GABA(A) receptors as molecular targets of general anesthetics: identification of binding sites provides clues to allosteric modulation

Abstract

Purpose: The purpose of this review is to summarize current knowledge of detailed biochemical evidence for the role of γ-aminobutyric acid type A receptors (GABA(A)-Rs) in the mechanisms of general anesthesia.

Principal findings: With the knowledge that all general anesthetics positively modulate GABA(A)-R-mediated inhibitory transmission, site-directed mutagenesis comparing sequences of GABA(A)-R subunits of varying sensitivity led to identification of amino acid residues in the transmembrane domain that are critical for the drug actions in vitro. Using a photo incorporable analogue of the general anesthetic, R(+)etomidate, we identified two transmembrane amino acids that were affinity labelled in purified bovine brain GABA(A)-R. Homology protein structural modelling positions these two residues, αM1-11' and βM3-4', close to each other in a single type of intersubunit etomidate binding pocket at the β/α interface. This position would be appropriate for modulation of agonist channel gating. Overall, available information suggests that these two etomidate binding residues are allosterically coupled to sites of action of steroids, barbiturates, volatile agents, and propofol, but not alcohols. Residue α/βM2-15' is probably not a binding site but allosterically coupled to action of volatile agents, alcohols, and intravenous agents, and α/βM1-(-2') is coupled to action of intravenous agents.

Conclusions: Establishment of a coherent and consistent structural model of the GABA(A)-R lends support to the conclusion that general anesthetics can modulate function by binding to appropriate domains on the protein. Genetic engineering of mice with mutation in some of these GABA(A)-R residues are insensitive to general anesthetics in vivo, suggesting that further analysis of these domains could lead to development of more potent and specific drugs.

Fig. 1

Three-dimensional views of the γ-aminobutyric acid type A receptors (GABA_AR) homology model from different perspectives (A,B) and helical wheel representation of the transmembrane regions at the etomidate binding domain at the β/α interface (C). A) Three-dimensional view of the GABA_AR homology model (β3, cyan; α1, yellow; γ2, green) from a perspective outside the membrane looking down the channel or B) from a perspective parallel to the membrane surface. Modified from Li et al. (2006) in which our homology model is based roughly on the nicotinic acetylcholine receptor cryo-EM derived structure (PDB:2BG9), modified by helical alignment data from Jansen & Akabas (2006). Pockets in the vicinity of the etomidate (maroon), GABA (purple), and benzodiazepine (red) binding sites are shown as Connolly surfaces. C) Helical wheel representation of the GABA_AR β/α interface in the trans-membrane domain illustrating the proposed binding site for etomidate (modified from Li et al., 2009). The model illustrates the orientation of residues from a homology model built on the nicotinic acetylcholine receptor cryo-EM derived structure, with the residues in αM1 and βM3 photolabelled by [³H]azietomidate (circled residues in green) contributing to a common binding pocket at the β/α interface. Also included: the position in βM2 (N265) that functions as a determinant of etomidate/azietomidate anesthetic potency in vivo (pink),,; the residues in αM1 and βM3 identified as sensitivity determinants for direct activation by neurosteroids (boxed residues in yellow); and the positions in αM1 and βM3 that can form intersubunit cross-links when mutated to Cys (red and orange),

Fig. 2

The intersubunit anesthetic binding pocket in the γ-aminobutyric acid type A (GABA_A) receptor transmembrane domain identified by photoaffinity labelling with [³H]azietomidate. The identified residue, M236, in the M1 domain of the α subunit is situated nearly adjacent to the identified residue, M286, in the M3 domain of the β subunit. Reproduced with permission from: Li GD, Chiara DC, Sawyer GW, Hussain SS, Olsen RW, Cohen JB. Identification of a GABA_A receptor anesthetic binding site at subunit interfaces by photolabelling with an etomidate analog. J Neurosci 2006; 45: 11599-605