Loop 2 Structure in Glycine and GABAA Receptors Plays a Key Role in Determining Ethanol Sensitivity
2009; Elsevier BV; Volume: 284; Issue: 40 Linguagem: Inglês
10.1074/jbc.m109.023598
ISSN1083-351X
AutoresDaya I. Perkins, James R. Trudell, Daniel K. Crawford, Liana Asatryan, Ronald L. Alkana, Daryl L. Davies,
Tópico(s)Neuroscience and Neuropharmacology Research
ResumoThe present study tests the hypothesis that the structure of extracellular domain Loop 2 can markedly affect ethanol sensitivity in glycine receptors (GlyRs) and γ-aminobutyric acid type A receptors (GABAARs). To test this, we mutated Loop 2 in the α1 subunit of GlyRs and in the γ subunit of α1β2γ2GABAARs and measured the sensitivity of wild type and mutant receptors expressed in Xenopus oocytes to agonist, ethanol, and other agents using two-electrode voltage clamp. Replacing Loop 2 of α1GlyR subunits with Loop 2 from the δGABAAR (δL2), but not the γGABAAR subunit, reduced ethanol threshold and increased the degree of ethanol potentiation without altering general receptor function. Similarly, replacing Loop 2 of the γ subunit of GABAARs with δL2 shifted the ethanol threshold from 50 mm in WT to 1 mm in the GABAA γ-δL2 mutant. These findings indicate that the structure of Loop 2 can profoundly affect ethanol sensitivity in GlyRs and GABAARs. The δL2 mutations did not affect GlyR or GABAAR sensitivity, respectively, to Zn2+ or diazepam, which suggests that these δL2-induced changes in ethanol sensitivity do not extend to all allosteric modulators and may be specific for ethanol or ethanol-like agents. To explore molecular mechanisms underlying these results, we threaded the WT and δL2 GlyR sequences onto the x-ray structure of the bacterial Gloeobacter violaceus pentameric ligand-gated ion channel homologue (GLIC). In addition to being the first GlyR model threaded on GLIC, the juxtaposition of the two structures led to a possible mechanistic explanation for the effects of ethanol on GlyR-based on changes in Loop 2 structure. The present study tests the hypothesis that the structure of extracellular domain Loop 2 can markedly affect ethanol sensitivity in glycine receptors (GlyRs) and γ-aminobutyric acid type A receptors (GABAARs). To test this, we mutated Loop 2 in the α1 subunit of GlyRs and in the γ subunit of α1β2γ2GABAARs and measured the sensitivity of wild type and mutant receptors expressed in Xenopus oocytes to agonist, ethanol, and other agents using two-electrode voltage clamp. Replacing Loop 2 of α1GlyR subunits with Loop 2 from the δGABAAR (δL2), but not the γGABAAR subunit, reduced ethanol threshold and increased the degree of ethanol potentiation without altering general receptor function. Similarly, replacing Loop 2 of the γ subunit of GABAARs with δL2 shifted the ethanol threshold from 50 mm in WT to 1 mm in the GABAA γ-δL2 mutant. These findings indicate that the structure of Loop 2 can profoundly affect ethanol sensitivity in GlyRs and GABAARs. The δL2 mutations did not affect GlyR or GABAAR sensitivity, respectively, to Zn2+ or diazepam, which suggests that these δL2-induced changes in ethanol sensitivity do not extend to all allosteric modulators and may be specific for ethanol or ethanol-like agents. To explore molecular mechanisms underlying these results, we threaded the WT and δL2 GlyR sequences onto the x-ray structure of the bacterial Gloeobacter violaceus pentameric ligand-gated ion channel homologue (GLIC). In addition to being the first GlyR model threaded on GLIC, the juxtaposition of the two structures led to a possible mechanistic explanation for the effects of ethanol on GlyR-based on changes in Loop 2 structure. Alcohol abuse and dependence are significant problems in our society, with ∼14 million people in the United States being affected (1McGinnis J.M. Foege W.H. Proc. Assoc. Am. Physicians. 1999; 111: 109-118Crossref PubMed Scopus (151) Google Scholar, 2Volpicelli J.R. J. Clin. Psychiatry. 2001; 62: 4-10PubMed Google Scholar). Alcohol causes over 100,000 deaths in the United States, and alcohol-related issues are estimated to cost nearly 200 billion dollars annually (2Volpicelli J.R. J. Clin. Psychiatry. 2001; 62: 4-10PubMed Google Scholar). To address this, considerable attention has focused on the development of medications to prevent and treat alcohol-related problems (3Heilig M. Egli M. Pharmacol. Ther. 2006; 111: 855-876Crossref PubMed Scopus (323) Google Scholar, 4Steensland P. Simms J.A. Holgate J. Richards J.K. Bartlett S.E. