Short report

Scanning mutagenesis of the I-II loop of the Ca_v2.2 calcium channel identifies residues Arginine 376 and Valine 416 as molecular determinants of voltage dependent G protein inhibition

Hugo W Tedford^* , Alexandra E Kisilevsky^* , Lucienne B Vieira , Diego Varela , Lina Chen and Gerald W Zamponi

Department of Physiology and Pharmacology, Hotchkiss Brain Institute, University of Calgary, Canada

author email corresponding author email* Contributed equally

Molecular Brain 2010, 3:6doi:10.1186/1756-6606-3-6

Published:	3 February 2010

Abstract

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.