Functional sites for anesthetics in GABAA receptors

A key target for the intravenous anesthetics propofol and etomidate is the γ-aminobutyric acid type A (GABAA) receptor [1]. GABAA receptors are the most important inhibitory neurotransmitter receptors in the central nervous system. They are composed of five subunits that surround a central Clselective ion channel. Each subunit has an extracellular domain, four trans-membrane domains, and a variable-size intracellular loop. The major receptor isoform is composed of α1, β2, and γ2 subunits, arranged counter clockwise γ2β2α1β2α1 as seen from the cell exterior [for review see 2 and references therein]. In α1β2γ2 GABAA receptors there are five subunit interfaces: two β+/αinterfaces, and one of each α+/β-, α+/γ-, and γ+/β-; where the sidedness of the subunits is designated + and -. Figure 1 shows a schematic representation of a cross section of the receptor near the extracellular surface of the membrane. Two GABA binding sites are located at the β+/αextracellular subunit interfaces. Earlier efforts to identify binding sites for intravenous anesthetics on GABAA receptors used mutational analysis, sometimes combined with cysteine modification, or photoaffinity labeling [for review see 3 and references therein]. Photoaffinity labeling using photo-reactive analogs of anesthetics is a powerful method to identify amino acid residues located in or close to the binding pocket of the anesthetic by an irreversible covalent reaction. All residues identified with this method were located at subunit interfaces in the trans-membrane domain. Although this method has the advantage that it is able to point out single amino acid residues involved in binding, it largely ignores their functional relevance. Until now mutational approaches have concentrated on a limited number of subunit interfaces, without resolving them. One of the residues identified here was β2N265. We used mutation of this and homologous residues in other subunits as reporter mutations to investigate the functional importance of all subunit interfaces for potentiation by the anesthetics etomidate and propofol [4]. The mutations were N265I, in the β2 subunit and S269I and S280I in the α1 and γ2 subunits. Receptors were expressed in Xenopus oocytes, and characterized using two electrode voltage-clamp electrophysiology. In the triply mutated receptor, which combines mutations at all interfaces, potentiation by both anesthetics was eliminated [4]. In receptors carrying the reporter mutation in the α+/βor α+/γinterfaces, i.e. α1S269Iβ2γ2 receptors, potentiation by propofol and etomidate was unaltered [4]. The γ2S280I mutation, which reports on the involvement of the γ+/βinterface, altered the potentiation of both anesthetics [4], thus indicating that these anesthetics are acting at this interface. Photoaffinity labeling propofol analogs has also identified residues in the γ+/βinterface [3, 5], but the importance for function was not clear. Introduction of the β2N265I reporter mutation located at both β+/αinterfaces, eliminated potentiation by etomidate and propofol [4]. Several point mutations of β2N265 have shown its importance of this residue for modulation by etomidate and propofol [see 3, 4 and references therein], though no photoaffinity labeling has ever been observed. Labeling of other residues at the β+/αinterface(s) has indicated presence of binding sites for etomidate and propofol [3, 5], while the contribution of individual sites could not be differentiated. By using receptor concatenation and the β2N265I mutation, we individually altered one of the two sites, and were able to dissect the functional contribution of the two β+/αinterfaces. Etomidate acted almost exclusively at the β+/αinterface flanked by γ and β subunits with a minor contribution of the β+/αinterface flanked by α and γ subunits [4]. In contrast to etomidate, both interfaces were similarly important for modulation by propofol.