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 12518-12523Crossref PubMed Scopus (300) Google Scholar, 5Johnson B.A. Rosenthal N. Capece J.A. Wiegand F. Mao L. Beyers K. McKay A. Ait-Daoud N. Anton R.F. Ciraulo D.A. Kranzler H.R. Mann K. O'Malley S.S. Swift R.M. JAMA. 2007; 298: 1641-1651Crossref PubMed Scopus (455) Google Scholar). The development of such medications would be aided by a clear understanding of the molecular structures on which ethanol acts and how these structures influence receptor sensitivity to ethanol. Ligand-gated ion channels (LGICs) 2The abbreviations used are: LGICligand-gated ion channelGLICG. violaceus pentameric ligand-gated ion channel homologueGlyRglycine receptorGABAAγ-aminobutyric acid type AGABAARGABAA receptornAChRnicotinic acetylcholine receptorTMtransmembraneWTwild typeANOVAanalysis of variance. 2The abbreviations used are: LGICligand-gated ion channelGLICG. violaceus pentameric ligand-gated ion channel homologueGlyRglycine receptorGABAAγ-aminobutyric acid type AGABAARGABAA receptornAChRnicotinic acetylcholine receptorTMtransmembraneWTwild typeANOVAanalysis of variance. have received substantial attention as putative sites of ethanol action that cause its behavioral effects (6Deitrich R.A. Dunwiddie T.V. Harris R.A. Erwin V.G. Pharmacol. Rev. 1989; 41: 489-537PubMed Google Scholar, 7Harris R.A. Alcohol. Clin. Exp. Res. 1999; 23: 1563-1570PubMed Google Scholar, 8Mihic 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 (1097) Google Scholar, 9Ye 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; 273: 3314-3319Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar, 10Zhou Q. Lovinger D.M. J. Pharmacol. Exp. Ther. 1996; 278: 732-740PubMed Google Scholar, 11Cardoso R.A. Brozowski S.J. Chavez-Noriega L.E. Harpold M. Valenzuela C.F. Harris R.A. J. Pharmacol. Exp. Ther. 1999; 289: 774-780PubMed Google Scholar, 12Davies D.L. Alkana R.L. Alcohol. Clin. Exp. Res. 2001; 25: 1098-1106Crossref PubMed Scopus (11) Google Scholar). Research in this area has focused on investigating the effects of ethanol on two large superfamilies of LGICs: 1) the Cys-loop superfamily of LGICs (13Ortells M.O. Lunt G.G. Trends Neurosci. 1995; 18: 121-127Abstract Full Text PDF PubMed Scopus (467) Google Scholar, 14Karlin A. Nat. Rev. Neurosci. 2002; 3: 102-114Crossref PubMed Scopus (770) Google Scholar), whose members include nicotinic acetylcholine, 5-hydroxytryptamine3, γ-aminobutyric acid type A (GABAA), γ-aminobutyric acid type C, and glycine receptors (GlyRs) (10Zhou Q. Lovinger D.M. J. Pharmacol. Exp. Ther. 1996; 278: 732-740PubMed Google Scholar, 11Cardoso R.A. Brozowski S.J. Chavez-Noriega L.E. Harpold M. Valenzuela C.F. Harris R.A. J. Pharmacol. Exp. Ther. 1999; 289: 774-780PubMed Google Scholar, 15Mihic S.J. Harris R.A. J. Pharmacol. Exp. Ther. 1996; 277: 411-416PubMed Google Scholar, 16Grant K.A. Drug Alcohol Depend. 1995; 38: 155-171Crossref PubMed Scopus (109) Google Scholar, 17Davies D.L. Machu T.K. Guo Y. 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Recent studies have also begun investigating ethanol action in the ATP-gated P2X superfamily of LGICs (23Weight F.F. Li C. Peoples R.W. Neurochem. Int. 1999; 35: 143-152Crossref PubMed Scopus (48) Google Scholar, 24Davies D.L. Kochegarov A.A. Kuo S.T. Kulkarni A.A. Woodward J.J. King B.F. Alkana R.L. Neuropharmacology. 2005; 49: 243-253Crossref PubMed Scopus (61) Google Scholar, 25Asatryan L. Popova M. Woodward J.J. King B.F. Alkana R.L. Davies D.L. Neuropharmacology. 2008; 55: 835-843Crossref PubMed Scopus (28) Google Scholar). ligand-gated ion channel G. violaceus pentameric ligand-gated ion channel homologue glycine receptor γ-aminobutyric acid type A GABAA receptor nicotinic acetylcholine receptor transmembrane wild type analysis of variance. ligand-gated ion channel G. violaceus pentameric ligand-gated ion channel homologue glycine receptor γ-aminobutyric acid type A GABAA receptor nicotinic acetylcholine receptor transmembrane wild type analysis of variance. A series of studies that employed chimeric and mutagenic strategies combined with sulfhydryl-specific labeling identified key regions within Cys-loop receptors that appear to be initial targets for ethanol action that also can determine the sensitivity of the receptors to ethanol (7Harris R.A. Alcohol. Clin. Exp. Res. 1999; 23: 1563-1570PubMed Google Scholar, 8Mihic 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 (1097) Google Scholar, 9Ye 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; 273: 3314-3319Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar, 10Zhou Q. Lovinger D.M. J. Pharmacol. Exp. Ther. 1996; 278: 732-740PubMed Google Scholar, 11Cardoso R.A. Brozowski S.J. Chavez-Noriega L.E. Harpold M. Valenzuela C.F. Harris R.A. J. Pharmacol. Exp. Ther. 1999; 289: 774-780PubMed Google Scholar, 12Davies D.L. Alkana R.L. Alcohol. Clin. Exp. Res. 2001; 25: 1098-1106Crossref PubMed Scopus (11) Google Scholar, 18Davies D.L. Crawford D.K. Trudell J.R. Mihic S.J. Alkana R.L. J. Neurochem. 