A single hydrophobic residue confers barbiturate sensitivity to gamma-aminobutyric acid type C receptor

Barbiturate sensitivity was imparted to the human rho(1) homooligomeric gam ma-aminobutyric acid (GABA) receptor channel by mutation of a tryptophan re sidue at position 328 (Trp328), which is located within the third transmemb rane domain. Substitutions of Trp328 with a spectrum of amino acids reveale d that nearly all hydrophobic residues produced receptor channels that were both directly activated and modulated by pentobarbital with similar sensit ivities. Previous studies with ligand-gated ion channels (including GABA) h ave demonstrated that even conservative amino acid substitution within the agonist-dependent activation domain (N-terminal extracellular domain) can m arkedly impair agonist sensitivity. Thus, the lack of significant variation in pentobarbital sensitivity among the Trp328 mutants attests to an intrin sic difference between pentobarbital- and the GABA-dependent activation dom ain. Compared with the heterooligomeric alpha beta gamma receptor channel, the mode of modulation for homooligomeric Trp328 mutants by pentobarbital w as more dependent on the GABA concentration, yielding potentiation only at low concentrations of GABA (fractions of their respective EC50 values), yet causing inhibition at higher concentrations. Agonist-related studies have also demonstrated that residue 328 plays an important role in agonist-depen dent activation, suggesting a functional interconnection between the GABA a nd pentobarbital activation domains.