A three-way inter-molecular network accounts for the CaVα2δ1-induced functional modulation of the pore-forming CaV1.2 subunit

Abstract

L-type Ca_V1.2 channels are essential for the excitation-contraction coupling in cardiomyocytes and are hetero-oligomers of a pore-forming Ca_Vα1C assembled with Ca_Vβ and Ca_Vα2δ1 subunits. A direct interaction between Ca_Vα2δ1 and Asp-181 in the first extracellular loop of Ca_Vα1 reproduces the native properties of the channel. A 3D model of the von Willebrand factor type A (VWA) domain of Ca_Vα2δ1 complexed with the voltage sensor domain of Ca_Vα1C suggests that Ser-261 and Ser-263 residues in the metal ion-dependent adhesion site (MIDAS) motif are determinant in this interaction, but this hypothesis is untested. Here, coimmunoprecipitation assays and patch-clamp experiments of single-substitution variants revealed that Ca_Vα2δ1 Asp-259 and Ser-261 are the two most important residues in regard to protein interactions and modulation of Ca_V1.2 currents. In contrast, mutating the side chains of Ca_Vα2δ1 Ser-263, Thr-331, and Asp-363 with alanine did not completely prevent channel function. Molecular dynamics simulations indicated that the carboxylate side chain of Ca_Vα2δ1 Asp-259 coordinates the divalent cation that is further stabilized by the oxygen atoms from the hydroxyl side chain of Ca_Vα2δ1 Ser-261 and the carboxylate group of Ca_Vα1C Asp-181. In return, the hydrogen atoms contributed by the side chain of Ser-261 and the main chain of Ser-263 bonded the oxygen atoms of Ca_V1.2 Asp-181. We propose that Ca_Vα2δ1 Asp-259 promotes Ca²⁺ binding necessary to produce the conformation of the VWA domain that locks Ca_Vα2δ1 Ser-261 and Ser-263 within atomic distance of Ca_Vα1C Asp-181. This three-way network appears to account for the Ca_Vα2δ1-induced modulation of Ca_V1.2 currents.

Keywords: MIDAS; Western blot; calcium channel; channel activation; coimmunoprecipitation; homology modeling; molecular dynamics; patch clamp; protein structure; protein–protein interaction; structure-function; von Willebrand factor type A domain.

Figure 1.

3D homology model of the S1 to S4 region from domain I of Ca_V1.2 in complex with the VWA domain of Ca_Vα2δ1. A homology model of the Ca_V1.2–Ca_Vα2δ1 interface was built based using the molecular coordinates of the cryo-EM structure of the skeletal muscle Ca_V1.1 complex (PDB code 5GJV). This model differs slightly from the 3D model of the same region published previously (19) in regard to the orientation of the IS3S4 extracellular loop. A, residues forming the protein interface are zoomed in with emphasis on the MIDAS motif. The main-chain groups of amino acids forming the MIDAS motif (Asp-259, Ser-261, Ser-263, Thr-331, and Asp-363) are shown as pale rose sticks with carboxyl groups in red, amine groups are blue, and hydrogen atoms are white. Residues Asp-259, Ser-261, and Thr-331 identified within full-lined black boxes are in the foreground, and Ser-263 and Asp-363 identified within the dot-lined boxes are in the background. MIDAS residues are facing the IS1S2 loop in Ca_V1.2. Ca_V1.2 Asp-181 is shown in stick representation. B, VWA domain of the rat Ca_Vα2δ1 is shown in schematic representation in which α-helices appear in cyan, and β-strands are shown in pink. A single Ca²⁺ ion (green) is shown as being coordinated by the MIDAS residues. The region spanning from the S1 to the S4 segments in repeat I (IS1S4) of Ca_V1.2 is shown in schematic representation, and the transmembrane helices S1 to S4 are colored from the darker (S1) to the paler shade of blue (S4). Modeled DPPC lipids are shown in stick representation with carbon, oxygen, and phosphorus atoms being shown in gray, red, and pale green, respectively. Modeling was achieved with Modeler 9.17. The figure was produced using PyMOL (DeLano Scientific).

Figure 2.

