Antimycin A (AMA)(n=3), the right panel is the quantitative analysis. Error bars represent mean ± s.d.; *P
    Figure Legend Snippet: Cav1.3 knock out decrease intra cellular calcium and subsequently result in reduction of complex I derived ROS inactivation. ( A ) intra-cellular ROS detection by flow cytometry (n=3). ( B – E ) the intra cellular calcium, intra cellular ROS, caspase-3/7-AAD staining and C12FDG staining of NC and KO HEI-OC1 cells with or without Ionmycin (n=3). ( F ) immunofluorescence of mitoSOX (red) and mitotrackor (green) in NC and KO HEI-OC1 cells, nuclei was visualized by DAPI (magnification, ×400, scal bar: 50μm), the right panels are the quantitative analysis of mitoSOX (top) and mitoTrackor (bottom). ( G ) the intra cellular ROS of KO HEI-OC1 cells with or without gradient Retenone (Ret) and Antimycin A (AMA)(n=3), the right panel is the quantitative analysis. Error bars represent mean ± s.d.; *P

    Techniques Used: Knock-Out, Derivative Assay, Flow Cytometry, Staining, Immunofluorescence

    Cav1.3 knock down aggravated the loss of hair cells after senescence induction and resulted in hearing impairment. ( A ) immunofluorescence of CaV1.3 (green) and mCherry (red) in the organ of Corti (left) and spiral ganglion (right) of control and CaV1.3 knock down AAV group, nuclei was visualized by DAPI (magnification, ×400, scale bar: 50μm), the right panels are the quantitative analysis of CaV1.3 expression in organ of corti (top) and spiral ganglion (bottom). ( B ) auditory brainstem response (ABR) (top) and the whole cochlear basilar membrane after DAPI staining (bottom) of NC, CaV1.3 knock down, NC+D-Gal and CaV1.3 knock down+D-Gal group (n=6). ( C ) phalloidine staining for control and negative control AAV or Cav1.3 knock down AAV infected OC segment explants with H 2 O 2 treatment (n=3), the right panel is the quantitative analysis. Error bars represent mean ± s.d.; *P
    Figure Legend Snippet: Cav1.3 knock down aggravated the loss of hair cells after senescence induction and resulted in hearing impairment. ( A ) immunofluorescence of CaV1.3 (green) and mCherry (red) in the organ of Corti (left) and spiral ganglion (right) of control and CaV1.3 knock down AAV group, nuclei was visualized by DAPI (magnification, ×400, scale bar: 50μm), the right panels are the quantitative analysis of CaV1.3 expression in organ of corti (top) and spiral ganglion (bottom). ( B ) auditory brainstem response (ABR) (top) and the whole cochlear basilar membrane after DAPI staining (bottom) of NC, CaV1.3 knock down, NC+D-Gal and CaV1.3 knock down+D-Gal group (n=6). ( C ) phalloidine staining for control and negative control AAV or Cav1.3 knock down AAV infected OC segment explants with H 2 O 2 treatment (n=3), the right panel is the quantitative analysis. Error bars represent mean ± s.d.; *P

    Techniques Used: Immunofluorescence, Expressing, Staining, Negative Control, Infection

    14) Product Images from "Down-regulation of Cav1.3 in auditory pathway promotes age-related hearing loss by enhancing calcium-mediated oxidative stress in male mice"

    Article Title: Down-regulation of Cav1.3 in auditory pathway promotes age-related hearing loss by enhancing calcium-mediated oxidative stress in male mice

    Journal: Aging (Albany NY)

    doi: 10.18632/aging.102203

    Age-related expression of Cav1.3 in auditory pathway. ( A ) The immunofluorescence of CaV1.3 in the auditory cortex (green, magnification, ×400). ( B ) the quantitative analysis of CaV1.3 expression in the auditory cortex. ( C ) the western-blotting analysis of CaV1.3 expression in auditory cortex (top), the bottom panel is the quantitative analysis. ( D ) the mRNA expression of CaV1.3 in auditory cortex, inferior colliculus and cochlear nucleus. ( E ) CaV1.3 expression in auditory cortex was analyzed by flow cytometry, the right panel is the quantitative analysis. ( F ) Cav1.3 expression in neurons of auditory cortex, the neuron cells were gated as NeuN+, the right panel is the quantitative analysis. Error bars represent mean ± s.d.; *P
    Figure Legend Snippet: Age-related expression of Cav1.3 in auditory pathway. ( A ) The immunofluorescence of CaV1.3 in the auditory cortex (green, magnification, ×400). ( B ) the quantitative analysis of CaV1.3 expression in the auditory cortex. ( C ) the western-blotting analysis of CaV1.3 expression in auditory cortex (top), the bottom panel is the quantitative analysis. ( D ) the mRNA expression of CaV1.3 in auditory cortex, inferior colliculus and cochlear nucleus. ( E ) CaV1.3 expression in auditory cortex was analyzed by flow cytometry, the right panel is the quantitative analysis. ( F ) Cav1.3 expression in neurons of auditory cortex, the neuron cells were gated as NeuN+, the right panel is the quantitative analysis. Error bars represent mean ± s.d.; *P

    Techniques Used: Expressing, Immunofluorescence, Western Blot, Flow Cytometry

    Age-related Cav1.3 expression in cochlea. ( A , B ) immunofluorescence of CaV1.3(green) and Myo7a (red) in the organ of Corti (left) and spiral ganglion (right) (magnification, ×400), nuclei was visualized by DAPI (blue). ( C ) the immunofluorescent staining for CaV1.3 (green) in the whole cochlear basilar membrane. ( D ) quantitative analysis of CaV1.3 expression in hair cells, spiral ganglion and cochlea basilar membrane.
    Figure Legend Snippet: Age-related Cav1.3 expression in cochlea. ( A , B ) immunofluorescence of CaV1.3(green) and Myo7a (red) in the organ of Corti (left) and spiral ganglion (right) (magnification, ×400), nuclei was visualized by DAPI (blue). ( C ) the immunofluorescent staining for CaV1.3 (green) in the whole cochlear basilar membrane. ( D ) quantitative analysis of CaV1.3 expression in hair cells, spiral ganglion and cochlea basilar membrane.

    Techniques Used: Expressing, Immunofluorescence, Staining

    Hair cells were vulnerable to ROS injury after Cav1.3 was knocked out. ( A ) the effect of CaV1.3 knock out in HEI-OC1 was analyzed by flow cytometry. ( B ) membrane potential (top) and non-linear capacitance (NLC) (bottom) studies in WT HEI-OC1 and CaV1.3 KO HEI-OC1 cells (n=5). ( C ) western-blotting analysis of CaV1.3 and p53 expression in control and senescence HEI-OC1 cells induced by D-galactose (D-Gal) or hydrogen peroxide (H 2 O 2 ), the bottom panel is the quantitative analysis. ( D ) β-Galactosidase staining (top) and C12FDG staining (bottom) of control and senescent HEI-OC1 cells induced by H 2 O 2 . ( E ) flow cytometry analysis of CaV1.3 in control and H 2 O 2 induced HEI-OC1 cells. ( F ) C12FDG staining (top) and β-Galactosidase staining (bottom) of NC (negative control) and KO (CaV1.3 knock out) HEI-OC1 cells after H 2 O 2 induction. ( G, H ) CFSE staining and red dot staining of NC and KO HEI-OC1 cells with or without H 2 O 2 induction. ( I ) LDH assay of NC and KO HEI-OC1 cells after H 2 O 2 induction (n=3). ( J ) caspase-3/7-AAD staining of NC and KO HEI-OC1 cells after H 2 O 2 induction (n=3). Error bars represent mean ± s.d.; *P
    Figure Legend Snippet: Hair cells were vulnerable to ROS injury after Cav1.3 was knocked out. ( A ) the effect of CaV1.3 knock out in HEI-OC1 was analyzed by flow cytometry. ( B ) membrane potential (top) and non-linear capacitance (NLC) (bottom) studies in WT HEI-OC1 and CaV1.3 KO HEI-OC1 cells (n=5). ( C ) western-blotting analysis of CaV1.3 and p53 expression in control and senescence HEI-OC1 cells induced by D-galactose (D-Gal) or hydrogen peroxide (H 2 O 2 ), the bottom panel is the quantitative analysis. ( D ) β-Galactosidase staining (top) and C12FDG staining (bottom) of control and senescent HEI-OC1 cells induced by H 2 O 2 . ( E ) flow cytometry analysis of CaV1.3 in control and H 2 O 2 induced HEI-OC1 cells. ( F ) C12FDG staining (top) and β-Galactosidase staining (bottom) of NC (negative control) and KO (CaV1.3 knock out) HEI-OC1 cells after H 2 O 2 induction. ( G, H ) CFSE staining and red dot staining of NC and KO HEI-OC1 cells with or without H 2 O 2 induction. ( I ) LDH assay of NC and KO HEI-OC1 cells after H 2 O 2 induction (n=3). ( J ) caspase-3/7-AAD staining of NC and KO HEI-OC1 cells after H 2 O 2 induction (n=3). Error bars represent mean ± s.d.; *P

    Techniques Used: Knock-Out, Flow Cytometry, Western Blot, Expressing, Staining, Negative Control, Lactate Dehydrogenase Assay

    Cav1.3 knock out decrease intra cellular calcium and subsequently result in reduction of complex I derived ROS inactivation. ( A ) intra-cellular ROS detection by flow cytometry (n=3). ( B – E ) the intra cellular calcium, intra cellular ROS, caspase-3/7-AAD staining and C12FDG staining of NC and KO HEI-OC1 cells with or without Ionmycin (n=3). ( F ) immunofluorescence of mitoSOX (red) and mitotrackor (green) in NC and KO HEI-OC1 cells, nuclei was visualized by DAPI (magnification, ×400, scal bar: 50μm), the right panels are the quantitative analysis of mitoSOX (top) and mitoTrackor (bottom). ( G ) the intra cellular ROS of KO HEI-OC1 cells with or without gradient Retenone (Ret) and Antimycin A (AMA)(n=3), the right panel is the quantitative analysis. Error bars represent mean ± s.d.; *P
    Figure Legend Snippet: Cav1.3 knock out decrease intra cellular calcium and subsequently result in reduction of complex I derived ROS inactivation. ( A ) intra-cellular ROS detection by flow cytometry (n=3). ( B – E ) the intra cellular calcium, intra cellular ROS, caspase-3/7-AAD staining and C12FDG staining of NC and KO HEI-OC1 cells with or without Ionmycin (n=3). ( F ) immunofluorescence of mitoSOX (red) and mitotrackor (green) in NC and KO HEI-OC1 cells, nuclei was visualized by DAPI (magnification, ×400, scal bar: 50μm), the right panels are the quantitative analysis of mitoSOX (top) and mitoTrackor (bottom). ( G ) the intra cellular ROS of KO HEI-OC1 cells with or without gradient Retenone (Ret) and Antimycin A (AMA)(n=3), the right panel is the quantitative analysis. Error bars represent mean ± s.d.; *P

