Transmembrane residues define the action of isoflurane at the GABA_A receptor alpha-3 subunit

Abstract

The γ-aminobutyric acid type A (GABA_A) receptor is the target of a structurally diverse group of sedative, hypnotic, and anesthetic drugs, including the volatile anesthetic isoflurane. Previous studies on the GABA_A receptor have suggested the existence of a cavity located between transmembrane (TM) segments 2 and 3 in both alpha-1 and alpha-2 subunits, within which volatile anesthetics might bind. In this study, we have used site-directed mutagenesis to investigate the involvement of homologous residues of the GABA_A alpha-3 subunit in allosteric modulation by isoflurane. Mutation of serine residue 294 within the TM2 to histidine or tyrosine increased the potency of GABA and decreased positive modulation by isoflurane. Mutation of alanine residue 315 within the TM3 to tryptophan increased the potency of GABA and abolished isoflurane modulation. The activity of the intravenous anesthetic propofol was unaltered from wild-type at these mutant receptors. These findings are consistent with the action of isoflurane on a critical site within the transmembrane domains of the receptor and suggest a degree of functional homology between the GABA_A alpha-1, -2, and -3 subunits.

Introduction

The γ-aminobutyric acid type A (GABA_A) receptor complex has emerged as a strong candidate to mediate the actions of volatile anesthetics. These receptors are ligand-gated ion channels that subserve fast inhibitory neurotransmission in the mammalian brain and are regulated allosterically by a structurally diverse group of compounds, which includes halogenated volatile anesthetics, n-alcohols, propofol, barbiturates, benzodiazepines, and steroids. The GABA_A receptor is a heteromeric complex assembled from different subunits (α1-6, β1-3, γ1-3, δ, ɛ, π, and θ), which combine to form a chloride channel. In vivo GABA_A receptors likely assemble into pentameric complexes in a stoichiometry of 2α:2β:1γ [4]. Immunohistochemical studies in the mammalian brain have identified heterogeneous patterns of GABA_A subunit expression [7]. The most prevalent receptor complex consists of the subunit combination α1β2γ2 and represents ∼50% of the total GABA_A population [8]. In contrast, the α3 subunit is expressed at lower levels and has been localized to the cell bodies of neurons in the cortex, amygdala, reticular nucleus of the thalamus, serotonergic neurons of the raphe nuclei [9], to cholinergic neurons of the medial septum-diagonal band complex of the basal forebrain [10] and noradrenergic neurons of the locus coeruleus [16]. The α3 subunit most frequently coassembles with β2/3 and γ2 subunits, and this minor receptor subtype accounts for ∼15% of the total complement of receptors [17].

Previous studies have demonstrated that specific point mutations in the GABA_A receptor can selectively abolish agonist potentiation by inhaled anesthetics [12], [18]. Site-directed mutagenesis has identified residues Ser-270 and Ala-291 within the transmembrane spanning segments (TM) of the α1 and α2 subunits as part of a proposed binding cavity for the volatile anesthetics isoflurane, halothane, and chloroform [12]. Extensive mutagenesis at Ser270 in the α2 subunit also suggests a role of this residue in channel gating [14].

To date, there have been few structure–function studies involving the GABA_A α3 subunit. The high degree of homology between GABA_A α subunits sequences would suggest that conserved residues might function in a common capacity. The TM2 residue α3(Ser294) is homologous to Ser270 in the α1 and α2 subunits, and the TM3 residue α3(Ala315) is homologous to Ala291 in the α1 and α2 subunits. In the present report, we have used site-directed mutagenesis at these residues in the GABA_A α3 subunit to examine receptor activation and modulation by anesthetics.