The γ subunit determines whether anesthetic-induced leftward shift is altered by a mutation at α1S270 in α1β2γ2L GABAA receptors

Background: 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 cu, subunit serine at position 270 to isoleucine [alpha (1)(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 alpha (1)(S270I) affects enhancement versus inhibition by volatile anesthetics. Furthermore, the majority of GABA,Rs in mammalian brain are thought to consist of alpha (1), beta (2), and gamma (2) subunits, and the alpha (1)(S270I) mutation has not been studied in the context of this combination. Methods: Recombinant GABA(A)Rs composed of alpha (1)beta (2) or alpha (1)beta (2)gamma (21) subunit mixtures were studied electrophysiologically in whole Xenopus oocytes in the voltage clamp configuration. Currents elicited by GABA (0.03 muM 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. Results: In wild-type alpha (1)beta (2)gamma (21) 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 alpha (1)(S270I) mutation itself rendered the GABA,R more sensitive to GABA and reduced the Hm coefficient. At low GABA concentrations (EC5), anesthetic enhancement of peak current was much smaller in alpha (1)(S270I)beta (2)gamma (21), 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 alpha (1)(S270I)beta (2)gamma (21), GABA(A)Rs. In parallel studies on alpha (1)beta (2) (gamma -less) GABA,Rs, anesthetic-induced leftward shifts in wild-type receptors were more than eightfold at 2 MAC, and the alpha (1)(S270I) mutation nearly eliminated anesthetic-induced leftward shift. Conclusions The results support a role for alpha (1)S270 in alpha (1)beta (2)gamma (21) GABA(A)R gating and sensitivity to inhibition by volatile anesthetics. The alpha (1)S270 locus also modulates anesthetic enhancement in alpha (1)beta (2) GABA(A)R, The presence of the gamma (21) subunit reduces anesthetic-induced left shift of wild-type GABA,R and nullifies the impact of the alpha (1)(S270I) mutation on anesthetic modulation, Thus, the gamma (21) subunit plays a significant role in (;ABA,R modulation by volatile anesthetic compounds.