    Techniques Used: Knock-Out, Derivative Assay, Flow Cytometry, Staining, Immunofluorescence

    Cav1.3 knock down aggravated the loss of hair cells after senescence induction and resulted in hearing impairment. ( A ) immunofluorescence of CaV1.3 (green) and mCherry (red) in the organ of Corti (left) and spiral ganglion (right) of control and CaV1.3 knock down AAV group, nuclei was visualized by DAPI (magnification, ×400, scale bar: 50μm), the right panels are the quantitative analysis of CaV1.3 expression in organ of corti (top) and spiral ganglion (bottom). ( B ) auditory brainstem response (ABR) (top) and the whole cochlear basilar membrane after DAPI staining (bottom) of NC, CaV1.3 knock down, NC+D-Gal and CaV1.3 knock down+D-Gal group (n=6). ( C ) phalloidine staining for control and negative control AAV or Cav1.3 knock down AAV infected OC segment explants with H 2 O 2 treatment (n=3), the right panel is the quantitative analysis. Error bars represent mean ± s.d.; *P
    Figure Legend Snippet: Cav1.3 knock down aggravated the loss of hair cells after senescence induction and resulted in hearing impairment. ( A ) immunofluorescence of CaV1.3 (green) and mCherry (red) in the organ of Corti (left) and spiral ganglion (right) of control and CaV1.3 knock down AAV group, nuclei was visualized by DAPI (magnification, ×400, scale bar: 50μm), the right panels are the quantitative analysis of CaV1.3 expression in organ of corti (top) and spiral ganglion (bottom). ( B ) auditory brainstem response (ABR) (top) and the whole cochlear basilar membrane after DAPI staining (bottom) of NC, CaV1.3 knock down, NC+D-Gal and CaV1.3 knock down+D-Gal group (n=6). ( C ) phalloidine staining for control and negative control AAV or Cav1.3 knock down AAV infected OC segment explants with H 2 O 2 treatment (n=3), the right panel is the quantitative analysis. Error bars represent mean ± s.d.; *P

    Techniques Used: Immunofluorescence, Expressing, Staining, Negative Control, Infection

    15) Product Images from "Different CaV1.3 Channel Isoforms Control Distinct Components of the Synaptic Vesicle Cycle in Auditory Inner Hair Cells"

    Article Title: Different CaV1.3 Channel Isoforms Control Distinct Components of the Synaptic Vesicle Cycle in Auditory Inner Hair Cells

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.2374-16.2017

    Ca 2+ imaging and immunohistochemistry mostly localize Ca v 1.3 Ca 2+ channels at the synaptic ribbons of WT IHCs but small Ca v 1.3 clusters are also seen extrasynaptically right below the cuticular plate region. A , B , Example of simultaneous recording of Ca 2+ current (top) and capacitance response (bottom) in control condition ( A ) in a WT IHC evoked during a single 500 ms step depolarization from −80 mV to −10 mV. A simultaneous intracellular Ca 2+ increase was recorded through the fast Ca 2+ indicator, GCamP6f ( B , C ). Ca 2+ signals were measured from the synaptic zone (black line), above the nucleus (orange line), and under the cuticular plate region (blue line) as indicated in C , showing a row of IHCs genetically expressing the Ca 2+ indicator GCamP6f. Boxes represent the ROI used for the Ca 2+ measurement shown in B . Δ t represents the time difference between the maximum peak of intracellular Ca 2+ measured at the synaptic area and the region above the nucleus. D , Typical example of Ca V 1.3 immunolabeling at the apical turn of a whole-mount OC from a P14 mouse. F-actin is labeled in pink, otoferlin in blue, CtBP2 (ribbon) in red, and Cav1.3 in green. Right top panel shows the cuticular plate region at higher magnification and the small clusters of Ca V 1.3 (arrowheads). Right bottom panel shows two synaptic active zones at higher magnification.
    Figure Legend Snippet: Ca 2+ imaging and immunohistochemistry mostly localize Ca v 1.3 Ca 2+ channels at the synaptic ribbons of WT IHCs but small Ca v 1.3 clusters are also seen extrasynaptically right below the cuticular plate region. A , B , Example of simultaneous recording of Ca 2+ current (top) and capacitance response (bottom) in control condition ( A ) in a WT IHC evoked during a single 500 ms step depolarization from −80 mV to −10 mV. A simultaneous intracellular Ca 2+ increase was recorded through the fast Ca 2+ indicator, GCamP6f ( B , C ). Ca 2+ signals were measured from the synaptic zone (black line), above the nucleus (orange line), and under the cuticular plate region (blue line) as indicated in C , showing a row of IHCs genetically expressing the Ca 2+ indicator GCamP6f. Boxes represent the ROI used for the Ca 2+ measurement shown in B . Δ t represents the time difference between the maximum peak of intracellular Ca 2+ measured at the synaptic area and the region above the nucleus. D , Typical example of Ca V 1.3 immunolabeling at the apical turn of a whole-mount OC from a P14 mouse. F-actin is labeled in pink, otoferlin in blue, CtBP2 (ribbon) in red, and Cav1.3 in green. Right top panel shows the cuticular plate region at higher magnification and the small clusters of Ca V 1.3 (arrowheads). Right bottom panel shows two synaptic active zones at higher magnification.

    Techniques Used: Imaging, Immunohistochemistry, Expressing, Immunolabeling, Labeling

    Transcript mRNA expression of Ca v 1.3 short and long isoforms. A , RT-PCR expression of myosin VIIa (Myo VIIa, top left), 42L isoform (long Cav1.3 isoform, bottom left) using 4664s/4995as, both 43S and 42L isoforms using 5010s/5637as (top right) and 42A (bottom right) using 4946s/5146as in P35 Otof +/− (heterozygous: H) and Otof −/− (KO) OCs. B , Schematic presentation of alternative splicing of Cav1.3 C terminus (c-term) domain for long isoform 42L (top), short isoform 43S (middle), and short isoform 42A (bottom). Black and blank rectangles represent constitutive and alternative splicing, respectively. Incorporation of exon 42A instead of exon 42 induces a premature nonsense codon leading to a truncated c-term domain. Orange box represents the lack of 155 nt in the 43S sequence inducing a premature nonsense codon and a truncated c-term domain. Under each sequence, a schematic drawing of the translated α 1D c-term domain controlling Ca 2+ -dependent inactivation is shown. The red arrow (top schematic drawing) indicates the formation of the CTM by the interaction between the PCRD and DCRD for the long 42L isoform. Such a regulatory domain cannot be formed in the case of the two short 43S and 42A isoforms. Note that the green arrows above and under each sequence represent the hybridization site for each used primer. C , D , Q-PCR from P35 Otof +/− (black) and Otof −/− (red) OCs for short isoforms 43S ( C ) and 42A ( D ). Green dotted line represents the Ct. E , RT-PCR from pooled WT IHCs shows the transcripts of short (43S and 42A) and long (42L) Cav1.3 isoforms. ES: Extracellular solution was used as negative control. F , G , Western blot analysis indicating a reduction of 43S and 42A Cav1.3 isoforms in Otof −/− OC.
    Figure Legend Snippet: Transcript mRNA expression of Ca v 1.3 short and long isoforms. A , RT-PCR expression of myosin VIIa (Myo VIIa, top left), 42L isoform (long Cav1.3 isoform, bottom left) using 4664s/4995as, both 43S and 42L isoforms using 5010s/5637as (top right) and 42A (bottom right) using 4946s/5146as in P35 Otof +/− (heterozygous: H) and Otof −/− (KO) OCs. B , Schematic presentation of alternative splicing of Cav1.3 C terminus (c-term) domain for long isoform 42L (top), short isoform 43S (middle), and short isoform 42A (bottom). Black and blank rectangles represent constitutive and alternative splicing, respectively. Incorporation of exon 42A instead of exon 42 induces a premature nonsense codon leading to a truncated c-term domain. Orange box represents the lack of 155 nt in the 43S sequence inducing a premature nonsense codon and a truncated c-term domain. Under each sequence, a schematic drawing of the translated α 1D c-term domain controlling Ca 2+ -dependent inactivation is shown. The red arrow (top schematic drawing) indicates the formation of the CTM by the interaction between the PCRD and DCRD for the long 42L isoform. Such a regulatory domain cannot be formed in the case of the two short 43S and 42A isoforms. Note that the green arrows above and under each sequence represent the hybridization site for each used primer. C , D , Q-PCR from P35 Otof +/− (black) and Otof −/− (red) OCs for short isoforms 43S ( C ) and 42A ( D ). Green dotted line represents the Ct. E , RT-PCR from pooled WT IHCs shows the transcripts of short (43S and 42A) and long (42L) Cav1.3 isoforms. ES: Extracellular solution was used as negative control. F , G , Western blot analysis indicating a reduction of 43S and 42A Cav1.3 isoforms in Otof −/− OC.

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Sequencing, Hybridization, Polymerase Chain Reaction, Negative Control, Western Blot

    16) Product Images from "Cell-type-specific tuning of Cav1.3 Ca2+-channels by a C-terminal automodulatory domain"

    Article Title: Cell-type-specific tuning of Cav1.3 Ca2+-channels by a C-terminal automodulatory domain

    Journal: Frontiers in Cellular Neuroscience

    doi: 10.3389/fncel.2015.00309

    Western blot analysis of α1-subunits in WT and mutant mouse brain homogenates. (A) Proteins (100 μg/lane) were separated on 5% SDS page and immunostained with polyclonal antibody Cav1.3 α1 CT . The Cav1.3 α1 subunit was specifically detected as a 231 kDa protein at expression levels indistinguishable between WT and homozygous mutants (HA) (see Results). KO, Cav1.3 -/- negative control. (B) Same separation as in A (100 μg/lane) but detection with anti-Cav1.3α1 NT . The migration of recombinant mCav1.3 L (L) and mCav1.343 S (43S) on the same gel (not shown) and their calculated molecular mass are indicated by arrows (left). Migration of molecular mass standards as well as the brain long and short α1-subunit species are also indicated (right). An unspecific ∼120 kDa band served as loading control. One representative experiment of at least three independent experiments is shown for all panels. KO, Cav1.3 -/- ; HA, Cav1.3DCRD HA/HA , WT, wild-type littermate.
    Figure Legend Snippet: Western blot analysis of α1-subunits in WT and mutant mouse brain homogenates. (A) Proteins (100 μg/lane) were separated on 5% SDS page and immunostained with polyclonal antibody Cav1.3 α1 CT . The Cav1.3 α1 subunit was specifically detected as a 231 kDa protein at expression levels indistinguishable between WT and homozygous mutants (HA) (see Results). KO, Cav1.3 -/- negative control. (B) Same separation as in A (100 μg/lane) but detection with anti-Cav1.3α1 NT . The migration of recombinant mCav1.3 L (L) and mCav1.343 S (43S) on the same gel (not shown) and their calculated molecular mass are indicated by arrows (left). Migration of molecular mass standards as well as the brain long and short α1-subunit species are also indicated (right). An unspecific ∼120 kDa band served as loading control. One representative experiment of at least three independent experiments is shown for all panels. KO, Cav1.3 -/- ; HA, Cav1.3DCRD HA/HA , WT, wild-type littermate.

