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 α1 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 receptor-mediated 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 co-injected with various ratios of wild-type (wt) to S267C α1 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. 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 α1 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 receptor-mediated 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 co-injected with various ratios of wild-type (wt) to S267C α1 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 (1Mihic S.J. Neurochem. Int. 1999; 35: 115-123Crossref PubMed Scopus (134) Google Scholar, 2Rudolph U. Antkowiak B. Nat. Rev. Neurosci. 2004; 5: 709-720Crossref PubMed Scopus (612) Google Scholar). Among the most studied molecular targets for these drugs are members of the Cys-loop family of ligand-gated ion channels, including the glycine receptor (GlyR), 2The abbreviations used are: GlyR, glycine receptor; PMTS, propyl methanethiosulfonate; TM, transmembrane domain; wt, wild-type. the primary inhibitory neurotransmitter receptor in the spinal cord and brain stem (3Legendre P. Cell. Mol. Life Sci. 2001; 58: 760-793Crossref PubMed Scopus (438) Google Scholar). Studies in rats have shown that GlyRs mediate the immobilizing effects of the anesthetics halothane, isoflurane, and cyclopropane (4Zhang Y. Laster M.J. Hara K. Harris R.A. Eger E.I. Stabernack C.R. Sonner J.M. Anesth. Analg. 2003; 96: 97-101Crossref PubMed Google Scholar). In transgenic mice expressing an alcohol-insensitive mutant GlyR, these receptors mediate, at least in part, the sedating and anesthetic effects of alcohol (5Findlay G.S. Wick M.J. Mascia M.P. Wallace D. Miller G.W. Harris R.A. Blednov Y.A. J. Pharmacol. Exp. Ther. 2002; 300: 526-534Crossref PubMed Scopus (58) Google Scholar). 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 (6Mascia M.P. Machu T.K. Harris R.A. Br. J. Pharmacol. 1996; 119: 1331-1336Crossref PubMed Scopus (194) Google Scholar, 7Downie D.L. Hall A.C. Lieb W.R. Franks N.P. Br. J. Pharmacol. 1996; 118: 493-502Crossref PubMed Scopus (130) Google Scholar, 8Beckstead M.J. Phelan R. Mihic S.J. J. Biol. Chem. 2001; 276: 24959-24964Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 9Daniels S. Roberts R.J. Toxicol. Lett. (Shannon). 1998; 100–101: 71-76Crossref PubMed Scopus (49) Google Scholar). 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 (10Langosch D. Thomas L. Betz H. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 7394-7398Crossref PubMed Scopus (309) Google Scholar). 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 (11Lynch J.W. Physiol. Rev. 2004; 84: 1051-1095Crossref PubMed Scopus (617) Google Scholar). Mihic et al. (12Mihic S.J. Ye Q. Wick M.J. Koltchine V.V. Krasowski M.D. Finn S.E. Mascia M.P. Valenzuela C.F. Hanson K.K. Greenblatt E.P. Harris R.A. Harrison N.L. Nature. 1997; 389: 385-389Crossref PubMed Scopus (1103) Google Scholar) identified two amino acids, serine 267 (Ser-267) in TM2 and alanine 288 (Ala-288) in TM3, that, when mutated, blocked alcohol and anesthetic enhancement of GlyR-mediated currents. Alcohols and anesthetics were hypothesized to potentiate GlyRs by binding in a water-filled pocket formed between TM2 and TM3. A subsequent study supported this hypothesis by showing that thiol reagents could covalently react with cysteine residues at position 267 (S267C) to irreversibly enhance receptor function (13Mascia M.P. Trudell J.R. Harris R.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 9305-9310Crossref PubMed Scopus (234) Google Scholar). Thiols, like volatile anesthetics, may enhance GlyR function by occupying space within the water-filled pocket. Interestingly, recent structural data for the nicotinic acetylcholine receptor, another Cys-loop family member, indicates that there is a water-filled crevice located between the transmembrane domains of each receptor subunit (14Miyazawa A. Fujiyoshi Y. Unwin N. Nature. 