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 beta 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 alpha(1) subunit, in which individual residues were replaced by either alanine or cysteine. We coexpressed wild type G beta(1 gamma 2) 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 Arg(376) and Val(416) of the alpha(1) subunit, residues which are surface-exposed in the presence of the calcium channel beta subunit, contribute significantly to the functional inhibition by G beta(1). To further characterize the roles of Arg(376) and Val(416) in this interaction, we performed secondary mutagenesis of these residues, coexpressing the resulting mutants with wild type G beta(1 gamma 2) subunits and with several isoforms of the auxiliary beta subunit of the N-type channel, again assessing VDI using patch clamp recordings. The results confirm the importance of Arg(376) 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.