    Techniques Used: Western Blot, Mutagenesis, SDS Page, Expressing, Negative Control, Migration, Recombinant

    Cav1.3 α1 transcripts containing exons 43 S and 43 L in mouse IHCs and OHCs, at P6 and P22 using nested PCR. Fragments containing 43S (403 bp) or 43L (557 bp) were amplified using nested PCR (see Materials and Methods) with primers specific for exon 42 (forward) and 45 (reverse) of mouse Cav1.3. S1–S14 represent samples from independent preparations. For each cell type and developmental stage at least three independent experiments were performed. Whole brain (WB) and heart (WH) served as positive controls, H 2 O (no template) as negative control. Specificity of PCR products was confirmed by sequencing. When two independent PCR reactions with three different RNA samples of each cell type were performed, the number of successful detections for each transcript was as follows: detection of 43L: 6 (out of six experiments) in IHC and OHC preparations of all developmental stages; detection of 43S: 4 (6) in IHC P06 and IHC P22, 6 (6) in OHC P06 and 5 (6) in OHC P22. Bp, basepair markers.
    Figure Legend Snippet: Cav1.3 α1 transcripts containing exons 43 S and 43 L in mouse IHCs and OHCs, at P6 and P22 using nested PCR. Fragments containing 43S (403 bp) or 43L (557 bp) were amplified using nested PCR (see Materials and Methods) with primers specific for exon 42 (forward) and 45 (reverse) of mouse Cav1.3. S1–S14 represent samples from independent preparations. For each cell type and developmental stage at least three independent experiments were performed. Whole brain (WB) and heart (WH) served as positive controls, H 2 O (no template) as negative control. Specificity of PCR products was confirmed by sequencing. When two independent PCR reactions with three different RNA samples of each cell type were performed, the number of successful detections for each transcript was as follows: detection of 43L: 6 (out of six experiments) in IHC and OHC preparations of all developmental stages; detection of 43S: 4 (6) in IHC P06 and IHC P22, 6 (6) in OHC P06 and 5 (6) in OHC P22. Bp, basepair markers.

    Techniques Used: Nested PCR, Amplification, Western Blot, Negative Control, Polymerase Chain Reaction, Sequencing, Immunohistochemistry

    Absence of smaller C-terminally–derived Cav1.3 α1 fragments in WT and Cav1.3DCRD HA/HA brain preparations. (A) Mouse brain homogenate (100 μg of protein/lane) prepared from WT or Cav1.3DCRD HA/HA (HA) mice were separated on 4–15% gradient SDS-PAGE and immunostained with anti-HA antibody. The blot was overexposed to also visualize less abundant smaller fragments. in separate experiments α1- associated HA-immunoreactivity could be detected with only 10% (10 μg/lane) of the protein amount used ( n = 3) demonstrating the sensitivity of the assay. (B) Mouse brain membranes (100 μg of protein/lane) were analyzed as in (A). (C) Mouse brain membranes (100 μg of protein/lane) from WT or Cav1.3 -/- (KO) mice were blotted as in (B) and stained with anti-Cav1.3α1 CT antibodies. To some WT samples (33 μg/lane) a 45 kDa recombinant C-terminal control peptide was added (arrow, amounts indicated) before separation to demonstrate successful transfer and sensitive detection as a positive control for sensitivity.
    Figure Legend Snippet: Absence of smaller C-terminally–derived Cav1.3 α1 fragments in WT and Cav1.3DCRD HA/HA brain preparations. (A) Mouse brain homogenate (100 μg of protein/lane) prepared from WT or Cav1.3DCRD HA/HA (HA) mice were separated on 4–15% gradient SDS-PAGE and immunostained with anti-HA antibody. The blot was overexposed to also visualize less abundant smaller fragments. in separate experiments α1- associated HA-immunoreactivity could be detected with only 10% (10 μg/lane) of the protein amount used ( n = 3) demonstrating the sensitivity of the assay. (B) Mouse brain membranes (100 μg of protein/lane) were analyzed as in (A). (C) Mouse brain membranes (100 μg of protein/lane) from WT or Cav1.3 -/- (KO) mice were blotted as in (B) and stained with anti-Cav1.3α1 CT antibodies. To some WT samples (33 μg/lane) a 45 kDa recombinant C-terminal control peptide was added (arrow, amounts indicated) before separation to demonstrate successful transfer and sensitive detection as a positive control for sensitivity.

    Techniques Used: Derivative Assay, Mouse Assay, SDS Page, Staining, Recombinant, Positive Control

    Comparison of the protein localization of anti-HA-labeled Cav1.3 with immunolabeled Ca v 1.3, Ca v β2 and CtBP2/RIBEYE in IHCs. (A–D) Whole-mount preparations of apical turns of the organ of Corti from adult Ca v 1.3DCRD HA/HA (A,C,D) and WT (B) mice were co-immunolabeled with anti-HA and anti-Ca v 1.3 ( A, B , 11 weeks), anti-HA and anti-Ca v β2 ( C , P28), or anti-HA and anti-CtBP2/RIBEYE antibodies ( D , P37). Every image shows the basolateral poles of two adjacent IHCs the nuclei of which are indicated by asterisks in the rightmost column, respectively. HA staining (A1,C1,D1) largely overlapped with Ca v 1.3 (A2) , Ca v ß2 (C2) and CtBP2 (D2) staining at the basal poles of IHCs as evident upon merging corresponding images (A3,C3,D3) . In the WT, no specific HA-labeling (B1) was present at the position of Ca v 1.3 labeling (B2,B3) . The weak ‘cloudy’ green anti-HA staining was present in all specimen investigated and therefore considered unspecific. Cell nuclei of IHCs were counterstained with DAPI (blue). 1 of 3 ( A , age: 2–3 months), 1 of 4 ( B , age: P25 – 3 month), 1 of 5 ( C , P25–P31) and 1 of 5 ( D , P28–P37) independent experiments is illustrated, respectively. Scale bars: 5 μm.
    Figure Legend Snippet: Comparison of the protein localization of anti-HA-labeled Cav1.3 with immunolabeled Ca v 1.3, Ca v β2 and CtBP2/RIBEYE in IHCs. (A–D) Whole-mount preparations of apical turns of the organ of Corti from adult Ca v 1.3DCRD HA/HA (A,C,D) and WT (B) mice were co-immunolabeled with anti-HA and anti-Ca v 1.3 ( A, B , 11 weeks), anti-HA and anti-Ca v β2 ( C , P28), or anti-HA and anti-CtBP2/RIBEYE antibodies ( D , P37). Every image shows the basolateral poles of two adjacent IHCs the nuclei of which are indicated by asterisks in the rightmost column, respectively. HA staining (A1,C1,D1) largely overlapped with Ca v 1.3 (A2) , Ca v ß2 (C2) and CtBP2 (D2) staining at the basal poles of IHCs as evident upon merging corresponding images (A3,C3,D3) . In the WT, no specific HA-labeling (B1) was present at the position of Ca v 1.3 labeling (B2,B3) . The weak ‘cloudy’ green anti-HA staining was present in all specimen investigated and therefore considered unspecific. Cell nuclei of IHCs were counterstained with DAPI (blue). 1 of 3 ( A , age: 2–3 months), 1 of 4 ( B , age: P25 – 3 month), 1 of 5 ( C , P25–P31) and 1 of 5 ( D , P28–P37) independent experiments is illustrated, respectively. Scale bars: 5 μm.

    Techniques Used: Labeling, Immunolabeling, Mouse Assay, Staining

    Activation and inactivation properties of I Ca through mCav1.3 L and mCav1.3 L -HA channels. (A) α1-subunits were heterologously expressed in tsA-201 cells together with β 3 and α 2 δ 1 (at least three independent transfections). Whole-cell patch-clamp current–voltage relationship obtained by depolarizations from a Vh of -80 mV to the indicated test potentials in cells transfected with mouse wild-type (WT) Cav1.3 (mCav1.3 L , black) and mCav1.3 L -HA (blue). All data were junction potential – corrected. (B) Percent I Ca inactivation (15 mM Ca 2+ ) during a test pulse from -80 mV to the V max . ∗∗∗ p
    Figure Legend Snippet: Activation and inactivation properties of I Ca through mCav1.3 L and mCav1.3 L -HA channels. (A) α1-subunits were heterologously expressed in tsA-201 cells together with β 3 and α 2 δ 1 (at least three independent transfections). Whole-cell patch-clamp current–voltage relationship obtained by depolarizations from a Vh of -80 mV to the indicated test potentials in cells transfected with mouse wild-type (WT) Cav1.3 (mCav1.3 L , black) and mCav1.3 L -HA (blue). All data were junction potential – corrected. (B) Percent I Ca inactivation (15 mM Ca 2+ ) during a test pulse from -80 mV to the V max . ∗∗∗ p

    Techniques Used: Activation Assay, Transfection, Patch Clamp

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    Alomone Labs rabbit anti cav1 3
    RS1 augments the current density of <t>Cav1.3-LTCC</t> in HEK cells. (A) Cells transfected with Cav1.3 subunit without other LTCC auxiliary subunits (Cav1.3 + EGFP) do not have functional LTCCs. Co-transfection with Cav1.3 and RS1 do not elicit LTCC currents carried by Ba 2+ ( I Ba ). (B) Cells transfected with Cav1.3 and β2 (Cav1.3 + β2 + EGFP), or Cav1.3, β2, and α2δ1 (Cav1.3 + β2 + α2δ1 + EGFP) display functional Cav1.3-LTCC currents. RS1 significantly enhances Cav1.3-LTCC when co-transfected with functional Cav1.3-LTCC (Cav1.3 + β2 + RS1, or Cav1.3 + β2 + α2δ1 + RS1). (C) The maximal current densities ( I Ba ) elicited at −20 mV are (in pA/pF): −9.22 ± 1.20 for EGFP (Cav1.3 + β2 + EGFP), −16.51 ± 1.27 for RS1 (Cav1.3 + β2 + RS1), −12.58 ± 1.40 for α2δ1 + EGFP (Cav1.3 + β2 + α2δ1 + EGFP), and −18.06 ± 1.13 for α2δ1 + RS1 (Cav1.3 + β2 + α2δ1 + RS1). *Indicates a significant difference between the groups (* p
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    RS1 augments the current density of Cav1.3-LTCC in HEK cells. (A) Cells transfected with Cav1.3 subunit without other LTCC auxiliary subunits (Cav1.3 + EGFP) do not have functional LTCCs. Co-transfection with Cav1.3 and RS1 do not elicit LTCC currents carried by Ba 2+ ( I Ba ). (B) Cells transfected with Cav1.3 and β2 (Cav1.3 + β2 + EGFP), or Cav1.3, β2, and α2δ1 (Cav1.3 + β2 + α2δ1 + EGFP) display functional Cav1.3-LTCC currents. RS1 significantly enhances Cav1.3-LTCC when co-transfected with functional Cav1.3-LTCC (Cav1.3 + β2 + RS1, or Cav1.3 + β2 + α2δ1 + RS1). (C) The maximal current densities ( I Ba ) elicited at −20 mV are (in pA/pF): −9.22 ± 1.20 for EGFP (Cav1.3 + β2 + EGFP), −16.51 ± 1.27 for RS1 (Cav1.3 + β2 + RS1), −12.58 ± 1.40 for α2δ1 + EGFP (Cav1.3 + β2 + α2δ1 + EGFP), and −18.06 ± 1.13 for α2δ1 + RS1 (Cav1.3 + β2 + α2δ1 + RS1). *Indicates a significant difference between the groups (* p

