The actions of sevoflurane and desflurane on the γ-aminobutyric acid receptor type A -: Effects of TM2 mutations in the α and β subunits

Background: Previous studies have shown that specific amino acid residues in the putative second transmembrane segment (TM2) of the gamma-aminobutyric acid receptor type A (GABA(A)) receptor play a critical role in the enhancement of GABA(A) receptor function by halothane, enflurane, and isoflurane. However, very little is known about the actions of sevoflurane and desflurane on recombinant GABA(A) receptors. The aim of this study was to examine the effects of sevoflurane and desflurane on potentiation of GABA-induced responses in the wild-type GABA(A) receptor and in receptors mutated in TM2 of the alpha1, alpha2, or beta2 subunits. Methods: GABA(A) receptor alpha1 or alpha2, beta2 or beta3, and gamma2s subunit cDNAs were expressed for pharmacologic study by transfection of human embryonic kidney 293 cells and assayed using the whole cell voltage clamp technique. Concentration-response curves and EC50 values for agonist were determined in the wild-type alpha1beta2gamma2s and alpha2beta3gamma2s receptors, and in receptors harboring mutations in TM2, such as alpha1(S270W)beta2gamma2s, alpha1beta2(N265W)gamma2s, and alpha2(S270I)beta3gamma2s. The actions of clinically relevant concentration of volatile anesthetics (isoflurane, sevoflurane, and desflurane) on GABA activated Cl- currents were compared in the wild-type and mutant GABA(A) receptors. Results: Both sevoflurane and desflurane potentiated submaximal GABA currents in the wild-type GABA(A) alpha1beta2gamma2s receptor and alpha2beta3gamma2s receptor. Substitution of Ser270 in TM2 of the alpha subunit by a larger amino acid, tryptophan (W) or isoleucine (I), as in alpha1(S270W)beta2gamma2s and alpha2(S270I)beta3gamma2s, completely abolished the potentiation of GABA-induced currents by these anesthetic agents. In contrast, mutation of Asn265 in TM2 of the beta subunit to tryptophan (W) did not prevent potentiation of GABA-induced responses. The actions of sevoflurane and desflurane in the wildtype receptor and in mutated receptors were qualitatively and quantitatively similar to those observed for isoflurane. Conclusions: Positions Ser270 of the GABA(A) alpha1 and alpha2 subunits, but not Asn265 in the TM2 of the beta2 subunit, are critical for regulation of the GABA(A) receptor by sevoflurane and desflurane, as well as isoflurane, consistent with the idea that these three volatile anesthetics share a common site of actions on the a subunit of the GABA(A) receptor.