Channel Gating of the Glycine Receptor Changes Accessibility to Residues Implicated in Receptor Potentiation by Alcohols and Anesthetics

Abstract

The glycine receptor is a target for both alcohols and anesthetics, and certain amino acids in the ␣1 subunit transmembrane segments (TM) are critical for drug effects. Introducing larger amino acids at these positions increases the potency of glycine, suggesting that introducing larger residues, or drug molecules, into the drugbinding cavity facilitates channel opening. A possible mechanism for these actions is that the volume of the cavity expands and contracts during channel opening and closing. To investigate this hypothesis, mutations for amino acids in TM1 (I229C) and TM2 (G256C, T259C, V260C, M263C, T264C, and A288C showed state-dependent reaction with MTS only in the presence of agonist. M263C and S270C were also accessible to MTS labeling. Mutated residues more intracellular than M263C did not react, indicating a floor of the cavity. These data demonstrate that the conformational changes accompanying channel gating increase accessibility to amino acids critical for drug action in TM1, TM2, and TM3, which may provide a mechanism by which alcohols and anesthetics can act on glycine (and likely other) receptors.The glycine receptor (GlyR) 1 is a target for both alcohols and anesthetics. Three amino acids were previously identified as critical for alcohol and/or volatile anesthetic action on glycine receptors (as well as the homologous residues on GABA A receptors): Ile 229 (in TM1), Ser 267 (in TM2), and Ala 288 (in TM3)(1-8). To study and identify water accessible residues of ion channels, such as those in drug binding pockets, methanethiosulfonate (MTS) reagents may be used as structural probes using the substituted cysteine accessibility method (9). MTS reagents rapidly react to form disulfide bonds with cysteines in the presence of water, and an irreversible change in receptor function is taken as evidence of disulfide bond formation. By use of this method, residues accessible in the presence and/or absence of neurotransmitter to sulfhydryl-specific reagents have been determined for TM2 in GABA A and acetylcholine receptors and for TM3 and the TM2-TM3 loop for GABA A receptors (10 -14 (19) found that covalent reaction of propyl methanethiosulfonate with a cysteine introduced in the putative alcohol/anesthetic binding site of the glycine receptor irreversibly enhanced receptor function and abolished further potentiation by alcohols and anesthetics (19). Glycine receptors predominate in the spinal cord and brain stem and are present in the ventral tegmental area, a brain region of importance in the rewarding effects of alcohol (20 -24). Clinically relevant concentrations of ethanol, longer chain alcohols, and volatile anesthetics enhance the function of the glycine receptor (and the homologous GABA A receptor) in heterologous expression systems (25)(26)(27). Numerous studies have shown ethanol potentiation of glycine activated currents in cultured cells, including neurons of the hippocampus and ventral tegmental area, brain synaptoneurosomes, and mouse and chick spinal cord neurons (21, 24, 28 -31)....

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

“…Such a large potentiating effect is seen because PMTS, unlike volatile anesthetics, binds covalently and thus is always present when glycine binds to activate the receptor. A similarly large effect was previously observed with benzyl-MTS labeling of homomeric S267C GlyRs (22). In addition, our estimate of the maximal effect of glycine (EC 100 ) is likely an underestimate because drug perfusion in oocytes occurs slowly and considerable desensitization may have occurred before the current peak height was achieved.…”

“…Using the substituted cysteine accessibility method with homomeric cysteine mutants, Lobo et al (22) recently found that the GlyR alcohol and anesthetic binding pocket undergoes a conformational change upon receptor gating to the open state. This suggests that alcohol or anesthetic occupancy of the binding pocket promotes the open state by favoring an enlarged pocket conformation.…”

The structural basis of function in Cys-loop receptors

“…Mutations in M1 have been shown to produce receptors that have altered desensitization, changes in EC 50 or are non-functional (Akabas & Karlin, 1995 ;Bianchi et al 2001 ;Dang et al 2000 ;Engblom et al 2002 ;England et al 1999 ;Greenfield et al 2002 ;Lobitz et al 2001 ;Lobo et al 2004 ;Spitzmaul et al 2004 ;Zhang & Karlin, 1997). M1 may therefore be a structural element involved in transmitting movement of the ligand-bound ECD into M2, possibly through direct interactions with the M2 helix following activation (Unwin et al 2002).…”

Conformational Variability of the Glycine Receptor M2 Domain in Response to Activation by Different Agonists

“…Propofol is an intravenous anesthetic that potentiates GlyRs by binding to the alcohol and anesthetic binding site located between TM2 and TM3 (26,27). Its potentiation, particularly of taurine-and ␤-alanine-gated currents, is facilitated by mutations to R19Ј in ␣1 GlyRs (28).…”