    Journal: Frontiers in Cellular Neuroscience

    Article Title: Retinoschisin Facilitates the Function of L-Type Voltage-Gated Calcium Channels

    doi: 10.3389/fncel.2017.00232

    Figure Lengend Snippet: RS1 augments the current density of Cav1.3-LTCC in HEK cells. (A) Cells transfected with Cav1.3 subunit without other LTCC auxiliary subunits (Cav1.3 + EGFP) do not have functional LTCCs. Co-transfection with Cav1.3 and RS1 do not elicit LTCC currents carried by Ba 2+ ( I Ba ). (B) Cells transfected with Cav1.3 and β2 (Cav1.3 + β2 + EGFP), or Cav1.3, β2, and α2δ1 (Cav1.3 + β2 + α2δ1 + EGFP) display functional Cav1.3-LTCC currents. RS1 significantly enhances Cav1.3-LTCC when co-transfected with functional Cav1.3-LTCC (Cav1.3 + β2 + RS1, or Cav1.3 + β2 + α2δ1 + RS1). (C) The maximal current densities ( I Ba ) elicited at −20 mV are (in pA/pF): −9.22 ± 1.20 for EGFP (Cav1.3 + β2 + EGFP), −16.51 ± 1.27 for RS1 (Cav1.3 + β2 + RS1), −12.58 ± 1.40 for α2δ1 + EGFP (Cav1.3 + β2 + α2δ1 + EGFP), and −18.06 ± 1.13 for α2δ1 + RS1 (Cav1.3 + β2 + α2δ1 + RS1). *Indicates a significant difference between the groups (* p

    Article Snippet: The primary antibodies used were rabbit anti-Cav1.3 (1:100; Alomone), mouse anti-Ribeye (1:100; EMD Millipore, Billerica, MA, USA), rabbit anti-Cav1.4 (1:1000; generated in Amy Lee’s laboratory) and rabbit anti-RS1 (1:100; Santa Cruz Biotechnology, Dallas, TX, USA).

    Techniques: Transfection, Functional Assay, Cotransfection

    Deletion of RS1 decreases the protein expression of Cav1.3 and Cav1.4. (A) The upper panel (a1-c5) contains retinal sections of wild type (WT), and the lower panel (d1-f5) contains retinal sections of RS1 −/− . (a1-a2) and (d1-d2) are the immunostaining for RS1. (b1-b5) and (e1-e5) are the double immunostaining for Cav1.3 and Ribeye; (c1-c5) and (f1-f5) are the double immunostaining for Cav1.4 and Ribeye. The scale bar represents 50 μm. (B) The same immunostained retinal sections are shown at a higher magnification (40×). The upper panel contains retinal sections from WT (a1-a5) and RS1 −/− (b1-b5) that were double-stained for Cav1.3 and Ribeye. Images in (a1-a5) and (b1-b5) include retinal layers of IS, ONL, OPL and INL. The lower panel contains retinal sections from WT (c1-c5) and RS1 −/− (d1-d5) that were double-stained for Cav1.4 and Ribeye. Images in (c1-c5) and (d1-d5) include retinal layers of ONL, OPL and INL. The scale bar represents 50 μm. 4′ s ,6-diamidino-2-phenylindole (DAPI) stains the nuclei. BF, bright field; IS, photoreceptor inner segments; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer.

    Journal: Frontiers in Cellular Neuroscience

    Article Title: Retinoschisin Facilitates the Function of L-Type Voltage-Gated Calcium Channels

    doi: 10.3389/fncel.2017.00232

    Figure Lengend Snippet: Deletion of RS1 decreases the protein expression of Cav1.3 and Cav1.4. (A) The upper panel (a1-c5) contains retinal sections of wild type (WT), and the lower panel (d1-f5) contains retinal sections of RS1 −/− . (a1-a2) and (d1-d2) are the immunostaining for RS1. (b1-b5) and (e1-e5) are the double immunostaining for Cav1.3 and Ribeye; (c1-c5) and (f1-f5) are the double immunostaining for Cav1.4 and Ribeye. The scale bar represents 50 μm. (B) The same immunostained retinal sections are shown at a higher magnification (40×). The upper panel contains retinal sections from WT (a1-a5) and RS1 −/− (b1-b5) that were double-stained for Cav1.3 and Ribeye. Images in (a1-a5) and (b1-b5) include retinal layers of IS, ONL, OPL and INL. The lower panel contains retinal sections from WT (c1-c5) and RS1 −/− (d1-d5) that were double-stained for Cav1.4 and Ribeye. Images in (c1-c5) and (d1-d5) include retinal layers of ONL, OPL and INL. The scale bar represents 50 μm. 4′ s ,6-diamidino-2-phenylindole (DAPI) stains the nuclei. BF, bright field; IS, photoreceptor inner segments; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer.

    Article Snippet: The primary antibodies used were rabbit anti-Cav1.3 (1:100; Alomone), mouse anti-Ribeye (1:100; EMD Millipore, Billerica, MA, USA), rabbit anti-Cav1.4 (1:1000; generated in Amy Lee’s laboratory) and rabbit anti-RS1 (1:100; Santa Cruz Biotechnology, Dallas, TX, USA).

    Techniques: Expressing, Immunostaining, Double Immunostaining, Staining

    Deletion of Cav1.3 decreases RS1 distribution in the retina. (A) Images were taken at a lower magnification (20×) of WT (a1-a3, b1-b3) and Cav1.3 −/− (c1-c3, d1-d3). Retinal sections were immunostained for Cav1.3 (a1-a3, c1-c3) and RS1 (b1-b3, d1-d3). The scale bar represents 50 μm. (B) Images were taken at a higher magnification (40×) of WT (a1-a3, c1-c3) and Cav1.3 −/− (b1-b3, d1-d3). Retinal sections were immunostained for RS1. The scale bar represents 50 μm. DAPI stains the nuclei. BF, bright field; IS, photoreceptor inner segments; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer.

    Journal: Frontiers in Cellular Neuroscience

    Article Title: Retinoschisin Facilitates the Function of L-Type Voltage-Gated Calcium Channels

    doi: 10.3389/fncel.2017.00232

    Figure Lengend Snippet: Deletion of Cav1.3 decreases RS1 distribution in the retina. (A) Images were taken at a lower magnification (20×) of WT (a1-a3, b1-b3) and Cav1.3 −/− (c1-c3, d1-d3). Retinal sections were immunostained for Cav1.3 (a1-a3, c1-c3) and RS1 (b1-b3, d1-d3). The scale bar represents 50 μm. (B) Images were taken at a higher magnification (40×) of WT (a1-a3, c1-c3) and Cav1.3 −/− (b1-b3, d1-d3). Retinal sections were immunostained for RS1. The scale bar represents 50 μm. DAPI stains the nuclei. BF, bright field; IS, photoreceptor inner segments; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer.

    Article Snippet: The primary antibodies used were rabbit anti-Cav1.3 (1:100; Alomone), mouse anti-Ribeye (1:100; EMD Millipore, Billerica, MA, USA), rabbit anti-Cav1.4 (1:1000; generated in Amy Lee’s laboratory) and rabbit anti-RS1 (1:100; Santa Cruz Biotechnology, Dallas, TX, USA).

    Techniques:

    There is a physical interaction between retinoschisin (RS1) and L-type voltage-gated calcium channel (LTCC)α1 subunits. (A) Anti-RS1 antibody (RS1 Ab) is able to co-immunoprecipitate Cav1.3 from the porcine retina. (B) RS1 Ab is able to co-immunoprecipitate Cav1.4 from the porcine retina. (C) The whole cell lysates as loading control for (A,B) . (D) Mammalian two-hybrid (luciferase reporter) assays show that hRS1 is able to interact with the first 500 amino acids from the N-terminus of Cav1.4 (hCav1.4-N) including the first motif (I). Cells co-transfected with hRS1 and hCav1.4-N (hRS1 + hCav1.4-N) have significantly higher luciferase activities than the other two control groups ( n = 6 for each group, * p

    Journal: Frontiers in Cellular Neuroscience

    Article Title: Retinoschisin Facilitates the Function of L-Type Voltage-Gated Calcium Channels

    doi: 10.3389/fncel.2017.00232

    Figure Lengend Snippet: There is a physical interaction between retinoschisin (RS1) and L-type voltage-gated calcium channel (LTCC)α1 subunits. (A) Anti-RS1 antibody (RS1 Ab) is able to co-immunoprecipitate Cav1.3 from the porcine retina. (B) RS1 Ab is able to co-immunoprecipitate Cav1.4 from the porcine retina. (C) The whole cell lysates as loading control for (A,B) . (D) Mammalian two-hybrid (luciferase reporter) assays show that hRS1 is able to interact with the first 500 amino acids from the N-terminus of Cav1.4 (hCav1.4-N) including the first motif (I). Cells co-transfected with hRS1 and hCav1.4-N (hRS1 + hCav1.4-N) have significantly higher luciferase activities than the other two control groups ( n = 6 for each group, * p

    Article Snippet: The primary antibodies used were rabbit anti-Cav1.3 (1:100; Alomone), mouse anti-Ribeye (1:100; EMD Millipore, Billerica, MA, USA), rabbit anti-Cav1.4 (1:1000; generated in Amy Lee’s laboratory) and rabbit anti-RS1 (1:100; Santa Cruz Biotechnology, Dallas, TX, USA).

    Techniques: Luciferase, Transfection

    Western blot analysis of α1-subunits in WT and mutant mouse brain homogenates. (A) Proteins (100 μg/lane) were separated on 5% SDS page and immunostained with polyclonal antibody Cav1.3 α1 CT . The Cav1.3 α1 subunit was specifically detected as a 231 kDa protein at expression levels indistinguishable between WT and homozygous mutants (HA) (see Results). KO, Cav1.3 -/- negative control. (B) Same separation as in A (100 μg/lane) but detection with anti-Cav1.3α1 NT . The migration of recombinant mCav1.3 L (L) and mCav1.343 S (43S) on the same gel (not shown) and their calculated molecular mass are indicated by arrows (left). Migration of molecular mass standards as well as the brain long and short α1-subunit species are also indicated (right). An unspecific ∼120 kDa band served as loading control. One representative experiment of at least three independent experiments is shown for all panels. KO, Cav1.3 -/- ; HA, Cav1.3DCRD HA/HA , WT, wild-type littermate.