Multiple mutations of MIDAS residues abolish the coimmunoprecipitation of Ca_V1.2 with Ca_Vα2δ1. A, schematic demonstrating the coimmunoprecipitation assay using Ca_Vβ3–c-Myc as the bait. B, HEKT cells were transiently transfected with pCMV-Ca_V1.2, pCMV-Ca_Vβ3–c-Myc, and pmCherry-Ca_Vα2δ1-HA WT or mutants as shown. Cell lysates were immunoprecipitated (IP) overnight with anti-c-Myc magnetic beads to capture Ca_Vβ3, eluted in a Laemmli buffer and fractionated by SDS-PAGE using 8% gels. B, immunoblotting (IB) was carried out on total proteins (20 μg) before coimmunoprecipitation assay (input lane) to confirm that all proteins have been correctly translated. Constructs are from left to right: mCherry-Ca_Vα2δ1-HA WT; mCherry-Ca_Vα2δ1-HA DSS/A (D259A/S261A/S263A); mCherry-Ca_Vα2δ1-HA DSST/A (D259A/S261A/S263A/T331A); and mCherry-Ca_Vα2δ1-HA DSSD/A (D259A/S261A/S263A/D363A). The signal for the housekeeping protein GAPDH is shown. C, immunoblotting was carried out after eluting the protein complexes (IP–c-Myc lane) from the beads with anti-Ca_V1.2, anti-Ca_Vα2δ1, and anti-Ca_Vβ3 antibodies (from top to bottom, as indicated). All immunoblots were carried out in parallel under the same transfection and extraction conditions. The signal for Ca_Vα2δ1 was revealed after three exposures times (1, 50, and 200 s). Ca_Vβ3 and Ca_V1.2 proteins migrated, respectively, at 60 and 250 kDa. All Ca_Vα2δ1 proteins migrated at ≈175 kDa, which is consistent with the molecular mass of the mCherry-Ca_Vα2δ1-HA WT reported in previous studies (19). These assays were repeated three times over the course of 6 months with different cell preparations. Assays carried out with anti-HA–coated beads to pull down the channel complex with Ca_Vα2δ1-HA as the bait (as seen in Fig. 6) yielded similar results. D, schematic illustrating the relative position of the extracellular HA epitope and the intracellular mCherry translated after the C terminus on the mCherry-Ca_Vα2δ1-HA construct used to carry the flow cytometry assays. The construct allows for detection of intracellular and extracellular fluorescence using, respectively, a FITC-conjugated anti-HA and the constitutive fluorescence of mCherry. E, representative two-dimensional plots of mCherry versus FITC fluorescence. The cell-surface expression of the mCherry-Ca_Vα2δ1-HA construct WT and mutants from the surface fluorescence emitted by the FITC-conjugated anti-HA as measured using a flow cytometry assay (10,000 intact cells) is shown. From top to bottom, the constructs that were tested are as follows: (a) mCherry-Ca_Vα2δ1-HA WT; (b) mCherry-Ca_Vα2δ1-HA DSS/A (D259A/S261A/S263A); (c) mCherry-Ca_Vα2δ1-HA DSSD/A (D259A/S261A/S263A/D363A); and (d) mCherry-Ca_Vα2δ1-HA DSST/A (D259A/S261A/S263A/T3331A). Left panel: relative intensity of the fluorescence signal produced by the FITC-conjugated anti-HA (x axis) and produced by the mCherry (y axis) yields an estimate of the cell surface and intracellular expression, respectively. The robust mCherry signal (y axis) confirms that the proteins were translated up to the end of the coding sequence, an observation also obtained from carrying out routine Western blotting. Middle panel: histogram of the relative fluorescence intensity for the FITC-conjugated anti-HA. Right panel: histogram of the relative fluorescence intensity for the constitutive mCherry signal. The cell-surface fluorescence for FITC, calculated as ΔMedFI, as explained under “Experimental procedures,” was significantly smaller for Ca_Vα2δ1-HA mutants than for the WT construct (see Table 1 for numerical values). Furthermore, the mCherry signal, which reflects the total protein density, was also decreased suggesting that these mutations impaired protein stability.

Figure 3.

Ca_Vβ3/Ca_V1.2 protein complex coimmunoprecipitated with Ca_Vα2δ1 V260R. HEKT cells were transiently transfected with pCMV-Ca_V1.2 and pCMV-Ca_Vβ3–c-Myc and either pmCherry-Ca_Vα2δ1-HA WT, or pmCherry-Ca_Vα2δ1-HA D259R, or pmCherry-Ca_Vα2δ1-HA V260R, or pmCherry-Ca_Vα2δ1-HA S261R, or pmCherry-Ca_Vα2δ1-HA G262R, or pmCherry-Ca_Vα2δ1-HA S263R. Cell lysates were immunoprecipitated (IP) overnight with anti-c-Myc magnetic beads to capture Ca_Vβ3, eluted in a Laemmli buffer, and fractionated by SDS-PAGE using 8% gels. A, immunoblotting (IB) was carried out on total proteins (20 μg) before the immunoprecipitation assay (input lane) to confirm that constructs were expressed at the expected molecular weight. The signal for the housekeeping protein GAPDH is shown. B, immunoblotting was carried out after eluting the protein complexes from the beads with anti-Ca_V1.2, anti-Ca_Vα2δ1, and anti-Ca_Vβ3 antibody (from top to bottom, as indicated). Images for Ca_Vα2δ1 were captured after shorter (1 s) or longer exposure times until the signal was saturated (20 s). All immunoblots were carried out in parallel under the same transfection and extraction conditions. These assays were successfully repeated three times with similar results.

