Direct interactions of anesthetics and nonanesthetics with the nicotinic acetylcholine receptor pore

Abstract

(1) We review evidence that anesthetics inhibit peripheral nAChR cation translocation by binding directly to a protein site in the transmembrane pore. (2) This site is near the middle of the pore-forming M2 domains on alpha and beta subunits, but further from the homologous portions of gamma and delta subunits. (3) Interactions between both anesthetics and nonanesthetics with the nAChR pore site are determined primarily by hydrophobic forces rather than steric factors. (4) Anesthetics and nonanesthetics display different state-dependent accessibility to this site, suggesting a mechanism for the different in vivo actions of these two classes of drugs.

Introduction

Neuronal ion channels are considered likely targets for anesthetics, since they underlie excitability in the CNS. Postsynaptic ligand-gated ion channels are particularly sensitive at concentrations that anesthetize experimental animals and humans (Franks and Lieb, 1994). General anesthetic interactions with peripheral nicotinic acetylcholine receptors (nAChRs) have been characterized in great detail, demonstrating that anesthetics produce a number of changes in nAChR function that reflect anesthetic effects on neuronal ligand-gated ion channels (Forman and Miller, 1989). This review focuses on the molecular mechanism of inhibition of nAChR-mediated cation translocation in the presence of clinical anesthetic concentrations. nAChR-mediated cation translocation is thought to occur via a transmembrane pore formed by homologous domains from each of five (stoichiometry α₂βγδ) subunits arranged around this water-filled passage (Devillers-Thiery et al., 1993). Enhanced electron micrographic analysis of nAChR from Torpedo demonstrates a roughly pentagonal protein structure with a central channel (Unwin, 1993). Photolabeling of Torpedo nAChR protein and site-directed mutagenesis of Torpedo nAChR and homologous vertebrate nAChRs identify a set of amino acids in the second of four predicted transmembrane domains (M2) in each receptor subunit that interact with translocated ions as well as blockers (Leonard et al., 1988, Imoto et al., 1991, Cohen et al., 1992, Pedersen et al., 1992). Most models of the nAChR pore incorporate a five-fold symmetry assumption such that the M2 domains surround the pore like barrel staves and homologous amino acid side-chains (aligned from 1′ at the cytoplasmic surface to 20′ at the extracellular surface) on each subunit M2 domain form distinct `rings' along the transmembrane axis (Imoto et al., 1988, Miller, 1989, Devillers-Thiery et al., 1993). A symmetrical pore is consistent with both the high degree of amino acid sequence homology among the nAChR subunit M2 domains (Table 1) and site-directed mutagenesis studies showing that homologous mutations on different subunits have similar functional effects.

Here, we review the evidence suggesting that both anesthetics and nonanesthetics inhibit the nAChR by binding to a discrete site within the transmembrane pore. The initial studies focus on the difficult job of demonstrating the existence of this site in a complex protein that functions within a membrane environment (Forman et al., 1995). With the pore site established, its shape and location were probed by evaluating the contributions of different M2 side-chains to the anesthetic binding energy (Forman, 1997). Finally, interactions of nonanesthetic volatiles with the pore site were investigated to determine whether this site discriminates between anesthetic and non-anesthetic drugs (Forman and Raines, 1998). Data is expressed as mean±standard error of the mean throughout this paper.