A three-way inter-molecular network accounts for the Ca V α2δ1-induced functional modulation of the pore-forming Ca V 1.2 subunit.

L-type CaV 1.2 channels are essential for the excitation-contraction coupling in cardiomyocytes and are hetero-oligomers of a pore-forming CaV α1C assembled with CaV β and CaV α2δ1 subunits. A direct interaction between CaV α2δ1 and Asp-181 in the first extracellular loop of CaV α1 reproduces the native properties of the channel. A 3D model of the von Willebrand factor type A (VWA) domain of CaV α2δ1 complexed with the voltage sensor domain of CaV α1C 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 CaV α2δ1 Asp-259 and Ser-261 are the two most important residues in regard to protein interactions and modulation of CaV 1.2 currents. In contrast, mutating the side chains of CaV α2δ1 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 CaV α2δ1 Asp-259 coordinates the divalent cation that is further stabilized by the oxygen atoms from the hydroxyl side chain of CaV α2δ1 Ser-261 and the carboxylate group of CaV α1C 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 CaV 1.2 Asp-181. We propose that CaV α2δ1 Asp-259 promotes Ca2+ binding necessary to produce the conformation of the VWA domain that locks CaV α2δ1 Ser-261 and Ser-263 within atomic distance of CaV α1C Asp-181. This three-way network appears to account for the CaV α2δ1-induced modulation of CaV 1.2 currents.