2003; 423: 949-955Crossref PubMed Scopus (1082) Google Scholar). Each of the five subunits in a homomeric GlyR possesses a possible TM2/TM3 anesthetic binding site. In previous studies of binding pocket mutants, the mutant subunits were expressed without wt subunits so that all five subunits in receptors bore the mutation; as a result it was unclear how many subunits had to be bound by anesthetics for their enhancing effects to be observed. In the present study, we tested the hypothesis that occupancy of a single anesthetic binding pocket per receptor pentamer is sufficient to potentiate GlyR currents. By co-injecting different ratios of the S267C mutant cDNA with wild-type α1 GlyR subunit cDNAs in Xenopus laevis oocytes, we generated heteromeric receptors containing one to four mutant subunits and then tested the effects of alcohols, anesthetics, and the thiol labeling reagent propyl methanethiosulfonate (PMTS) on these receptors. Our results suggest for the first time that occupancy of even a single alcohol or anesthetic binding pocket in a GlyR pentamer can result in enhancement of GlyR function. Two-electrode voltage clamp experiments were conducted on X. laevis oocytes expressing α1 GlyR subunits. Frogs (Xenopus Express) were maintained and oocytes harvested and isolated as described previously (15Beckstead M.J. Weiner J.L. Eger E.I. Gong D.H. Mihic S.J. Mol. Pharmacol. 2000; 57: 1199-1205PubMed Google Scholar). A cDNA encoding the wild-type (wt) α1 GlyR subunit was previously subcloned into the pBK-CMV expression vector (12Mihic S.J. Ye Q. Wick M.J. Koltchine V.V. Krasowski M.D. Finn S.E. Mascia M.P. Valenzuela C.F. Hanson K.K. Greenblatt E.P. Harris R.A. Harrison N.L. Nature. 1997; 389: 385-389Crossref PubMed Scopus (1103) Google Scholar). Using the QuikChange mutagenesis kit (Stratagene), the codon for serine 267 was mutated to a cysteine codon (S267C) or a methionine codon (S267M) and the codon for proline 250 was mutated to a threonine codon (P250T). cDNA concentrations and purities were ascertained through UV absorption spectra on a DU 640 spectrophotometer (Beckman). Wild-type, S267C, S267M, and P250T cDNAs were injected into the nuclei of isolated oocytes, either individually to express homomeric GlyRs or in a range of wt:S267C, S267M:S267C, or P250T:S267C cDNA ratios so as to promote the expression of heteromeric GlyRs. Assuming that wt and S267C subunits are equally likely to assemble into receptor pentamers, the predicted distribution of receptor compositions can be described by the formula shown in Equation 1 P=100×pwtX×pS267C(5−X)×5!X!(5−X)!(Eq. 1) where P represents the percentage of receptors containing x wt subunits and (5 – x) S267C subunits, pwt represents the fraction of wt cDNAs injected, and pS267C represents the fraction of S267C cDNAs injected. Calculated receptor composition percentage distributions for the cDNA ratios tested are shown in Table 1.TABLE 1Predicted prevalence of receptor subunit compositions according to the ratio of wt to S267C α1 GlyR cDNAs injected Open table in a new tab Recordings were made 1–7 days after cDNA injection. Oocytes were perfused with modified Barth's solution, impaled with two electrodes filled with 3 m KCl, and voltage clamped at –70 mV. Glycine and drug solutions were prepared in modified Barth's solution at the indicated concentrations and were applied through the bath perfusion. At the beginning of each experiment, maximal GlyR currents were determined by a 30-s application of 10 mm glycine. All subsequent glycine and glycine+ drug solutions were applied for 45 s and were followed by 10-min modified Barth's solution washouts to ensure receptor recovery from desensitization. In most experiments, the concentration of glycine producing roughly a 10% maximal effect (EC10) was determined next, and the oocyte was considered suitable for recording if the EC10 current magnitude changed less than 20% over a series of three applications. For the PMTS labeling experiments, EC10 glycine was established, the oocyte was washed for 5 PMTS was applied for and a was the glycine concentrations of the volatile anesthetics the concentration and were determined by E.I. P. Laster M.J. Gong D. Harris R.A. Trudell J.R. A. Anesth. Analg. 1999; PubMed Google Scholar). All were from for and PMTS and of and from were conducted as described previously G.S. Phelan R. Roberts S.E. Mihic S.J. Blednov Y.A. Harris R.A. J. Neurosci. 2003; PubMed Google Scholar). were to that the electrodes had to promote the of were perfused with an to a to a solution time of Significant were by or of with or with or The S267C mutation in TM2 of the α1 subunit of the GlyR receptor enhancement by ethanol Q. Koltchine V.V. Mihic S.J. Mascia M.P. Wick M.J. Finn S.E. Harrison N.L. Harris R.A. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar) potentiation by isoflurane, or (13Mascia M.P. Trudell J.R. Harris R.