    Journal: Frontiers in Cellular Neuroscience

    Article Title: Cell-type-specific tuning of Cav1.3 Ca2+-channels by a C-terminal automodulatory domain

    doi: 10.3389/fncel.2015.00309

    Figure Lengend Snippet: Western blot analysis of α1-subunits in WT and mutant mouse brain homogenates. (A) Proteins (100 μg/lane) were separated on 5% SDS page and immunostained with polyclonal antibody Cav1.3 α1 CT . The Cav1.3 α1 subunit was specifically detected as a 231 kDa protein at expression levels indistinguishable between WT and homozygous mutants (HA) (see Results). KO, Cav1.3 -/- negative control. (B) Same separation as in A (100 μg/lane) but detection with anti-Cav1.3α1 NT . The migration of recombinant mCav1.3 L (L) and mCav1.343 S (43S) on the same gel (not shown) and their calculated molecular mass are indicated by arrows (left). Migration of molecular mass standards as well as the brain long and short α1-subunit species are also indicated (right). An unspecific ∼120 kDa band served as loading control. One representative experiment of at least three independent experiments is shown for all panels. KO, Cav1.3 -/- ; HA, Cav1.3DCRD HA/HA , WT, wild-type littermate.

    Article Snippet: The close corresponding staining of anti-Cav1.3 and anti-HA suggests that in adult IHCs all Cav1.3 clusters contain long Cav1.3 ( Figure ).

    Techniques: Western Blot, Mutagenesis, SDS Page, Expressing, Negative Control, Migration, Recombinant

    Cav1.3 α1 transcripts containing exons 43 S and 43 L in mouse IHCs and OHCs, at P6 and P22 using nested PCR. Fragments containing 43S (403 bp) or 43L (557 bp) were amplified using nested PCR (see Materials and Methods) with primers specific for exon 42 (forward) and 45 (reverse) of mouse Cav1.3. S1–S14 represent samples from independent preparations. For each cell type and developmental stage at least three independent experiments were performed. Whole brain (WB) and heart (WH) served as positive controls, H 2 O (no template) as negative control. Specificity of PCR products was confirmed by sequencing. When two independent PCR reactions with three different RNA samples of each cell type were performed, the number of successful detections for each transcript was as follows: detection of 43L: 6 (out of six experiments) in IHC and OHC preparations of all developmental stages; detection of 43S: 4 (6) in IHC P06 and IHC P22, 6 (6) in OHC P06 and 5 (6) in OHC P22. Bp, basepair markers.

    Journal: Frontiers in Cellular Neuroscience

    Article Title: Cell-type-specific tuning of Cav1.3 Ca2+-channels by a C-terminal automodulatory domain

    doi: 10.3389/fncel.2015.00309

    Figure Lengend Snippet: Cav1.3 α1 transcripts containing exons 43 S and 43 L in mouse IHCs and OHCs, at P6 and P22 using nested PCR. Fragments containing 43S (403 bp) or 43L (557 bp) were amplified using nested PCR (see Materials and Methods) with primers specific for exon 42 (forward) and 45 (reverse) of mouse Cav1.3. S1–S14 represent samples from independent preparations. For each cell type and developmental stage at least three independent experiments were performed. Whole brain (WB) and heart (WH) served as positive controls, H 2 O (no template) as negative control. Specificity of PCR products was confirmed by sequencing. When two independent PCR reactions with three different RNA samples of each cell type were performed, the number of successful detections for each transcript was as follows: detection of 43L: 6 (out of six experiments) in IHC and OHC preparations of all developmental stages; detection of 43S: 4 (6) in IHC P06 and IHC P22, 6 (6) in OHC P06 and 5 (6) in OHC P22. Bp, basepair markers.

    Article Snippet: The close corresponding staining of anti-Cav1.3 and anti-HA suggests that in adult IHCs all Cav1.3 clusters contain long Cav1.3 ( Figure ).

    Techniques: Nested PCR, Amplification, Western Blot, Negative Control, Polymerase Chain Reaction, Sequencing, Immunohistochemistry

    Absence of smaller C-terminally–derived Cav1.3 α1 fragments in WT and Cav1.3DCRD HA/HA brain preparations. (A) Mouse brain homogenate (100 μg of protein/lane) prepared from WT or Cav1.3DCRD HA/HA (HA) mice were separated on 4–15% gradient SDS-PAGE and immunostained with anti-HA antibody. The blot was overexposed to also visualize less abundant smaller fragments. in separate experiments α1- associated HA-immunoreactivity could be detected with only 10% (10 μg/lane) of the protein amount used ( n = 3) demonstrating the sensitivity of the assay. (B) Mouse brain membranes (100 μg of protein/lane) were analyzed as in (A). (C) Mouse brain membranes (100 μg of protein/lane) from WT or Cav1.3 -/- (KO) mice were blotted as in (B) and stained with anti-Cav1.3α1 CT antibodies. To some WT samples (33 μg/lane) a 45 kDa recombinant C-terminal control peptide was added (arrow, amounts indicated) before separation to demonstrate successful transfer and sensitive detection as a positive control for sensitivity.

    Journal: Frontiers in Cellular Neuroscience

    Article Title: Cell-type-specific tuning of Cav1.3 Ca2+-channels by a C-terminal automodulatory domain

    doi: 10.3389/fncel.2015.00309

    Figure Lengend Snippet: Absence of smaller C-terminally–derived Cav1.3 α1 fragments in WT and Cav1.3DCRD HA/HA brain preparations. (A) Mouse brain homogenate (100 μg of protein/lane) prepared from WT or Cav1.3DCRD HA/HA (HA) mice were separated on 4–15% gradient SDS-PAGE and immunostained with anti-HA antibody. The blot was overexposed to also visualize less abundant smaller fragments. in separate experiments α1- associated HA-immunoreactivity could be detected with only 10% (10 μg/lane) of the protein amount used ( n = 3) demonstrating the sensitivity of the assay. (B) Mouse brain membranes (100 μg of protein/lane) were analyzed as in (A). (C) Mouse brain membranes (100 μg of protein/lane) from WT or Cav1.3 -/- (KO) mice were blotted as in (B) and stained with anti-Cav1.3α1 CT antibodies. To some WT samples (33 μg/lane) a 45 kDa recombinant C-terminal control peptide was added (arrow, amounts indicated) before separation to demonstrate successful transfer and sensitive detection as a positive control for sensitivity.

    Article Snippet: The close corresponding staining of anti-Cav1.3 and anti-HA suggests that in adult IHCs all Cav1.3 clusters contain long Cav1.3 ( Figure ).

    Techniques: Derivative Assay, Mouse Assay, SDS Page, Staining, Recombinant, Positive Control

    Comparison of the protein localization of anti-HA-labeled Cav1.3 with immunolabeled Ca v 1.3, Ca v β2 and CtBP2/RIBEYE in IHCs. (A–D) Whole-mount preparations of apical turns of the organ of Corti from adult Ca v 1.3DCRD HA/HA (A,C,D) and WT (B) mice were co-immunolabeled with anti-HA and anti-Ca v 1.3 ( A, B , 11 weeks), anti-HA and anti-Ca v β2 ( C , P28), or anti-HA and anti-CtBP2/RIBEYE antibodies ( D , P37). Every image shows the basolateral poles of two adjacent IHCs the nuclei of which are indicated by asterisks in the rightmost column, respectively. HA staining (A1,C1,D1) largely overlapped with Ca v 1.3 (A2) , Ca v ß2 (C2) and CtBP2 (D2) staining at the basal poles of IHCs as evident upon merging corresponding images (A3,C3,D3) . In the WT, no specific HA-labeling (B1) was present at the position of Ca v 1.3 labeling (B2,B3) . The weak ‘cloudy’ green anti-HA staining was present in all specimen investigated and therefore considered unspecific. Cell nuclei of IHCs were counterstained with DAPI (blue). 1 of 3 ( A , age: 2–3 months), 1 of 4 ( B , age: P25 – 3 month), 1 of 5 ( C , P25–P31) and 1 of 5 ( D , P28–P37) independent experiments is illustrated, respectively. Scale bars: 5 μm.

    Journal: Frontiers in Cellular Neuroscience

    Article Title: Cell-type-specific tuning of Cav1.3 Ca2+-channels by a C-terminal automodulatory domain

    doi: 10.3389/fncel.2015.00309

    Figure Lengend Snippet: Comparison of the protein localization of anti-HA-labeled Cav1.3 with immunolabeled Ca v 1.3, Ca v β2 and CtBP2/RIBEYE in IHCs. (A–D) Whole-mount preparations of apical turns of the organ of Corti from adult Ca v 1.3DCRD HA/HA (A,C,D) and WT (B) mice were co-immunolabeled with anti-HA and anti-Ca v 1.3 ( A, B , 11 weeks), anti-HA and anti-Ca v β2 ( C , P28), or anti-HA and anti-CtBP2/RIBEYE antibodies ( D , P37). Every image shows the basolateral poles of two adjacent IHCs the nuclei of which are indicated by asterisks in the rightmost column, respectively. HA staining (A1,C1,D1) largely overlapped with Ca v 1.3 (A2) , Ca v ß2 (C2) and CtBP2 (D2) staining at the basal poles of IHCs as evident upon merging corresponding images (A3,C3,D3) . In the WT, no specific HA-labeling (B1) was present at the position of Ca v 1.3 labeling (B2,B3) . The weak ‘cloudy’ green anti-HA staining was present in all specimen investigated and therefore considered unspecific. Cell nuclei of IHCs were counterstained with DAPI (blue). 1 of 3 ( A , age: 2–3 months), 1 of 4 ( B , age: P25 – 3 month), 1 of 5 ( C , P25–P31) and 1 of 5 ( D , P28–P37) independent experiments is illustrated, respectively. Scale bars: 5 μm.

    Article Snippet: The close corresponding staining of anti-Cav1.3 and anti-HA suggests that in adult IHCs all Cav1.3 clusters contain long Cav1.3 ( Figure ).

    Techniques: Labeling, Immunolabeling, Mouse Assay, Staining

    Activation and inactivation properties of I Ca through mCav1.3 L and mCav1.3 L -HA channels. (A) α1-subunits were heterologously expressed in tsA-201 cells together with β 3 and α 2 δ 1 (at least three independent transfections). Whole-cell patch-clamp current–voltage relationship obtained by depolarizations from a Vh of -80 mV to the indicated test potentials in cells transfected with mouse wild-type (WT) Cav1.3 (mCav1.3 L , black) and mCav1.3 L -HA (blue). All data were junction potential – corrected. (B) Percent I Ca inactivation (15 mM Ca 2+ ) during a test pulse from -80 mV to the V max . ∗∗∗ p

    Journal: Frontiers in Cellular Neuroscience

    Article Title: Cell-type-specific tuning of Cav1.3 Ca2+-channels by a C-terminal automodulatory domain

    doi: 10.3389/fncel.2015.00309

    Figure Lengend Snippet: Activation and inactivation properties of I Ca through mCav1.3 L and mCav1.3 L -HA channels. (A) α1-subunits were heterologously expressed in tsA-201 cells together with β 3 and α 2 δ 1 (at least three independent transfections). Whole-cell patch-clamp current–voltage relationship obtained by depolarizations from a Vh of -80 mV to the indicated test potentials in cells transfected with mouse wild-type (WT) Cav1.3 (mCav1.3 L , black) and mCav1.3 L -HA (blue). All data were junction potential – corrected. (B) Percent I Ca inactivation (15 mM Ca 2+ ) during a test pulse from -80 mV to the V max . ∗∗∗ p

    Article Snippet: The close corresponding staining of anti-Cav1.3 and anti-HA suggests that in adult IHCs all Cav1.3 clusters contain long Cav1.3 ( Figure ).

