Scanning mutagenesis of the I-II loop of the Cav2.2 calcium channel identifies residues Arginine 376 and Valine 416 as molecular determinants of voltage dependent G protein inhibition

Direct interaction with the β subunit of the heterotrimeric G protein complex causes voltage-dependent inhibition of N-type calcium channels. To further characterize the molecular determinants of this interaction, we performed scanning mutagenesis of residues 372-387 and 410-428 of the N-type channel α1 subunit, in which individual residues were replaced by either alanine or cysteine. We coexpressed wild type Gβ1γ2 subunits with either wild type or point mutant N-type calcium channels, and voltage-dependent, G protein-mediated inhibition of the channels (VDI) was assessed using patch clamp recordings. The resulting data indicate that Arg376 and Val416 of the α1 subunit, residues which are surface-exposed in the presence of the calcium channel β subunit, contribute significantly to the functional inhibition by Gβ1. To further characterize the roles of Arg376 and Val416 in this interaction, we performed secondary mutagenesis of these residues, coexpressing the resulting mutants with wild type Gβ1γ2 subunits and with several isoforms of the auxiliary β subunit of the N-type channel, again assessing VDI using patch clamp recordings. The results confirm the importance of Arg376 for G protein-mediated inhibition and show that a single amino acid substitution to phenylalanine drastically alters the abilities of auxiliary calcium channel subunits to regulate G protein inhibition of the channel.