A CACNA1C variant associated with cardiac arrhythmias provides mechanistic insights in the calmodulation of L-type Ca2+ channels

Figures

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  Figure 1. The CaV1.2 variant is located in the intracellular linker before the binding site for the CaVβ subunit. The LQTS-related CaVα1C mutation G449 is located before the α-interacting domain (AID). A, the cryo-EM 3D structure of the rabbit CaV1.1 oligomeric complex at 3.6 Å for CaVα1S and at 3.9 Å for CaVβ (Protein Data Bank code: 5GJV). CaVα1C and CaVα1S share 81% homology in their primary protein sequence. L-type calcium channels share similar structure, being composed of the pore-forming subunit CaVα1 in red and CaVβ in blue and an intracellular subunit bound to CaVα1 through the intracellular helix linking domains I and II of CaVα1 (shown in dark green). The first transmembrane domain of CaVα1 (DI) is shown in yellow. The human LQTS-related CaVα1C G419R variant is similar to the rabbit CaVα1C G449R and is equivalent to Gly358 in CaVα1S. Image was produced by Discovery Studio 2020 (BIOVIA Pipeline Pilot 2020). B, cartoon of the corresponding secondary structure for the CaVα1C pore-forming subunit of the L-type CaV1.2 channel showing the four homologous domains (domains I to IV) with the N and C termini located into the cytoplasm. The CaVβ subunit–binding site on the CaVα1C subunit…

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  Figure 2. G449R interacts with CaVβ proteins. HEKT cells were transiently transfected with CaV1.2 WT (WT) or CaV1.2 G449R (GR) with cMyc-tagged CaVβ3 or cMyc-tagged CaVβ2a. CaVα2δ1 was present throughout. Cell lysates were immunoprecipitated overnight with anti-cMyc magnetic beads (Pierce Anti-c-Myc Magnetic Beads; catalog no.: 88842, Thermo Fisher Scientific) to capture the CaVβ, eluted in a 2× Laemmli buffer and fractionated by 8% SDS-PAGE gels. A, immunoblotting was carried out on total proteins (20 μg) collected from the cell lysates before the immunoprecipitation assay (total proteins). The signal for the housekeeping protein GAPDH is shown below each blot. B, immunoblotting of “IP-proteins” was carried out after eluting the protein complexes from the beads. All immunoblots were carried out in parallel under the same transfection and extraction conditions. Western blotting was carried out with either anti-CaVβ3 (Alomone; catalog no.: ACC008, 1:10,000 dilution), anti-CaVβ2a (Alomone; catalog no.: ACC105, 1:1000 dilution), anti-CaV1.2 directed against CaVα1C (Alomone; catalog no.: ACC003, 1:250 dilution), anti-CaVα2δ1 (Alomone; catalog no.: ACC015, 1:1000 dilution), and GAPDH (Sigma;…

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  Figure 3. CaV1.2 G449R is insensitive to W-13.A, representative CaV1.2 WT current traces recorded in the presence of native/endogenous CaM from HEKT cells before (left) and after (middle) the application of W-13. Peak current densities of CaV1.2 WT currents are plotted against the applied voltages and fitted by a Boltzmann equation (right). Incubation with 10 μM W-13 for 15 min reduced the CaV1.2 WT current density by approximately 50%, from −15 ± 4 pA/pF under control conditions versus −8 ± 2 pA/pF in the presence of W-13. B, representative CaV1.2 G449R current traces recorded in the presence of native/endogenous CaM from HEKT cells before (left) and after (middle) W-13 treatment. In contrast to CaV1.2 WT, CaV1.2 G449R currents were unaffected by the W-13 and did not display any inhibition in the peak current density (right). C, representative CaV1.2 WT current traces cotransfected with CaM WT recorded from HEKT cells before (left) and after (middle) W-13 treatment. Overexpression of CaM WT significantly enhanced the current density of CaV1.2 WT, whereas only approximately, 10% of peak CaV1.2 currents remained following W-13 treatment (right). Peak current densities of CaV1.2 WT…

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  Figure 4. Calmodulin (CaM) pulls down the L-type calcium channel. HEKT cells were transiently transfected with CaVβ2a, CaM WT, and either CaV1.2 WT (WT) or G449R (GR). CaM was captured by the anti-His–coated beads. A, proteins were homogenized, and a fraction of this solution (referred to as total) was set aside to validate protein expression. B, coimmunoprecipitation was carried out with anti-His magnetic beads. The bound proteins were eluted (referred to as pull-down) and electrophoresed on a 6% SDS-polyacrylamide gel or a 10% SDS-polyacrylamide gel for CaM and GAPDH before being transferred onto a nitrocellulose membrane. Western blotting was carried out with anti-CaVβ2 (Alomone; catalog no.: ACC105, 1:1000 dilution), anti-CaV1.2 (Alomone; catalog no.: ACC003, 1:250 dilution) with an anti-rabbit as secondary antibody (Jackson ImmunoResearch, 1:10,000 dilution), and anti-CaM (Millipore; catalog no.: 05-193, 1:1000 dilution) with an anti-mouse as secondary antibody (Jackson ImmunoResearch, 1:10,000 dilution). Molecular weights were estimated using Image Lab software, version 5.2 (Bio-Rad) by linear regression of standard molecular weight markers. CaV1.2 WT and G449R, CaVβ2a, and CaM…

