The gamma subunit determines whether anesthetic-induced leftward shift is altered by a mutation at alpha1S270 in alpha1beta2gamma2L GABA(A) receptors.

Abstract

A major action of volatile anesthetics is enhancement of gamma-aminobutyric acid receptor type A (GABA(A)R) currents. In recombinant GABA(A)Rs consisting of several subunit mixtures, mutating the alpha1 subunit serine at position 270 to isoleucine [alpha1(S270I)] was reported to eliminate anesthetic-induced enhancement at low GABA concentrations. In the absence of studies at high GABA concentrations, it remains unclear whether alpha1(S270I) affects enhancement versus inhibition by volatile anesthetics. Furthermore, the majority of GABA(A)Rs in mammalian brain are thought to consist of alpha1, beta2, and gamma2 subunits, and the alpha1(S270I) mutation has not been studied in the context of this combination. Recombinant GABA(A)Rs composed of alpha1beta2 or alpha1beta2gamma2L subunit mixtures were studied electrophysiologically in whole Xenopus oocytes in the voltage clamp configuration. Currents elicited by GABA (0.03 microM to 1 mM) were measured in the absence and presence of isoflurane or halothane. Anesthetic effects on GABA concentration responses were evaluated for individual oocytes. In wild-type alpha1beta2gamma2L GABA(A), anesthetics at approximately 2 minimum alveolar concentration (MAC) shifted GABA concentration response curves to the left approximately threefold, decreased the Hill coefficient, and enhanced currents at all GABA concentrations. The alpha1(S270I) mutation itself rendered the GABA(A)R more sensitive to GABA and reduced the Hill coefficient. At low GABA concentrations (EC5), anesthetic enhancement of peak current was much smaller in alpha1(S270I)beta2gamma2L versus wild-type channels. Paradoxically, the leftward shift of the whole GABA concentration-response relation by anesthetics was the same in both mutant and wild-type channels. At high GABA concentrations, volatile anesthetics reduced currents in alpha1(S270I)beta2gammaL GABA(A)Rs. In parallel studies on alpha1beta2 (gamma-less) GABA(A)Rs, anesthetic-induced leftward shifts in wild-type receptors were more than eightfold at 2 MAC, and the alpha1(S270I) mutation nearly eliminated anesthetic-induced leftward shift. The results support a role for alpha1S270 in alpha1beta2gamma2L GABA(A)R gating and sensitivity to inhibition by volatile anesthetics. The alpha1S270 locus also modulates anesthetic enhancement in alpha1beta2 GABA(A)R. The presence of the gamma2L subunit reduces anesthetic-induced left shift of wild-type GABA(A)R and nullifies the impact of the alpha1(S2701) mutation on anesthetic modulation. Thus, the gamma2L subunit plays a significant role in GABA(A)R modulation by volatile anesthetic compounds.