Scanning mutagenesis of the I-II loop of the Ca.sub.v 2.2 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].sub.1 subunit, in which individual residues were replaced by either alanine or cysteine. We coexpressed wild type G[beta].sub.1 [gamma].sub.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.sup.376 .sup.and Val.sup.416 .sup.of the [alpha].sub.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].sub.1 . To further characterize the roles of Arg.sup.376 .sup.and Val.sup.416 .sup.in this interaction, we performed secondary mutagenesis of these residues, coexpressing the resulting mutants with wild type G[beta].sub.1 [gamma].sub.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.sup.376 .sup.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.