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

Voltage-gated L-type Ca(V)1.2 channels in cardiomyocytes exist as heteromeric complexes. Co-expression of Ca-V alpha 2 delta 1 with Ca-V beta/Ca-V alpha 1 proteins reconstitutes the functional properties of native L-type currents, but the interacting domains at the Ca(V)1.2/ Ca-V alpha 2 delta 1 interface are unknown. Here, a homology- based model of Ca(V)1.2 identified protein interfaces between the extracellular domain of Ca-V alpha 2 delta 1 and the extracellular loops of the Ca-V alpha 1 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 Ca-V alpha 2 delta 1. 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 Ca-V alpha 2 delta 1. Substitutions of CaV1.2 Asp-181 impaired the co-immunoprecipitation of Ca-V beta/Ca(V)1.2 with Ca-V alpha 2 delta 1 and the Ca-V alpha 2 delta 1-dependent shift in voltage-dependent activation gating. In contrast, single substitutions in Ca(V)1.2 in neighboring positions in the same loop (179, 180, and 182-184) did not significantly alter the functional up-regulation of Ca(V)1.2 whole-cell currents. However, a negatively charged residue at position 180 was necessary to convey the Ca-V alpha 2 delta 1-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 Ca(V)1.2. We conclude that Ca(V)1.2 Asp-181 anchors the physical interaction that facilitates the Ca-V alpha 2 delta 1-mediated functional modulation of Ca(V)1.2 currents. By stabilizing the first extracellular loop of Ca(V)1.2, Ca-V alpha 2 delta 1 may up-regulate currents by promoting conformations of the voltage sensor that are associated with the channel's open state.