    Techniques: Activation Assay, Transfection, Patch Clamp

    A cartoon representing normal and hypothyroid adult IHC synapses. (a) WT IHC. (b) Hypothyroid IHC. (c) Magnification of the box in b. A hypothyroid IHC is characterized by an excess of afferent synapses caused by the abnormal retention of afferent neurite branches and terminals, calcium channels being more widely distributed around the hair cell, and higher glutamate buildup at the synaptic cleft, owing to lower GLAST levels (b and c). In comparison, a normal IHC shows fewer but well-organized afferent terminals and a more clustered pattern of CaV1.3 calcium channel expression at the basal region of the hair cell. a, afferent fibre; e, efferent fibre; r, ribbon synapse.

    Journal: The European journal of neuroscience

    Article Title: Thyroid hormone is required for pruning, functioning and long-term maintenance of afferent inner hair cell synapses

    doi: 10.1111/ejn.13081

    Figure Lengend Snippet: A cartoon representing normal and hypothyroid adult IHC synapses. (a) WT IHC. (b) Hypothyroid IHC. (c) Magnification of the box in b. A hypothyroid IHC is characterized by an excess of afferent synapses caused by the abnormal retention of afferent neurite branches and terminals, calcium channels being more widely distributed around the hair cell, and higher glutamate buildup at the synaptic cleft, owing to lower GLAST levels (b and c). In comparison, a normal IHC shows fewer but well-organized afferent terminals and a more clustered pattern of CaV1.3 calcium channel expression at the basal region of the hair cell. a, afferent fibre; e, efferent fibre; r, ribbon synapse.

    Article Snippet: The following polyclonal primary antibodies were used: goat anti-CtBP2 (1 : 200; Santa Cruz Biotechnology) for RIBEYE, rabbit anti-SHANK1 (1 : 200; Neuromics), rabbit anti-CaV1.3 (1 : 500; Alomone), goat anti-GLAST (1 : 200; Cell Signaling), and rabbit anti-myosin VIIa (1 : 300; Proteus Biosciences).

    Techniques: Immunohistochemistry, Expressing

    Abnormal CaV1.3 puncta clustering at the synapses of Pit1 dw IHCs. (a and b) Projection of confocal sections obtained from the mid-turn of cochlear whole mounts stained with the afferent presynaptic marker RIBEYE (green) and the calcium channel CaV1.3 (red) at P7 in WT mice (a) and Pit1 dw mice (b). (c–f) The same markers at P14 for WT (c) and Pit1 dw mice (d), and at P24 for WT (e) and Pit1 dw mice (f). (g–i) Box plots of the quantification of RIBEYE (g), CaV1.3 (h) and RIBEYE–CaV1.3 (i) puncta from the mid-turn of the cochlea in WT and Pit1 dw mice. significant comparisons ( P

    Journal: The European journal of neuroscience

    Article Title: Thyroid hormone is required for pruning, functioning and long-term maintenance of afferent inner hair cell synapses

    doi: 10.1111/ejn.13081

    Figure Lengend Snippet: Abnormal CaV1.3 puncta clustering at the synapses of Pit1 dw IHCs. (a and b) Projection of confocal sections obtained from the mid-turn of cochlear whole mounts stained with the afferent presynaptic marker RIBEYE (green) and the calcium channel CaV1.3 (red) at P7 in WT mice (a) and Pit1 dw mice (b). (c–f) The same markers at P14 for WT (c) and Pit1 dw mice (d), and at P24 for WT (e) and Pit1 dw mice (f). (g–i) Box plots of the quantification of RIBEYE (g), CaV1.3 (h) and RIBEYE–CaV1.3 (i) puncta from the mid-turn of the cochlea in WT and Pit1 dw mice. significant comparisons ( P

    Article Snippet: The following polyclonal primary antibodies were used: goat anti-CtBP2 (1 : 200; Santa Cruz Biotechnology) for RIBEYE, rabbit anti-SHANK1 (1 : 200; Neuromics), rabbit anti-CaV1.3 (1 : 500; Alomone), goat anti-GLAST (1 : 200; Cell Signaling), and rabbit anti-myosin VIIa (1 : 300; Proteus Biosciences).

    Techniques: Staining, Marker, Mouse Assay

    Cav1 channel deletion compensated by the increased expression of other Cav channel types. Pharmacological dissection of Ca 2+ channels in mouse chromaffin cells from WT and Cav1.3 −/− cells. (a and b) Time course of the Ca 2+ charge density obtained after sequentially and cumulatively adding the different Ca 2+ channel blockers, in WT and Cav1.3 −/− cells, respectively: 3 μM nifedipine was used to block Cav1 channels, 1 μM ω‐CTX‐GVIA to block Cav2.2 channels, 3 μM ω‐CTX‐MVIIC to block Cav2.1 channels, and 200 μM Cd 2+ to block the residual Ca 2+ current. (c and d) Original traces of the Ca 2+ currents recorded at the stationary stage using each Ca 2+ channel blocker (corresponding to points a–f in panels 3a and b, where a : control, b : after 3 μM nifedipine perfusion, c : after 3 μM nifedipine and 1 μM ω‐CTX‐GVIA perfusion and d : after 3 μM nifedipine, ω‐CTX‐GVIA and 3 μM ω‐CTX‐MVIIC perfusion). (e) Ca 2+ charge density of the different Ca 2+ channel types for WT (black columns) and Cav1.3 −/− cells (white columns), respectively. (f) Total Ca 2+ charge obtained under control conditions for WT (black column) and Cav1.3 −/− cells (white column). (g) Sizes of chromaffin cells obtained from WT (black column) and Cav1.3 −/− mice (white column). Experiments were performed on nine paired cultures of WT ( n = 18 cells) and Cav1.3 −/− cells ( n = 17 cells), using 1–2 mice of each strain. Bars represent means ± SEM. ** p

    Journal: Journal of Neurochemistry

    Article Title: Different roles attributed to Cav1 channel subtypes in spontaneous action potential firing and fine tuning of exocytosis in mouse chromaffin cells

    doi: 10.1111/j.1471-4159.2010.07089.x

    Figure Lengend Snippet: Cav1 channel deletion compensated by the increased expression of other Cav channel types. Pharmacological dissection of Ca 2+ channels in mouse chromaffin cells from WT and Cav1.3 −/− cells. (a and b) Time course of the Ca 2+ charge density obtained after sequentially and cumulatively adding the different Ca 2+ channel blockers, in WT and Cav1.3 −/− cells, respectively: 3 μM nifedipine was used to block Cav1 channels, 1 μM ω‐CTX‐GVIA to block Cav2.2 channels, 3 μM ω‐CTX‐MVIIC to block Cav2.1 channels, and 200 μM Cd 2+ to block the residual Ca 2+ current. (c and d) Original traces of the Ca 2+ currents recorded at the stationary stage using each Ca 2+ channel blocker (corresponding to points a–f in panels 3a and b, where a : control, b : after 3 μM nifedipine perfusion, c : after 3 μM nifedipine and 1 μM ω‐CTX‐GVIA perfusion and d : after 3 μM nifedipine, ω‐CTX‐GVIA and 3 μM ω‐CTX‐MVIIC perfusion). (e) Ca 2+ charge density of the different Ca 2+ channel types for WT (black columns) and Cav1.3 −/− cells (white columns), respectively. (f) Total Ca 2+ charge obtained under control conditions for WT (black column) and Cav1.3 −/− cells (white column). (g) Sizes of chromaffin cells obtained from WT (black column) and Cav1.3 −/− mice (white column). Experiments were performed on nine paired cultures of WT ( n = 18 cells) and Cav1.3 −/− cells ( n = 17 cells), using 1–2 mice of each strain. Bars represent means ± SEM. ** p

    Article Snippet: The primary antibodies (dilution 1 : 200) were goat polyclonal anti‐Cav1.1 and anti‐Cav1.4 (Santa Cruz Biotechnology, Heidelberg, Germany), and rabbit polyclonal anti‐Cav1.2 and anti‐Cav1.3 (Alomone Labs, Jerusalem, Israel).

    Techniques: Expressing, Dissection, Blocking Assay, Mouse Assay

    Contribution of Cav1 channel subtypes to pacemaking activity, and shaping of action potential waveform. (a–b) Recordings of the spontaneous firing of action potentials performed in the current clamp configuration in WT (a) or Cav1.3 −/− cells (b) under control conditions, and after perfusion with 300 nM nifedipine and 3 μM nifedipine. (c–d) Mean action potential obtained by averaging the action potentials recorded over 10 s under control conditions or after 300 nM and 3 μM nifedipine application to WT ( n = 6) (c) or Cav1.3 −/− cells ( n = 4) (d). Data were obtained in two paired cultures of WT and Cav1.3 −/− cells, using 1–2 mice of each strain.

    Journal: Journal of Neurochemistry

    Article Title: Different roles attributed to Cav1 channel subtypes in spontaneous action potential firing and fine tuning of exocytosis in mouse chromaffin cells

    doi: 10.1111/j.1471-4159.2010.07089.x

    Figure Lengend Snippet: Contribution of Cav1 channel subtypes to pacemaking activity, and shaping of action potential waveform. (a–b) Recordings of the spontaneous firing of action potentials performed in the current clamp configuration in WT (a) or Cav1.3 −/− cells (b) under control conditions, and after perfusion with 300 nM nifedipine and 3 μM nifedipine. (c–d) Mean action potential obtained by averaging the action potentials recorded over 10 s under control conditions or after 300 nM and 3 μM nifedipine application to WT ( n = 6) (c) or Cav1.3 −/− cells ( n = 4) (d). Data were obtained in two paired cultures of WT and Cav1.3 −/− cells, using 1–2 mice of each strain.

    Article Snippet: The primary antibodies (dilution 1 : 200) were goat polyclonal anti‐Cav1.1 and anti‐Cav1.4 (Santa Cruz Biotechnology, Heidelberg, Germany), and rabbit polyclonal anti‐Cav1.2 and anti‐Cav1.3 (Alomone Labs, Jerusalem, Israel).

