Negatively charged residues in the first extracellular loop of the L-type CaV1.2 channel anchor the interaction with the CaVα2δ1 auxiliary subunit

Abstract

Voltage-gated L-type CaV1.2 channels in cardiomyocytes exist as heteromeric complexes. Co-expression of CaVα2δ1 with CaVβ/CaVα1 proteins reconstitutes the functional properties of native L-type currents, but the interacting domains at the CaV1.2/CaVα2δ1 interface are unknown. Here, a homology-based model of CaV1.2 identified protein interfaces between the extracellular domain of CaVα2δ1 and the extracellular loops of the CaVα1 protein in repeats I (IS1S2 and IS5S6), II (IIS5S6), and III (IIIS5S6). Insertion of a 9-residue hemagglutinin epitope in IS1S2, but not in IS5S6 or in IIS5S6, prevented the co-immunoprecipitation of CaV1.2 with CaVα2δ1. IS1S2 contains a cluster of three conserved negatively charged residues Glu-179, Asp-180, and Asp-181 that could contribute to non-bonded interactions with CaVα2δ1. Substitutions of CaV1.2 Asp-181 impaired the co-immunoprecipitation of CaVβ/CaV1.2 with CaVα2δ1 and the CaVα2δ1-dependent shift in voltage-dependent activation gating. In contrast, single substitutions in CaV1.2 in neighboring positions in the same loop (179, 180, and 182–184) did not significantly alter the functional up-regulation of CaV1.2 whole-cell currents. However, a negatively charged residue at position 180 was necessary to convey the CaVα2δ1-mediated shift in the activation gating. We also found a more modest contribution from the positively charged Arg-1119 in the extracellular pore region in repeat III of CaV1.2. We conclude that CaV1.2 Asp-181 anchors the physical interaction that facilitates the CaVα2δ1-mediated functional modulation of CaV1.2 currents. By stabilizing the first extracellular loop of CaV1.2, CaVα2δ1 may up-regulate currents by promoting conformations of the voltage sensor that are associated with the channel's open state.

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

“…A homology model of the Ca V 1.2-Ca V ␣2␦1 interface was built based using the molecular coordinates of the cryo-EM structure of the skeletal muscle Ca V 1.1 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.…”

“…We recently identified the negatively charged Asp-181 in the first extracellular loop of Ca V 1.2 as an essential determinant for the interaction with the extracellular Ca V ␣2␦1 protein (19). To identify the molecular determinants of the interface, we have produced a 3D model of the IS1S4 region using the molecular coordinates of the cryo-EM structure of the homologous Ca V 1.1 channel complex (PDB code 5GJV) (16).…”

“…The ablation of the negative or polar side chain in the MIDAS residues prevented the coimmunoprecipitation of any of the multiple mutants in Ca V ␣2␦1, even after a 200-s exposure. Two-color flow-cytometry assays (17)(18)(19) were performed to determine whether disrupting MIDAS also altered the cell-surface expression of Ca V ␣2␦1. As detailed elsewhere (17,18), the cell-surface expression of the constructs, relative to mCherry- A, schematic demonstrating the coimmunoprecipitation assay using Ca V ␤3c-Myc as the bait.…”

“…Although Ca V 1.2 is responsible for the passage of Ca 2ϩ through the pore, its association with Ca V ␣2␦1 is necessary to reproduce the biophysical properties of the native channel (17,18), in particular the channel activation at negative membrane potentials. We have recently shown that two adjacent negatively charged residues Asp-180 and Asp-181 in the S1S2 loop in the repeat I of Ca V 1.2 are required to recapitulate the complex characteristics of the Ca V ␣2␦1-induced modulation of Ca V 1.2 currents (19). Whereas Asp-180 confers the voltage-dependent shift in channel activation, Ca V 1.2 Asp-181 anchors the physical interaction with Ca V ␣2␦1, a necessary step to exert its function.…”

“…Based on the recent cryo-electron microscopy (cryo-EM) structure of the homologous Ca V 1.1 channel complex (Ca V ␣1S with auxiliary subunits) (16), we have built a three-dimensional (3D) model of the VWA domain of the rat Ca V ␣2␦1 in complex with the first voltage sensor (IS1S4) of Ca V 1.2 (19). Within the VWA domain of Ca V ␣2␦1 lies a so-called "perfect" MIDAS motif with the typical Asp-Xaa-Ser-Xaa-Asp signature (20).…”