A single amino acid in γ-aminobutyric acid ρ1 receptors affects competitive and noncompetitive components of picrotoxin inhibition

A class of bicuculline-insensitive γ-aminobutyric acid (GABA) receptors, GABA(C), has been identified in retina. Several lines of evidence indicate that GABA(C) receptors are formed partially or wholly of GABA ρ subunits. These receptors generate a Cl^- current in response to GABA but differ from GABA(A) receptors in a number of ways. Picrotoxin, widely accepted as a noncompetitive antagonist of GABA(A) receptors, displays competitive and noncompetitive antagonism of GABA(C) receptors in perch and bovine retina and GABA ρ1 receptors expressed in Xenopus oocytes. The aim of this study was to identify the molecular basis of the two components of picrotoxin inhibition of GABA ρ1 receptors. By using a domain-swapping and mutagenesis strategy, a difference in picrotoxin sensitivity between ρ1 and ρ2 receptors was localized to a single amino acid in the putative second transmembrane domain. Substitution of this amino acid with residues found in the analogous position in highly picrotoxin-sensitive glycine α and GABA(A) subunits increased the sensitivity of ρ1 mutants 10- to 500-fold. Importantly, the competitive component of picrotoxin inhibition of the ρ1 mutant receptors was almost eliminated. These findings demonstrate that an amino acid in the putative channel domain of GABA ρ1 receptors influences picrotoxin sensitivity and mediates agonist binding by an allosteric mechanism.