A SINGLE AMINO-ACID IN GAMMA-AMINOBUTYRIC-ACID P1 RECEPTORS AFFECTS COMPETITIVE AND NONCOMPETITIVE COMPONENTS OF PICROTOXIN INHIBITION

A class of bicuculline-insensitive gamma-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 rho 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 rho 1 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 rho 1 receptors. By using a domain-swapping and mutagenesis strategy, a difference in picrotoxin sensitivity between rho 1 and rho 2 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 alpha and GABA(A) subunits increased the sensitivity of rho 1 mutants 10- to 500-fold. Importantly, the competitive component of picrotoxin inhibition of the rho 1 mutant receptors was almost eliminated. These findings demonstrate that an amino acid in the putative channel domain of GABA rho 1 receptors influences picrotoxin sensitivity and mediates agonist binding by an allosteric mechanism.