A single glycine residue at the entrance to the first membrane-spanning domain of the gamma-aminobutyric acid type a receptor beta 2 subunit affects allosteric sensitivity to GABA and anesthetics

Site-directed mutagenesis of the gamma-aminobutyric acid type A (GABA(A)) r eceptor beta 2 subunit has demonstrated that conversion of a conserved glyc ine residue located at the entrance to the first transmembrane domain into the homologous rho(1) residue phenylalanine alters the modulating effects o f four different i.v. anesthetics: pentobarbital, alphaxalone, etomidate, a nd propofol. Using the baculovirus expression system in Spodoptera frugiper da 9 cells, anesthetic-induced enhancement of [H-3]muscimol and [H-3]flunit razepam binding in receptors containing the beta 2(G219F) point mutation di splayed a significantly reduced efficacy in modulation by all four i.v. ane sthetics tested. Furthermore, GABA(A) receptors containing the alpha(1)(G22 3F) point mutation also significantly decreased the maximal effect of etomi date- and propofol-induced enhancement of ligand binding. Conversely, the h omologous point mutation in rho(1) receptors (F261G) changed the i.v. anest hetic-insensitive receptor to confer anesthetic modulation of [H-3]muscimol binding. Consistent with the binding, functional analysis of pentobarbital -enhanced GABA currents recorded with whole-cell patch clamp demonstrated t he beta 2(G219F) subunit mutation eliminated the potentiating effect of the anesthetic. Similarly, propofol-enhanced GABA currents were potentiated le ss in alpha(1)beta(2)(G219F)gamma(2) receptors than in alpha(1)beta(2)gamma (2) receptors. Although ligand binding displayed comparable K-D values for muscimol among wild-type, alpha(1)beta(2)gamma(2), and mutant receptors, pa tch-clamp recordings showed that alpha(1)beta(2)(G219F)gamma(2) receptors h ad a significantly more potent response to GABA than did alpha(1)beta(2)gam ma(2) or alpha(1)(G223F)beta(2)gamma(2). The alpha(1)beta(2)(G219F)gamma(2) receptors also were more sensitive to direct channel activation by pentoba rbital and propofol in the absence of GABA. These results suggest that the first transmembrane glycine residue on the beta(2) subunit may be important for conformational or allosteric interactions of channel gating by both GA BA and anesthetics.