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  Figure 5. CaM promotes the cell surface localization of CaV1.2 WT but not CaV1.2 G449R.A, HEKT cells were transiently transfected with CaV1.2 WT + CaVα2δ1 + CaVβ2a (left) and CaV1.2 G449R + CaVα2δ1 + CaVβ2a (right) in the presence of native/endogenous CaM. B, HEKT cells were transiently transfected with CaV1.2 WT + CaVα2δ1 + CaVβ2a + CaM WT (left) and CaV1.2 G449R + CaVα2δ1 + CaVβ2a + CaM WT (right). Two days after transfection, the cells were lysed, and cell fractions were obtained through preparative ultracentrifugation as described in the Experimental procedures section. Western blotting was carried out for the four protein fractions found in lanes 1 to 4; lane 1: total proteins; lane 2: cytoplasmic proteins; lane 3: total membrane proteins; and lane 4: plasma membrane proteins. The proteins were probed with the following antibodies: CaV1.2 (Alomone; catalog no.: ACC003, 1:250 dilution) with anti-rabbit (1:10,000 dilution); CaVα2δ1 (Alomone; catalog no.: ACC015, 1:1000 dilution) with anti-rabbit (1:10,000 dilution); CaVβ2a (Alomone; catalog no.: ACC105, 1:1000 dilution) with anti-rabbit (1:10,000 dilution); CaM (Millipore; catalog no.: 05-193, 1:1000 dilution) with anti-mouse (1:10,000…

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  Figure 6. Phosphomimetic CaM T79D and S81D upregulate CaV1.2 WT channels.A and C, middle, CaV1.2 WT was coexpressed with the “phosphoresistant” CaM mutations T79A or S81A. Overexpression of CaM T79A or S81A failed to enhance the currents and was insensitive to W-13. A and C, right, average I–V curves of CaV1.2 WT coexpressed with CaM T79A or S81A. The peak current densities were not different between control and W-13 treatment. B and D, left, middle, CaV1.2 WT current traces recorded from HEKT cells after coexpression with phosphomimetic CaM T79D or S81D. Overexpression of CaM T79D or CaM S81D boosted CaV1.2 peak currents that were sharply abolished by the extracellular application of W-13. The vertical scale bars are 10 pA/pF, and the horizontal scale bars are 100 ms throughout. B and D, right, average I–V curves of CaV1.2 WT coexpressed with CaM T79D or S81D for control and W-13 treatment. E and F, the distribution of the peak current densities and E0.5,act for control and W-13 are summarized as filled circles for CaV1.2 WT coexpressed with either CaM WT (black), T79A (red), T79D (blue), S81A (green), or S81D (light purple). The mean data ± SD are shown as gray hyphens. The values of the…

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  Figure 7. CaV1.2 G449R is not modulated by CaM or CaM inhibitor W-13. Representative CaV1.2 G449R current traces were recorded from HEKT cells in the presence of 2 mM Ca2+. A–D, left, CaV1.2 G449R was coexpressed with CaM WT, with the phosphoresistant CaM (T79A or S81A) or with phosphomimetic CaM (T79D or S81D) as shown. A–D, middle, CaV1.2 G449R channels coexpressed with either CaM WT, T79A, T79D, S81A, or S81D are resistant to block by W-13. The vertical scale bars are 10 pA/pF, and the horizontal scale bars are 100 ms throughout. A–D, right, average I–V curves of CaV1.2 G449R coexpressed with CaM T79A, T79D, S81A, or S81D. The peak current densities were not different between control and W-13 treatment. E and F, the distribution of the peak current densities and E0.5,act for control conditions and after W-13 treatment are summarized individually as filled circles for Cav1.2 G449R coexpressed with either CaM WT (black), T79A (red), T79D (blue), S81A (green), or S81D (light purple). The mean data ± SD are shown as gray hyphens. The complete set of values is found in Table 3. CaM, calmodulin; HEKT, human embryonic kidney 293T cell line.

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  Figure 8. CaM T79A and T79D coimmunoprecipitate CaV1.2 WT and G449R. HEKT cells were transiently transfected with CaVβ2a in the presence of CaV1.2 WT or CaV1.2 G449R and either CaM WT, CaM T79A, or CaM T79D. A, total proteins are shown. B, coimmunoprecipitation was carried out with anti-His magnetic beads. Immunoblotting was carried out after elution of the bound proteins using the antibodies described in the legend of Figure 4. As seen, CaV1.2 WT and G449R, CaVβ2a, and CaM proteins were translated at the expected molecular masses of 250, 70, and 18 to 24 kDa, respectively. There was no significant difference between the signals measured in the presence of either CaM WT, CaM T79A, or CaM T79D. The signals were nonetheless systematically stronger for CaV1.2 G449R than for CaV1.2 WT despite equivalent loading and similar signals for the total proteins. Similar data were obtained from three independent transfections carried out over the course of 2 months with protein extraction carried out with digitonin or CHAPS. CaM, calmodulin; HEKT, human embryonic kidney 293T cell line.

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  Figure 9. CaV1.2 G449P is modulated by CaM phosphorylation surrogates.A, whole-cell currents were recorded from HEKT cells transiently transfected with CaV1.2 G449P coexpressed with Cavβ2a and Cavα2δ1 and either CaM WT, with the phosphoresistant CaM (T79A or S81A), or with phosphomimetic CaM (T79D or S81D) as indicated. Exemplar traces are shown (from left to right) for CaV1.2 G449P + CaM WT, G449P + CaM T79A, G449P + CaM T79D, G449P + CaM S81A, and G449P + CaM S81D. The vertical scale bars are 10 pA/pF, and the horizontal scale bars are 100 ms throughout. B, the corresponding peak current densities are plotted as a function of applied voltage. C and D, the summarized distribution of the peak current densities and the midpotential of activation E0.5,act. Peak whole-cell currents and E0.5,act are reported individually as black circles. The mean data ± SD are shown as red hyphens. Values of peak current densities and E0.5,act are reported in Table 6. CaM, calmodulin; HEKT, human embryonic kidney 293T cell line.