Exploring the Role of the First Extracellular Loop of CaV2.3 in Mediating the Interaction with Auxiliary Subunits

Abstract

Presynaptic calcium entry occurs through voltage-gated calcium (CaV) channels which are activated by membrane depolarization. The presynaptic CaV2 channel gene family includes the pharmaco-resistant CaV2.3 or R-type Ca2+ channel. Like other high-voltage-activated CaV2 channels, CaV2.3 is formed by the association of the pore-forming CaVα1 subunit with auxiliary CaVβ and CaVα2δ1. CaVβ promotes cell surface expression, and co-expression with CaVα2δ1 increases by 2-3 fold the peak current density of CaV2.3. In contrast to CaV1.2, CaVα2δ1 does not modulate the activation potential of CaV2.3. Herein we explored the molecular basis for these differences. Homology-based model of CaV2.3 identified the first extracellular loop of CaVα1 (IS1S2) as a potential interface with the VWA domain of CaVα2δ1 (Briot et al., JBC 2018). IS1S2 of CaV2.3 contains a cluster of three consecutive negatively charged residues 113-PEDDK-117 that bears strong homology with the IS1S2 loop of CaV1.2 (178-PEDDS-182). Previous studies from our group have identified CaV1.2 Asp-181 (equivalent to CaV2.3 Asp-116) as anchoring the interaction between CaV1.2 and CaVα2δ1 (Bourdin et al., JBC 2017). We are herein showing that substitutions of Asp-116 disrupt the co-immunoprecipitation of CaVβ3/CaV2.3 with CaVα2δ1 whereas single substitutions to alanine residues in neighboring positions (Pro-113, Glu-114, Asp-115, and Lys-117) did not impair the interaction between CaVα2δ1 and CaV2.3. We are currently carrying out patch-clamp experiments to investigate the functional modulation of single-substituted CaV2.3 proteins by CaVα2δ1. Preliminary data suggest that channels substituted at positions Asp-115 or Lys-117 displayed altered activation properties when coexpressed with CaVα2δ1. We conclude that the functional modulation of CaV2.3 by CaVα2δ1 involves a broader range of residues than previously described for the L-type CaV1.2 channel and suggests family specific differences in channel modulation.