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Gamma-aminobutyric acid type A (GABA(A) )receptors are targets for allosteric modulation by general anesthetics. Mutation of Ser270 within the second transmembrane domain of the GABA(A) receptor alpha subunit can ablate the modulation of the receptor by the anesthetic ether isoflurane. To investigate further the function of this critical amino acid residue, we made multiple amino acid substitutions at Ser270 and analyzed the concentration-dependent gating by GABA and regulation by isoflurane in each mutant receptor. There is a strong negative correlation between the EC(50) for GABA and the molecular volume of the amino acid residue at position 270. Replacement of Ser by large residues such as His and Trp produced a shift of the GABA concentration-response curve to the left, whereas replacement of Ser with Gly had the opposite effect. There also was a strong negative association between the molecular volume of the amino acid residue at 270 and the degree of enhancement of submaximal GABA responses by isoflurane. These results indicate the significance of the amino acid at position alpha270 in gating of the GABA(A) receptor. In addition, the data on isoflurane are consistent with the existence of a cavity of finite size in the region of alpha270 that may be filled by the anesthetic molecule or by the side chain of a larger residue at alpha270. The introduction of isoflurane, or of a large residue, into this cavity may stabilize the open state of the GABA(A) receptor relative to the closed state.

Both volatile and intravenous general anesthetics allosterically enhance gamma-aminobutyric acid (GABA)-evoked chloride currents at the GABA type A (GABAA) receptor. Recent work has revealed that two specific amino acid residues within transmembrane domain (TM)2 and TM3 are necessary for positive modulation of GABAA and glycine receptors by the volatile anesthetic enflurane. We now report that mutation of these residues within either GABAA alpha2 (S270 or A291) or beta1 (S265 or M286) subunits resulted in receptors that retain normal or near-normal gating by GABA but are insensitive to clinically relevant concentrations of another inhaled anesthetic, isoflurane. To determine whether receptor modulation by intravenous general anesthetics also was affected by these point mutations, we examined the effects of propofol, etomidate, the barbiturate methohexital, and the steroid alphaxalone on wild-type and mutant GABAA receptors expressed in human embryonic kidney 293 cells. In most cases, these mutations had little or no effect on the actions of these intravenous anesthetics. However, a point mutation in the beta1 subunit (M286W) abolished potentiation of GABA by propofol but did not alter direct activation of the receptor by high concentrations of propofol. These data indicate that the receptor structural requirements for positive modulation by volatile and intravenous general anesthetics may be quite distinct.

Glycine and gamma-aminobutyric acid (GABA)A receptors are members of the "superfamily" of ion channels, and are sensitive to allosteric modulation by n-alcohols such as ethanol and butanol. We recently demonstrated that the mutation of Ser-267 to Ile in the alpha1 subunit abolished ethanol regulation of glycine receptors (Gly-R). In the present study, a pair of chimeric receptors was studied, in which a 45-amino acid domain comprising transmembrane domains 2 and 3 was exchanged between the Gly-Ralpha1 and gamma-aminobutyric acid rho1 subunits. Detailed pharmacologic analysis of these chimeras confirmed that this domain of the Gly-R confers enhancement of receptor function by ethanol and butanol. An extensive series of mutations at Ser-267 in the Gly-Ralpha1 subunit was also prepared, and the resulting homomeric receptors were expressed and tested for sensitivity to glycine, and allosteric modulation by alcohols. All of the mutant receptors expressed successfully in Xenopus oocytes. Mutation of Ser-267 to small amino acid residues such as Gly or Ala produced receptors in which glycine responses were potentiated by ethanol. As we have reported previously, the mutant Gly-Ralpha1 (Ser-267 --Ile) was completely insensitive to ethanol; mutation of Ser-267 to Val had a similar effect. Mutation of Ser-267 to large residues such as His, Cys, or Tyr resulted in inhibition of Gly-R function by ethanol. These results demonstrate that the size of the amino acid residue at position alpha267 plays a crucial role in determining the functional consequences of allosteric modulation of the Gly-R by alcohols.

GABAA and glycine receptors are close relatives in the "gene superfamily" of ligand-gated ion channels, but have distinctly different pharmacology. For example, barbiturates have two effects on GABAA receptors (GABAA-R): at low micromolar concentrations (2-5 microM), the anesthetic barbiturate methohexital potentiates submaximal chloride current responses to GABA; at higher concentrations (20-50 microM), the barbiturate causes direct gating of the channel in the absence of agonist. Neither of these barbiturate effects is seen on the glycine receptor (Gly-R). In order to study the structural parts of the GABAA-R involved in this barbiturate pharmacology, two unique restriction sites were introduced into the cDNAs encoding the alpha 2 and beta 1 subunits of the human GABAA-R and the alpha 1 subunit of the human gly-R. The first site ('X') corresponded to the C-terminal end of the third transmembrane domain (M3) in each subunit and enabled exchange of C-terminal fragment of approximately 100 amino acids (which includes the large 'cytoplasmic loop' and M4 segment) between GABAA-R and Gly-R subunits. The second site ('S') was approximately 30 amino acids 3'- from the N-terminal end of each subunit and enabled exchange of a small N-terminal fragment between GABAA-R and Gly-R subunits. Several chimeric receptor subunit cDNAs were constructed and the resulting receptors tested for their ability to respond to GABA and glycine and for sensitivity to the barbiturate methohextial (MTX). The results show that neither the large C-terminal fragment nor the smaller N-terminal fragment is associated with the enhancement or direct activation of the GABAA-R by MTX. These results demonstrate the viability of chimeric GABAA/Gly-R and suggest that the method will be suitable for further investigation of the molecular basis of the barbiturate pharmacology of the GABA-R.