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 9305-9310Crossref PubMed Scopus (234) Google Scholar). We and these by the effects of the alcohols ethanol and and the volatile anesthetics isoflurane, and on currents in oocytes expressing either homomeric wt α1 GlyRs or homomeric S267C α1 GlyRs. all these potentiation of wt GlyR currents when with EC10 glycine In oocytes expressing the mutant receptor, the S267C mutation blocked GlyR potentiation by 200 mm ethanol and changed the effect of 1.5 mm chloroform into a A and S267C had effect on receptor potentiation by mm isoflurane, mm and mm We hypothesized that the number of mutant a between receptor composition and the of receptor potentiation by ethanol and express heteromeric oocytes were injected with of wt and S267C cDNAs prepared in ratios of and the that wt and S267C cDNAs receptor subunits equally well and that the S267C mutation or effect on pentamer these cDNA to receptors with an of two to and four mutant subunits, Table 1 distributions of receptor subunit compositions for each of the cDNA ratios injected. In these oocytes, receptor enhancement by 200 mm ethanol and 1.5 mm chloroform as the of S267C subunits For GlyR potentiation was when an of of the receptor subunits mutant binding for potentiation was an of four subunits bore the mutant binding pocket. with the homomeric potentiation of the heteromeric receptors by mm isoflurane, mm and mm on receptor subunit indicating that the heteromeric receptors the to be by alcohols and anesthetics how many binding be to induce receptor we covalently modified the anesthetic binding pocket. PMTS is a small, that with the thiol of cysteine residues to a propyl through a was previously that of homomeric S267C mutant GlyRs with PMTS results in labeling of the mutant cysteine and potentiation of receptor function after PMTS (13Mascia M.P. Trudell J.R. Harris R.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 9305-9310Crossref PubMed Scopus (234) Google Scholar). this to binding pocket we that 5 mm PMTS GlyR function like alcohols or anesthetics when to wt and S267C GlyRs with an EC10 concentration of glycine a 10-min washout, the wt to EC10 glycine was from indicating that PMTS with these receptors. In the S267C significantly after PMTS washout, indicating that PMTS covalently the mutant cysteine in S267C receptors that the binding pocket by the propyl to the mutant We asked PMTS labeling of a single mutant cysteine in receptor containing four wt subunits and one S267C subunit to receptor express receptors containing one mutant oocytes were injected with cDNAs in the ratio of on the shown in Table this ratio to a of receptors in which are homomeric wt and the are the heteromeric were to one mutant subunit and the to two S267C subunits. For each an EC10 concentration of glycine was established, PMTS was perfused for and the to EC10 glycine was 5 A and from oocytes injected with the ratio of cDNAs were significantly following PMTS application and washout, as were currents from oocytes injected with S267C GlyR In currents from wt oocytes were enhanced by the Because PMTS labeling of the oocytes a we hypothesized that we could the of S267C cDNAs injected and a potentiation of GlyR function after PMTS By the of S267C subunits in the subunit the of than one mutant subunit into a receptor pentamer be this oocytes were injected with ratios of subunit cDNAs and as the to EC10 glycine was determined and after PMTS the potentiation was for each cDNA and the data a between the predicted percentage of heteromeric receptors to receptors and the of receptor enhancement This the hypothesis that occupancy of a single alcohol and anesthetic binding pentamer is sufficient to induce receptor Because drug binding to one pentamer was sufficient to enhance receptor we tested the hypothesis that the subunits binding drug could the enhancing effects of the drug bound to the first subunit. we a serine to methionine mutation at position 267 (S267M) that covalently react with GlyR potentiation by most alcohols and volatile anesthetics when expressed Oocytes were injected with and ratios of or α1 GlyR subunit and current to EC10 glycine were determined and after application of PMTS At both the and cDNA the mutation significantly the enhancing effects of PMTS when with currents from oocytes injected with cDNAs at the ratios These results suggest that anesthetic enhancement of GlyR function can be by subunits in the pentamer that are with the S267C mutation significantly GlyR expression or we homomeric and heteromeric GlyRs in two currents from oocytes application of a maximal concentration of glycine were The of these provide a of the expression and function of receptors G.S. Phelan R. Roberts S.E. Mihic S.J. Blednov Y.A. Harris R.A. J. Neurosci. 