    Techniques: Activity Assay, Mouse Assay

    Contribution of Cav1 channel subtypes to pacemaking activity. (a–b) Phase‐plane plot obtained by plotting dV/dt versus the voltage stimulus in WT and Cav1.3 −/− , respectively. Arrows indicate the points at which dV/dt increased from the initial baseline (threshold potential). Estimated threshold potentials were −27 mV and −22 mV for WT and Cav1.3 −/− cells, respectively; (c–d) action potential clamp experiments were performed by applying the mean voltage stimulus obtained under control conditions in Fig. 8(g and h) every 30 s. Starting from ‘Solution 3’ (see Material and Methods ), different blockers were sequentially added to that solution: 2 μM TTX (Solution 3 + TTX), 45 mM TEA (Solution 3 + TTX + TEA), 3 μM nifedipine (Solution 3 + TTX + TEA + Nife) and 200 μM CdCl 2 (Solution 3 + TTX + TEA + Nife + Cd). Perfusion with each solution was continued for at least 2 min so that the currents reached the steady‐state. Number of cell: 22 WT cells, 25 Cav1.3 −/− cells. Data were obtained in four paired cultures of WT and Cav1.3 −/− cells, using two mice of each strain. (e–f) Ion currents were calculated from the recordings of panels (c) and (d), for WT and Cav1.3 −/− cells, respectively. To obtain the Na + current, the current obtained after perfusion with ‘Solution 3 + TTX’ was subtracted from the ‘Solution 3’ current. The K + current was calculated as the difference in the current yielded under ‘Solution 3 + TTX + TEA’ minus ‘Solution 3 + TTX’. The Cav1 current was obtained as the difference between ‘Solution 3 + TTX + TEA’ and ‘Solution 3 + TTX + TEA + Nife’ and the total Ca 2+ current as the difference between ‘Solution 3 + TTX + TEA’ and ‘Solution 3 + TTX + TEA + Cd’. The voltage stimulus, obtained from Fig. 8(g and h) , control conditions, was superimposed on the ion currents. Data were obtained in 4 paired cultures of WT and Cav1.3 −/− cells, using two mice of each strain.

    Journal: Journal of Neurochemistry

    Article Title: Different roles attributed to Cav1 channel subtypes in spontaneous action potential firing and fine tuning of exocytosis in mouse chromaffin cells

    doi: 10.1111/j.1471-4159.2010.07089.x

    Figure Lengend Snippet: Contribution of Cav1 channel subtypes to pacemaking activity. (a–b) Phase‐plane plot obtained by plotting dV/dt versus the voltage stimulus in WT and Cav1.3 −/− , respectively. Arrows indicate the points at which dV/dt increased from the initial baseline (threshold potential). Estimated threshold potentials were −27 mV and −22 mV for WT and Cav1.3 −/− cells, respectively; (c–d) action potential clamp experiments were performed by applying the mean voltage stimulus obtained under control conditions in Fig. 8(g and h) every 30 s. Starting from ‘Solution 3’ (see Material and Methods ), different blockers were sequentially added to that solution: 2 μM TTX (Solution 3 + TTX), 45 mM TEA (Solution 3 + TTX + TEA), 3 μM nifedipine (Solution 3 + TTX + TEA + Nife) and 200 μM CdCl 2 (Solution 3 + TTX + TEA + Nife + Cd). Perfusion with each solution was continued for at least 2 min so that the currents reached the steady‐state. Number of cell: 22 WT cells, 25 Cav1.3 −/− cells. Data were obtained in four paired cultures of WT and Cav1.3 −/− cells, using two mice of each strain. (e–f) Ion currents were calculated from the recordings of panels (c) and (d), for WT and Cav1.3 −/− cells, respectively. To obtain the Na + current, the current obtained after perfusion with ‘Solution 3 + TTX’ was subtracted from the ‘Solution 3’ current. The K + current was calculated as the difference in the current yielded under ‘Solution 3 + TTX + TEA’ minus ‘Solution 3 + TTX’. The Cav1 current was obtained as the difference between ‘Solution 3 + TTX + TEA’ and ‘Solution 3 + TTX + TEA + Nife’ and the total Ca 2+ current as the difference between ‘Solution 3 + TTX + TEA’ and ‘Solution 3 + TTX + TEA + Cd’. The voltage stimulus, obtained from Fig. 8(g and h) , control conditions, was superimposed on the ion currents. Data were obtained in 4 paired cultures of WT and Cav1.3 −/− cells, using two mice of each strain.

    Article Snippet: The primary antibodies (dilution 1 : 200) were goat polyclonal anti‐Cav1.1 and anti‐Cav1.4 (Santa Cruz Biotechnology, Heidelberg, Germany), and rabbit polyclonal anti‐Cav1.2 and anti‐Cav1.3 (Alomone Labs, Jerusalem, Israel).

    Techniques: Activity Assay, Mouse Assay

    Cav1 channel subtypes expressed in mouse chromaffin cells. Sensitivity of Cav1 channel subtypes to DHPs. Square‐step depolarizing pulses of 50 ms duration were applied every 30 s to the peak current voltage. (a, c and e) Ca 2+ charge density blockade obtained after perfusion with 300 nM (black columns) and 3 μM (white columns) nifedipine (Nife) in WT, Cav1.3 −/− and Cav1.2DHP −/− cells, respectively. A large fraction of Cav1.3 −/− cells ( n = 21) did not respond to 300 nM nifedipine. (b, d and f) Original Ca 2+ current traces under control conditions or after perfusion with 300 nM or 3 μM nifedipine in WT, Cav1.3 −/− and Cav1.2DHP −/− cells, respectively. Experiments were performed on seven paired cultures of WT and Cav1.3 −/− cells and five paired cultures of WT and Cav1.2DHP −/− cells, using two mice from each strain. Numbers of cells indicated in parentheses. Bars represent means ± SEM. *** p

    Journal: Journal of Neurochemistry

    Article Title: Different roles attributed to Cav1 channel subtypes in spontaneous action potential firing and fine tuning of exocytosis in mouse chromaffin cells

    doi: 10.1111/j.1471-4159.2010.07089.x

    Figure Lengend Snippet: Cav1 channel subtypes expressed in mouse chromaffin cells. Sensitivity of Cav1 channel subtypes to DHPs. Square‐step depolarizing pulses of 50 ms duration were applied every 30 s to the peak current voltage. (a, c and e) Ca 2+ charge density blockade obtained after perfusion with 300 nM (black columns) and 3 μM (white columns) nifedipine (Nife) in WT, Cav1.3 −/− and Cav1.2DHP −/− cells, respectively. A large fraction of Cav1.3 −/− cells ( n = 21) did not respond to 300 nM nifedipine. (b, d and f) Original Ca 2+ current traces under control conditions or after perfusion with 300 nM or 3 μM nifedipine in WT, Cav1.3 −/− and Cav1.2DHP −/− cells, respectively. Experiments were performed on seven paired cultures of WT and Cav1.3 −/− cells and five paired cultures of WT and Cav1.2DHP −/− cells, using two mice from each strain. Numbers of cells indicated in parentheses. Bars represent means ± SEM. *** p

    Article Snippet: The primary antibodies (dilution 1 : 200) were goat polyclonal anti‐Cav1.1 and anti‐Cav1.4 (Santa Cruz Biotechnology, Heidelberg, Germany), and rabbit polyclonal anti‐Cav1.2 and anti‐Cav1.3 (Alomone Labs, Jerusalem, Israel).

    Techniques: Mouse Assay

    Voltage dependent activation of Ca 2+ channels. (a) I – V curves obtained under control conditions in WT and Cav1.3 −/− cells ( n = 8–12). 200 ms square‐step depolarizing pulses at increasing potentials (voltage increments of 10 mV), from −50 mV to 80 mV, were applied every 1 min. Data were obtained from four paired cultures of WT and Cav1.3 −/− cells, using 1–2 mice from each strain and normalized as the percentage of current in control conditions at 10 mV, plotted as the mean ± SEM. * p

    Journal: Journal of Neurochemistry

    Article Title: Different roles attributed to Cav1 channel subtypes in spontaneous action potential firing and fine tuning of exocytosis in mouse chromaffin cells

    doi: 10.1111/j.1471-4159.2010.07089.x

    Figure Lengend Snippet: Voltage dependent activation of Ca 2+ channels. (a) I – V curves obtained under control conditions in WT and Cav1.3 −/− cells ( n = 8–12). 200 ms square‐step depolarizing pulses at increasing potentials (voltage increments of 10 mV), from −50 mV to 80 mV, were applied every 1 min. Data were obtained from four paired cultures of WT and Cav1.3 −/− cells, using 1–2 mice from each strain and normalized as the percentage of current in control conditions at 10 mV, plotted as the mean ± SEM. * p

    Article Snippet: The primary antibodies (dilution 1 : 200) were goat polyclonal anti‐Cav1.1 and anti‐Cav1.4 (Santa Cruz Biotechnology, Heidelberg, Germany), and rabbit polyclonal anti‐Cav1.2 and anti‐Cav1.3 (Alomone Labs, Jerusalem, Israel).

    Techniques: Activation Assay, Mouse Assay

    Cav1 channel deletion compensated by the increased expression of other Ca 2+ channel types. Contribution of Cav channels to the exocytosis of neurotransmitters in mouse chromaffin cells from WT and Cav1.3 −/− mice. (a and b) C m traces recorded simultaneously to the Ca 2+ currents of Fig. 2(c and d) in WT and Cav1.3 −/− cells, respectively. (c) Percentage of total secretion attributed to each Ca 2+ channel type in WT (black columns) and Cav1.3 −/− cells (white columns). (d) Total secretion attained under control conditions for WT (black column) and Cav1.3 −/− cells (white column). Experiments were performed on seven paired cultures of WT ( n = 15 cells) and Cav1.3 −/− cells ( n = 11 cells), using 1–2 mice from each strain. Bars represent means ± SEM. * p

    Journal: Journal of Neurochemistry

    Article Title: Different roles attributed to Cav1 channel subtypes in spontaneous action potential firing and fine tuning of exocytosis in mouse chromaffin cells

    doi: 10.1111/j.1471-4159.2010.07089.x

    Figure Lengend Snippet: Cav1 channel deletion compensated by the increased expression of other Ca 2+ channel types. Contribution of Cav channels to the exocytosis of neurotransmitters in mouse chromaffin cells from WT and Cav1.3 −/− mice. (a and b) C m traces recorded simultaneously to the Ca 2+ currents of Fig. 2(c and d) in WT and Cav1.3 −/− cells, respectively. (c) Percentage of total secretion attributed to each Ca 2+ channel type in WT (black columns) and Cav1.3 −/− cells (white columns). (d) Total secretion attained under control conditions for WT (black column) and Cav1.3 −/− cells (white column). Experiments were performed on seven paired cultures of WT ( n = 15 cells) and Cav1.3 −/− cells ( n = 11 cells), using 1–2 mice from each strain. Bars represent means ± SEM. * p

    Article Snippet: The primary antibodies (dilution 1 : 200) were goat polyclonal anti‐Cav1.1 and anti‐Cav1.4 (Santa Cruz Biotechnology, Heidelberg, Germany), and rabbit polyclonal anti‐Cav1.2 and anti‐Cav1.3 (Alomone Labs, Jerusalem, Israel).

