Occupancy of a Single Anesthetic Binding Pocket Is Sufficient to Enhance Glycine Receptor Function

Abstract

Alcohols and volatile anesthetics enhance the function of inhibitory glycine receptors (GlyRs). This is hypothesized to occur by their binding to a pocket formed between the transmembrane domains of individual ␣1 GlyR subunits. Because GlyRs are pentameric, it follows that each GlyR contains up to five alcohol/anesthetic binding sites, with one in each subunit. We asked how many subunits per pentamer need be bound by drug in order to enhance receptormediated currents. A cysteine mutation was introduced at amino acid serine 267 (S267C) in the transmembrane 2 domain as a tool to block GlyR potentiation by some anesthetic drugs and to provide a means for covalent binding by the small, anesthetic-like thiol reagent propyl methanethiosulfonate. Xenopus laevis oocytes were coinjected with various ratios of wild-type (wt) to S267C ␣1 GlyR cDNAs in order to express heteromeric receptors with a range of wt:mutant subunit stoichiometries. The enhancement of GlyR currents by 200 mM ethanol and 1.5 mM chloroform was positively correlated with the number of wt subunits found in heteromeric receptors. Furthermore, currents from oocytes injected with high ratios of wt to S267C cDNAs (up to 200:1) were significantly and irreversibly enhanced following propyl methanethiosulfonate labeling and washout, demonstrating that drug binding to a single subunit in the receptor pentamer is sufficient to induce enhancement of GlyR currents.Although volatile anesthetics were long believed to have nonspecific, lipid-based mechanisms of action, accumulating evidence led to a shift in research focus to the study of protein sites of anesthetic action and especially to the effects of these drugs on ion channels (1, 2). Among the most studied molecular targets for these drugs are members of the Cysloop family of ligand-gated ion channels, including the glycine receptor (GlyR), 2 the primary inhibitory neurotransmitter receptor in the spinal cord and brain stem (3). Studies in rats have shown that GlyRs mediate the immobilizing effects of the anesthetics halothane, isoflurane, and cyclopropane (4). In transgenic mice expressing an alcohol-insensitive mutant GlyR, these receptors mediate, at least in part, the sedating and anesthetic effects of alcohol (5). These in vivo data agree with functional studies indicating that pharmacologically relevant concentrations of alcohols and volatile anesthetics potentiate GlyRs in both brain slices and heterologous expression systems (6 -9).Significant advances have been made in the understanding of the molecular mechanisms of alcohol and volatile anesthetic enhancement of GlyR function. The GlyR consists of a pentameric assembly of subunits surrounding a central, anion-conducting pore (10). Each of these subunits contains a large, N-terminal extracellular domain responsible for agonist binding, as well as four transmembrane (TM) domains, of which TM2 lines the channel pore and forms the channel gate (11). Mihic et al. (12) identified two amino acids, serine 267 (Ser-267) in TM2 and alanine 288 (Ala-288) in TM...

Assessment of Homology Templates and an Anesthetic Binding Site within the γ-Aminobutyric Acid Receptor

“…This propofol was optimized into position using molecular mechanics with the CHARMm force field, followed by molecular dynamics, 26 while allowing for sidechain flexibility with only backbone atoms fixed. While the work of Mihic et al has demonstrated that occupancy of only one binding site is sufficient to enhance the homologous glycine receptor’s function, 27 the work of Forman et al . 28–31 shows that propofol’s enhancing action in GABAaR requires occupancy of perhaps three propofol sites.…”

Structure and Pharmacologic Modulation of Inhibitory Glycine Receptors

“…From mutagenesis and physiologic studies, it is believed that the binding site for volatile anesthetics on GlyR consists of amino acids located in TM2 and TM3 that form an intrasubunit cavity (Mihic et al, 1997;Roberts et al, 2006;Duret et al, 2011) and include residues S267 (TM2) and A288 (TM3) (Horani et al, 2015) (Fig. 2A,D).…”

Molecular targets and mechanisms for ethanol action in glycine receptors

“…In addition, ethanol, in the absence of agonist, causes a conformational change in the receptor that can change accessibility of TM residues (Jung et al , 2005;Jung and Harris, 2006). Finally, PMTS occupancy of a single anesthetic binding pocket within the GlyR pentamer is sufficient to enhance receptor function (Roberts et al , 2005). Taken together, these studies support the notion that TM position 267 is capable of initiating the actions of ethanol.…”