2003; PubMed Google Scholar). was a between the maximal currents by homomeric wt and S267C receptors maximal currents from oocytes injected with the cDNA ratios in this significantly from wt or S267C homomeric receptors. Because the magnitude of maximal currents on the and and expression of we wt and S267C receptor function in from and to the S267C mutation of these studies that the time were for both wt and S267C homomeric receptors and that the receptors in their of in to a of 10 mm glycine The of wt α1 GlyR in to application of 10 glycine was for homomeric S267C α1 GlyR it was The in currents from wt and S267C in oocytes can likely be by the of S267C receptors. if channel between wt and S267C receptors individual wt and S267C receptors and can be the maximal currents be of channel on the is likely that the ratios of subunit cDNAs injected the protein of subunits found on the glycine data were from oocytes injected with wt or S267C cDNAs or a of cDNAs of the function that the S267C mutation had a effect on glycine in both heteromeric and homomeric mutant receptors. the S267C mutation subunit expression we an by and Neurosci. Lett. 2002; PubMed Scopus Google Scholar). These identified a GlyR α1 mutation that results in receptors with significantly glycine various ratios of wt to P250T found that the glycine of receptors as the of P250T cDNAs We that if this P250T mutant were co-injected with the S267C which evidence of a and if the S267C mutant were expressed at a than we then one P250T:S267C to glycine than P250T This was the studies suggest that alcohols and anesthetics GlyR function by binding to formed between the transmembrane domains of individual GlyR subunits. Because GlyRs are pentameric, each GlyR contains five binding In the present study, we asked how many of these binding be by drug in order to enhance GlyR currents. this GlyR subunits containing the S267C binding pocket mutation were with wild-type subunits in various ratios to heteromeric receptors containing different of mutant binding The of these mutant binding were then used to the effects of alcohols, anesthetics, and the thiol reagent PMTS on these receptors. In homomeric mutant the S267C mutation blocked GlyR potentiation by 1.5 mm chloroform and 200 mm ethanol block potentiation by mm mm halothane, and mm These data that S267C the enhancing effects of the chloroform and ethanol of This may be the of anesthetics to with residues in the binding pocket than ethanol and to the effects of the mutant in the binding pocket. be that the concentration of ethanol used in this study represents an anesthetic concentration and is than the mm alcohol concentration that the for alcohol receptor may the GlyR to ethanol at concentrations to the Chem. 2002; PubMed Scopus Google Scholar, M.P. Wick M.J. Harris R.A. Br. J. Pharmacol. 1998; PubMed Scopus Google Scholar). oocytes were injected with and ratios of chloroform and ethanol potentiation of receptor currents in a as the of S267C subunits These data suggest that receptor currents could be enhanced even when chloroform or ethanol could with all possible binding in the receptors. The of alcohols and anesthetics from these drugs to the number of binding that to be for enhancement of receptor function. labeling by PMTS, a to alcohols and anesthetics that enhanced receptor currents as an alcohol or anesthetic to this We that co-injecting oocytes with a high ratio of GlyR cDNAs the of homomeric wt GlyRs. A percentage of receptors be into containing four wt subunits and one mutant and S267C subunits. In oocytes injected with a ratio of PMTS followed by a washout, in enhancement of GlyR currents. this we the ratio of cDNAs injected to and Each of these cDNA ratios receptors that were significantly and irreversibly by At the of receptors were to be of wt subunits, the receptors a single S267C subunit and four wt subunits. Because PMTS had enhancing effect on homomeric wt the enhancement of receptor currents could be to PMTS occupancy of the single mutant binding pocket in heteromeric receptors containing one S267C subunit. the of potentiation in these receptors was correlated with the cDNA ratio hypothesis that S267C subunits were expressed when less S267C cDNA was injected. of PMTS in potentiation of GlyR function in receptors containing S267C subunits. For after of a ratio of one of expressed receptors to a single S267C subunit and the to be of wt subunits. The in current following PMTS application have been to a in the current generated by this of receptors. A be than the predicted glycine for these receptors. a effect is PMTS, volatile anesthetics, covalently and is present when glycine to the A effect was previously with labeling of homomeric S267C GlyRs Mascia M.P. Trudell J.R. Harris R.A. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). In of the maximal effect of glycine is likely an drug in oocytes and may have the current was the for glycine in these experiments are likely PMTS binding to a single S267C is sufficient to enhance GlyR ethanol and chloroform enhancement of GlyR function as the number of S267C subunits in heteromeric receptors A is that the of all subunits in a receptor and the with anesthetics the GlyR to anesthetics, all five subunits to the of the channel gate and the We tested this hypothesis by producing receptors that had at most a single S267C subunit with four subunits. labeling with PMTS, we found that these receptors were significantly less enhanced than their the of ethanol and chloroform to enhance receptors containing one or two wt subunits may be blocked by the S267C subunits the of the receptor Our of results on the hypothesis that the ratio of GlyR subunit cDNAs injected into oocytes is in the ratio of subunits found in the receptors. cDNAs injected in high ratios promote the of heteromeric receptors containing a single mutant subunit. lines of evidence that this is the S267C mutation is made by one in the GlyR α1 subunit it that there are in the of and between wt and S267C previous studies determined that the responsible for GlyR assembly are in the N-terminal of the from the S267C in TM2 N. A. J. Betz H. J. 1999; PubMed Scopus Google Scholar). the S267C mutation the of homomeric S267C then the of the subunit with S267C enhancement of S267C receptors by PMTS Our data that when and S267C cDNAs were the effects of PMTS labeling were significantly the of a cysteine for serine is both and it a for a it the glycine in either heteromeric or homomeric S267C receptors it receptor enhancement by some of the drugs at the cDNA where it was predicted that of the heteromeric receptors one mutant the of wt cDNA made it that than one S267C subunit could be into single receptor, and PMTS significantly enhanced the currents by these receptors. Our results to the that drug occupancy of a single alcohol or anesthetic binding pocket in a GlyR pentamer is sufficient to enhance receptor function. suggest that have a sites at which anesthetics can For three binding sites for were identified in S. Franks N.P. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google two binding sites for in N.P. A. Lieb W.R. P. J. 1998; Full Text Full Text PDF PubMed Scopus Google and there is a single alcohol binding in the protein R. Nat. Biol. 2003; PubMed Scopus Google Scholar). Interestingly, in the of it was found that the binding of a single alcohol the of the protein R. Nat. Biol. 2003; PubMed Scopus Google Scholar). In the of the glycine receptor, alcohol binding pocket a with the alcohol binding there is some for the that the of even a single anesthetic at position 267 is sufficient to GlyR by GlyR function. This effect is to the of to both the channel gate in TM2 and the TM2/TM3 that agonist binding to the gate A. Trudell J.R. Harrison N.L. Nature. 2003; PubMed Scopus Google Scholar). Our data binding of drug to a GlyR can to potentiation of receptor currents. likely that this is the studies that binding of a single glycine to one agonist binding on a GlyR can to channel binding of glycine both the and of D. J. Neurosci. 2004; PubMed Scopus Google Scholar, D. J. Neurosci. 2004; PubMed Scopus Google Scholar). one binding is sufficient to binding may so This to which that, to the of in one receptor subunit induce in the subunits Br. J. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar, S.J. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). Using the cysteine with homomeric cysteine mutants, et al. Mascia M.P. Trudell J.R. Harris R.A. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar) found that the GlyR alcohol and anesthetic binding pocket a receptor to the This that alcohol or anesthetic occupancy of the binding pocket the by an pocket on these studies and current we that the binding of one alcohol or anesthetic to the TM2/TM3 binding pocket of a single GlyR subunit can induce that to enhancement of channel We R. Harris for