    Techniques: Expressing, Mouse Assay

    Contribution of Cav1 channel subtypes to the Ca 2+ charge and exocytosis of chromaffin vesicles. Ca 2+ charge density (a) and the corresponding exocytosis (b) versus the voltage in WT and Cav1.3 −/− cells. Data were obtained in the same experiments as in Fig. 3 ( n = 8–12, from four paired cultures of WT and Cav1.3 −/− cells, using 1–2 mice of each strain) and normalized as the percentage of charge density in control conditions at 10 mV, plotted as the mean ± SEM. (c) Original traces of Ca 2+ current density and the corresponding exocytosis elicited at −20 mV, −10 mV and 0 mV in WT and Cav1.3 −/− cells. * p

    Journal: Journal of Neurochemistry

    Article Title: Different roles attributed to Cav1 channel subtypes in spontaneous action potential firing and fine tuning of exocytosis in mouse chromaffin cells

    doi: 10.1111/j.1471-4159.2010.07089.x

    Figure Lengend Snippet: Contribution of Cav1 channel subtypes to the Ca 2+ charge and exocytosis of chromaffin vesicles. Ca 2+ charge density (a) and the corresponding exocytosis (b) versus the voltage in WT and Cav1.3 −/− cells. Data were obtained in the same experiments as in Fig. 3 ( n = 8–12, from four paired cultures of WT and Cav1.3 −/− cells, using 1–2 mice of each strain) and normalized as the percentage of charge density in control conditions at 10 mV, plotted as the mean ± SEM. (c) Original traces of Ca 2+ current density and the corresponding exocytosis elicited at −20 mV, −10 mV and 0 mV in WT and Cav1.3 −/− cells. * p

    Article Snippet: The primary antibodies (dilution 1 : 200) were goat polyclonal anti‐Cav1.1 and anti‐Cav1.4 (Santa Cruz Biotechnology, Heidelberg, Germany), and rabbit polyclonal anti‐Cav1.2 and anti‐Cav1.3 (Alomone Labs, Jerusalem, Israel).

    Techniques: Mouse Assay

    Kinetics of the Cav1 channel subtypes. One‐second square‐step depolarizing pulses were applied at −10 mV every 5 min. (a) Inactivation kinetics. Left, Ca 2+ current remaining at the end of a 1‐s pulse expressed as a percentage of the peak current ( I 1000 / I peak ) in WT (black column) and Cav1.3 −/− cells (grey column); middle, percentage of cells whose inactivation kinetics could be well fitted to a single (τ inact single , black columns) or to a double (τ inact double , grey columns) exponential function in WT and Cav1.3 −/− ; right, the average τ inact single yielded by the single exponential fitting, and τ inact double , which exhibited two components, a fast component (τ inact fast ) and a slow component (τ inact slow ), were plotted for WT and Cav1.3 −/− cells (black and grey columns, respectively). (b) Original traces of the Cav1 channel currents recorded in WT and Cav1.3 −/− cells were averaged, superimposed and scaled to the peak WT Cav1 channel current. Number of cells indicated in parentheses. Data were obtained in three paired cultures of WT and Cav1.3 −/− cells, using two mice of each strain.

    Journal: Journal of Neurochemistry

    Article Title: Different roles attributed to Cav1 channel subtypes in spontaneous action potential firing and fine tuning of exocytosis in mouse chromaffin cells

    doi: 10.1111/j.1471-4159.2010.07089.x

    Figure Lengend Snippet: Kinetics of the Cav1 channel subtypes. One‐second square‐step depolarizing pulses were applied at −10 mV every 5 min. (a) Inactivation kinetics. Left, Ca 2+ current remaining at the end of a 1‐s pulse expressed as a percentage of the peak current ( I 1000 / I peak ) in WT (black column) and Cav1.3 −/− cells (grey column); middle, percentage of cells whose inactivation kinetics could be well fitted to a single (τ inact single , black columns) or to a double (τ inact double , grey columns) exponential function in WT and Cav1.3 −/− ; right, the average τ inact single yielded by the single exponential fitting, and τ inact double , which exhibited two components, a fast component (τ inact fast ) and a slow component (τ inact slow ), were plotted for WT and Cav1.3 −/− cells (black and grey columns, respectively). (b) Original traces of the Cav1 channel currents recorded in WT and Cav1.3 −/− cells were averaged, superimposed and scaled to the peak WT Cav1 channel current. Number of cells indicated in parentheses. Data were obtained in three paired cultures of WT and Cav1.3 −/− cells, using two mice of each strain.

    Article Snippet: The primary antibodies (dilution 1 : 200) were goat polyclonal anti‐Cav1.1 and anti‐Cav1.4 (Santa Cruz Biotechnology, Heidelberg, Germany), and rabbit polyclonal anti‐Cav1.2 and anti‐Cav1.3 (Alomone Labs, Jerusalem, Israel).

    Techniques: Mouse Assay

    Cav1 channel subtypes expressed in mouse chromaffin cells. Immunocytochemical characterization of Cav1 channel subtypes. (a–b) Confocal images of isolated mouse chromaffin cells from WT (a) or Cav1.3 −/− mice (b) labeled with antibodies against Cav1.1, Cav1.2, Cav1.3 and Cav1.4 channels (dilution 1 : 200) and the corresponding secondary antibody (dilution 1 : 200) Alexa Fluor excited at a wavelength of 594 nm (dilution 1 : 200). Experiments were performed on four paired cultures of WT and Cav1.3 −/− cells. Calibration bar: 75 microns.

    Journal: Journal of Neurochemistry

    Article Title: Different roles attributed to Cav1 channel subtypes in spontaneous action potential firing and fine tuning of exocytosis in mouse chromaffin cells

    doi: 10.1111/j.1471-4159.2010.07089.x

    Figure Lengend Snippet: Cav1 channel subtypes expressed in mouse chromaffin cells. Immunocytochemical characterization of Cav1 channel subtypes. (a–b) Confocal images of isolated mouse chromaffin cells from WT (a) or Cav1.3 −/− mice (b) labeled with antibodies against Cav1.1, Cav1.2, Cav1.3 and Cav1.4 channels (dilution 1 : 200) and the corresponding secondary antibody (dilution 1 : 200) Alexa Fluor excited at a wavelength of 594 nm (dilution 1 : 200). Experiments were performed on four paired cultures of WT and Cav1.3 −/− cells. Calibration bar: 75 microns.

    Article Snippet: The primary antibodies (dilution 1 : 200) were goat polyclonal anti‐Cav1.1 and anti‐Cav1.4 (Santa Cruz Biotechnology, Heidelberg, Germany), and rabbit polyclonal anti‐Cav1.2 and anti‐Cav1.3 (Alomone Labs, Jerusalem, Israel).

    Techniques: Isolation, Mouse Assay, Labeling

    Contribution of Cav1 channel subtypes to pacemaking activity. (a–b) In a different set of experiments performed under the current‐clamp configuration, the spontaneous oscillatory activity resistant to TTX, obtained in half the cells treated with this toxin, was reversibly abolished by 300 nM nifedipine in WT (a) or Cav1.3 −/− cells (b). In the other half of the cells, reversible blockade of spontaneous action potentials by 2 μM TTX in WT (c) or Cav1.3 −/− (d) cells was achieved. Data were obtained in two paired cultures of WT and Cav1.3 −/− cells using two mice of each strain.

    Journal: Journal of Neurochemistry

    Article Title: Different roles attributed to Cav1 channel subtypes in spontaneous action potential firing and fine tuning of exocytosis in mouse chromaffin cells

    doi: 10.1111/j.1471-4159.2010.07089.x

    Figure Lengend Snippet: Contribution of Cav1 channel subtypes to pacemaking activity. (a–b) In a different set of experiments performed under the current‐clamp configuration, the spontaneous oscillatory activity resistant to TTX, obtained in half the cells treated with this toxin, was reversibly abolished by 300 nM nifedipine in WT (a) or Cav1.3 −/− cells (b). In the other half of the cells, reversible blockade of spontaneous action potentials by 2 μM TTX in WT (c) or Cav1.3 −/− (d) cells was achieved. Data were obtained in two paired cultures of WT and Cav1.3 −/− cells using two mice of each strain.

    Article Snippet: The primary antibodies (dilution 1 : 200) were goat polyclonal anti‐Cav1.1 and anti‐Cav1.4 (Santa Cruz Biotechnology, Heidelberg, Germany), and rabbit polyclonal anti‐Cav1.2 and anti‐Cav1.3 (Alomone Labs, Jerusalem, Israel).

    Techniques: Activity Assay, Mouse Assay

    Coupling of Cav1 channel subtypes to BK channels. (a) Upper section: double‐pulse protocol used to recruit BK channels. This included a 400 ms test pulse ( V t ) to 140 mV or above that potential (trace 1), followed by a 10‐ms pre‐pulse applied at 0 mV before V t (trace 2). The Ca 2+ dependent K + currents activated using this protocol were BK channels. Lower section: original K + current traces recorded using the above protocol under control conditions and after perfusion with different K + channel blockers, added sequentially and cumulatively: first, 200 nM apamin, 100 nM charibdotoxin (ChTx), and finally 45 mM TEA. Pulses were applied every 2 min. Numbers of cells are indicated in parentheses. (b) The K + charge density was averaged and normalized for each condition with respect to the current in the absence of a pre‐pulse. (c–d) Effects of 3 μM nifedipine on BK channel currents in WT (c) and Cav1.3 −/− cells (d). Number of cells: 14 WT cells, 11 Cav1.3 −/− cells. Data were obtained in four paired cultures of WT and Cav1.3 −/− cells, using two mice of each strain.

    Journal: Journal of Neurochemistry

    Article Title: Different roles attributed to Cav1 channel subtypes in spontaneous action potential firing and fine tuning of exocytosis in mouse chromaffin cells

    doi: 10.1111/j.1471-4159.2010.07089.x

    Figure Lengend Snippet: Coupling of Cav1 channel subtypes to BK channels. (a) Upper section: double‐pulse protocol used to recruit BK channels. This included a 400 ms test pulse ( V t ) to 140 mV or above that potential (trace 1), followed by a 10‐ms pre‐pulse applied at 0 mV before V t (trace 2). The Ca 2+ dependent K + currents activated using this protocol were BK channels. Lower section: original K + current traces recorded using the above protocol under control conditions and after perfusion with different K + channel blockers, added sequentially and cumulatively: first, 200 nM apamin, 100 nM charibdotoxin (ChTx), and finally 45 mM TEA. Pulses were applied every 2 min. Numbers of cells are indicated in parentheses. (b) The K + charge density was averaged and normalized for each condition with respect to the current in the absence of a pre‐pulse. (c–d) Effects of 3 μM nifedipine on BK channel currents in WT (c) and Cav1.3 −/− cells (d). Number of cells: 14 WT cells, 11 Cav1.3 −/− cells. Data were obtained in four paired cultures of WT and Cav1.3 −/− cells, using two mice of each strain.

    Article Snippet: The primary antibodies (dilution 1 : 200) were goat polyclonal anti‐Cav1.1 and anti‐Cav1.4 (Santa Cruz Biotechnology, Heidelberg, Germany), and rabbit polyclonal anti‐Cav1.2 and anti‐Cav1.3 (Alomone Labs, Jerusalem, Israel).

    Techniques: Mouse Assay