A Single Glycine Residue at the Entrance to the First Membrane-Spanning Domain of the γ-Aminobutyric Acid Type A Receptor β2Subunit Affects Allosteric Sensitivity to GABA and Anesthetics

Abstract

Site-directed mutagenesis of the γ-aminobutyric acid type A (GABA A ) receptor β 2 subunit has demonstrated that conversion of a conserved glycine residue located at the entrance to the first transmembrane domain into the homologous ρ 1 residue phenylalanine alters the modulating effects of four different i.v. anesthetics: pentobarbital, alphaxalone, etomidate, and propofol. Using the baculovirus expression system in Spodoptera frugiperda 9 cells, anesthetic-induced enhancement of [ 3 H]muscimol and [ 3 H]flunitrazepam binding in receptors containing the β 2 (G219F) point mutation displayed a significantly reduced efficacy in modulation by all four i.v. anesthetics tested. Furthermore, GABA A receptors containing the α 1 (G223F) point mutation also significantly decreased the maximal effect of etomidate- and propofol-induced enhancement of ligand binding. Conversely, the homologous point mutation in ρ 1 receptors (F261G) changed the i.v. anesthetic-insensitive receptor to confer anesthetic modulation of [ 3 H]muscimol binding. Consistent with the binding, functional analysis of pentobarbital-enhanced GABA currents recorded with whole-cell patch clamp demonstrated the β 2 (G219F) subunit mutation eliminated the potentiating effect of the anesthetic. Similarly, propofol-enhanced GABA currents were potentiated less in α 1 β 2 (G219F)γ 2 receptors than in α 1 β 2 γ 2 receptors. Although ligand binding displayed comparable K D values for muscimol among wild-type, α 1 β 2 γ 2 , and mutant receptors, patch-clamp recordings showed that α 1 β 2 (G219F)γ 2 receptors had a significantly more potent response to GABA than did α 1 β 2 γ 2 or α 1 (G223F)β 2 γ 2 . The α 1 β 2 (G219F)γ 2 receptors also were more sensitive to direct channel activation by pentobarbital and propofol in the absence of GABA. These results suggest that the first transmembrane glycine residue on the β 2 subunit may be important for conformational or allosteric interactions of channel gating by both GABA and anesthetics.