Figure 4.

Ca_Vα2δ1 V260R stimulates Ca_V1.2 whole-cell currents. A, representative whole-cell Ca²⁺ current traces recorded after recombinant expression of Ca_V1.2 + Ca_Vβ3 and either pmCherry-Ca_Vα2δ1-HA WT or mutants D259R, V260R, S261R, G262R, S263R, T331R, and D363R (from left to right). Currents were recorded in the presence of 2 mm Ca²⁺ from a holding potential of −100 mV. Time scale was 100 ms throughout. The current density scale is 5 pA/pF, as indicated. B, averaged current-voltage relationships. Coexpression with mCherry-Ca_Vα2δ1-HA V260R produced whole-cell Ca_V1.2 currents with properties similar to currents measured in the presence of mCherry-Ca_Vα2δ1-HA WT. Coexpression with mCherry-Ca_Vα2δ1-HA D259R, S261R, G262R, S263R, T331R, and D363R impaired current up-regulation to different degrees. Statistical analyses were carried out with a one-way ANOVA test. 1 indicates that the mutant Ca_Vα2δ1 proteins are significantly different from the mock vector at p < 0.01, and 2 indicates that the mutant Ca_Vα2δ1 proteins are significantly different from mCherry-Ca_Vα2δ1-HA WT at p < 0.05. The complete set of values is shown in Table 2. C, bar graph reporting the distribution of the free energies of activation (ΔG_act) measured with each Ca_Vα2δ1 mutant. The distribution of the ΔG_act values for mCherry-Ca_Vα2δ1-HA D259R, S261R, G262R, and D363R was right-shifted as compared with mCherry-Ca_Vα2δ1-HA WT and overlapped with the ΔG_act values measured in the presence of mock vector (pmCherry-no Ca_Vα2δ1).

Figure 5.

Ca_Vα2δ1 S263A and T331A stimulate Ca_V1.2 whole-cell currents. A, representative whole-cell Ca²⁺ current traces recorded after recombinant expression of Ca_V1.2 + Ca_Vβ3 and either pmCherry-Ca_Vα2δ1-HA WT, D259A, S261A, G262A, S263A, G262A/S263A, T331A, or D363A (from left to right). Currents were recorded in the presence of 2 mm Ca²⁺ from a holding potential of −100 mV. Time scale is 100 ms throughout. The current density scale ranged from 5 to 10 pA/pF, as indicated. B, averaged current-voltage relationships. Coexpression with mCherry-Ca_Vα2δ1-HA S263A or T331A produced whole-cell Ca_V1.2 currents with properties similar to currents measured in the presence of mCherry-Ca_Vα2δ1-HA WT. Coexpression with mCherry-Ca_Vα2δ1-HA D259A, S261A, G262A, G262A/S263A, and D363A impaired current up-regulation to different degrees. Statistical analyses were carried out with a one-way ANOVA test. 1 indicates that the mutant Ca_Vα2δ1 proteins are significantly different from the mock vector at p < 0.01, and 2 indicates that the mutant Ca_Vα2δ1 proteins are significantly different from mCherry-Ca_Vα2δ1-HA WT at p < 0.05. Complete set of values is shown in Table 2. C, bar graph reporting the distribution of the free energies of activation (ΔG_act) measured with each Ca_Vα2δ1 mutant. The distribution of the ΔG_act values for mCherry-Ca_Vα2δ1-HA D259A, S261A, G262A, and G262A/S263A was right-shifted as compared with Ca_Vα2δ1 WT and overlapped with the ΔG_act values measured in the presence of mock vector (pmCherry-no Ca_Vα2δ1). Note that the distribution for the ΔG_act obtained with mCherry-Ca_Vα2δ1-HA S263A was very wide and may reflect different conformations of the mutant.

Figure 6.

Single alanine substitutions of Asp-259, Ser-261, and Ser-263 in Ca_Vα2δ1 impair coimmunoprecipitation of Ca_V1.2 and Ca_V2.3 proteins. A and B, HEKT cells were transiently transfected with pCMV-Ca_V1.2 and pCMV-Ca_Vβ3–c-Myc and either pmCherry-Ca_Vα2δ1-HA WT or pmCherry-Ca_Vα2δ1-HA D259A, V260A, S261A, G262A, S263A, T331A, or D363A. Cell lysates were immunoprecipitated (IP) overnight with anti-HA magnetic beads to capture Ca_Vα2δ1-HA, eluted in a Laemmli buffer and fractionated by SDS-PAGE using 8% gels. A, immunoblotting (IB) was carried out on total proteins (20 μg) before the immunoprecipitation assay to confirm protein expression in all samples (input lane). The signal for the housekeeping protein GAPDH is shown below. B, immunoblotting was carried out after eluting the protein complexes from the beads with anti-Ca_V1.2 and anti-Ca_Vα2δ1 antibodies (from top to bottom, as indicated). All immunoblots were carried out in parallel under the same transfection and extraction conditions. In these experiments, Ca_Vα2δ1-HA is bound to the beads such that the interaction is probed by investigating the signal for its Ca_V1.2 partner. The image was obtained using the automatic mode of the ChemiDoc Touch system, which optimizes the signal. As shown, the signal for Ca_V1.2 was significantly reduced for mCherry-Ca_Vα2δ1-HA D259A, S261A, and S263A. C and D, HEKT cells were transiently transfected with pcDNA3-Ca_V2.3 and pCMV-Ca_Vβ3–c-Myc and either pmCherry-Ca_Vα2δ1-HA WT or pmCherry-Ca_Vα2δ1-HA D259A, S261A, S263A, T331A, or D363A. Cell lysates were immunoprecipitated overnight with anti-c-Myc magnetic beads to capture Ca_Vβ3–c-Myc, eluted in a Laemmli buffer and fractionated by SDS-PAGE using 8% gels. C, immunoblotting was carried out on total proteins (20 μg) before the immunoprecipitation assay (input lane). The signal for the housekeeping protein GAPDH is shown. D, immunoblotting was carried out after eluting the protein complexes from the beads with anti-Ca_V2.3, anti-Ca_Vα2δ1, and anti-Ca_Vβ3 antibodies (from top to bottom, as indicated). In these experiments, Ca_Vβ3/Ca_V2.3 is bound to the beads such that the interaction is probed by investigating the signal for Ca_Vα2δ1. As shown, the signal for Ca_Vα2δ1 was significantly reduced for mCherry-Ca_Vα2δ1-HA D259A, S261A, S263A, and D363A. All immunoblots were carried out in parallel under the same transfection and extraction conditions.

Figure 7.

Ca_Vα2δ1 D259A could destabilize Ca²⁺ binding within MIDAS. Ca_Vα2δ1 MIDAS residues are shown in the lowest energy conformations obtained from three 25-ns MD trajectories produced with the 3D virtual models of Ca_Vα2δ1 WT (A) and Ca_Vα2δ1 D259A (B). Asp-181 in Ca_V1.2 and Ca_Vα2δ1 MIDAS residues are shown in stick representation and are colored in yellow and gray, respectively. The Ca²⁺ ion is shown as a green sphere, and water molecules are visualized as red dots. Within residues, oxygen atoms are in red, nitrogen atoms are in blue, and hydrogen atoms are in white. Potential interactions between atoms are indicated by black dashed lines. The 3D model was relatively stable with little fluctuations in the backbone atoms over the 25-ns simulation. In particular, the r.m.s.d. of the Cα for the whole complex (calculated after equilibration and between each time point of the 25-ns simulation) fluctuated in average 2.52 ± 0.59 Å (mean ± S.D.) and ≈80% of the data points deviate less than 3 Å when compared with the structure shown in A. As shown, the oxygen atoms from Ca_V1.2 Asp-181 are within a few angstroms of the side-chain hydrogen atom of Ca_Vα2δ1 Ser-261 and the main-chain hydrogen atom of Ser-263. Elimination of the negatively charged side chain at position 259 in Ca_Vα2δ1 D259A (B) produced a substantial alteration in the conformation of MIDAS, whereas the hydrogen atom from the hydroxyl group of Ca_Vα2δ1 Ser-261 switches from coordinating the Ca²⁺ ion and Ca_V1.2 Asp-181 to facing the carboxyl group in the side chain from Ca_Vα2δ1 Asp-363. In the Ca_Vα2δ1 D259A mutant, the Ca²⁺ ion is being more freely exposed to the solvent and becomes coordinated by five water molecules (B) instead of two water molecules in the WT protein complex (A). This conformational change disrupts the network of stabilizing interactions between Ca_Vα2δ1 and Ca_V1.2 Asp-181. PDB files are available upon request.