2004; 89: 1175-1185Crossref PubMed Scopus (27) Google Scholar, 19Crawford D.K. Trudell J.R. Bertaccini E.J. Li K. Davies D.L. Alkana R.L. J. Neurochem. 2007; 102: 2097-2109Crossref PubMed Scopus (59) Google Scholar, 26Lobo I.A. Harris R.A. Trudell J.R. J. Neurochem. 2008; 104: 1649-1662Crossref PubMed Scopus (26) Google Scholar, 27Mascia M.P. Machu T.K. Harris R.A. Br. J. Pharmacol. 1996; 119: 1331-1336Crossref PubMed Scopus (193) Google Scholar, 28Valenzuela C.F. Cardoso R.A. Wick M.J. Weiner J.L. Dunwiddie T.V. Harris R.A. Alcohol. Clin. Exp. Res. 1998; 22: 1132-1136Crossref PubMed Scopus (36) Google Scholar, 29Ye J.H. Tao L. Zhu L. Krnjević K. McArdle J.J. 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Neurochem. 2008; 104: 1649-1662Crossref PubMed Scopus (26) Google Scholar, 31Mascia M.P. Trudell J.R. Harris R.A. Proc. Natl. Acad. Sci. U.S.A. 2000; 97: 9305-9310Crossref PubMed Scopus (233) Google Scholar). Growing evidence from GlyRs indicates that ethanol also acts on the extracellular domain. The initial findings came from studies demonstrating that α1GlyRs are more sensitive to ethanol than are α2GlyRs despite the high (∼78%) sequence homology between α1GlyRs and α2GlyRs (32Mascia M.P. Mihic S.J. Valenzuela C.F. Schofield P.R. Harris R.A. Mol. Pharmacol. 1996; 50: 402-406PubMed Google Scholar). Further work found that an alanine to serine exchange at position 52 (A52S) in Loop 2 can eliminate the difference in ethanol sensitivity between α1GlyRs and α2GlyRs (18Davies D.L. Crawford D.K. Trudell J.R. Mihic S.J. Alkana R.L. J. Neurochem. 2004; 89: 1175-1185Crossref PubMed Scopus (27) Google Scholar, 20Perkins D.I. Trudell J.R. Crawford D.K. Alkana R.L. Davies D.L. J. Neurochem. 2008; 106: 1337-1349Crossref PubMed Scopus (30) Google Scholar, 33Davies D.L. Trudell J.R. Mihic S.J. Crawford D.K. Alkana R.L. Alcohol. Clin. Exp. Res. 2003; 27: 743-755Crossref PubMed Scopus (29) Google Scholar). These studies also demonstrated that mutations at position 52 in α1GlyRS and the homologous position 59 in α2GlyRs controlled the sensitivity of these receptors to a novel mechanistic ethanol antagonist (20Perkins D.I. Trudell J.R. Crawford D.K. Alkana R.L. Davies D.L. J. Neurochem. 2008; 106: 1337-1349Crossref PubMed Scopus (30) Google Scholar). Collectively, these studies suggest that there are multiple sites of ethanol action in α1GlyRs, with one site located in the TM domain (e.g. position 267) and another in the extracellular domain (e.g. position 52). Subsequent studies revealed that the polarity of the residue at position 52 plays a key role in determining the sensitivity of GlyRs to ethanol (20Perkins D.I. Trudell J.R. Crawford D.K. Alkana R.L. Davies D.L. J. Neurochem. 2008; 106: 1337-1349Crossref PubMed Scopus (30) Google Scholar). The findings with polarity in the extracellular domain contrast with the findings at position 267 in the TM domain, where molecular volume, but not polarity, significantly affected ethanol sensitivity (9Ye 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; 273: 3314-3319Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). Taken together, these findings indicate that the physical-chemical parameters of residues at positions in the extracellular and TM domains that modulate ethanol effects and/or initiate ethanol action in GlyRs are not uniform. Thus, knowledge regarding the physical-chemical properties that control agonist and ethanol sensitivity is key for understanding the relationship between the structure and the actions of ethanol in LGICs (19Crawford D.K. Trudell J.R. Bertaccini E.J. Li K. Davies D.L. Alkana R.L. J. 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Chem. 2007; 282: 26210-26216Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar, 39Crawford D.K. Perkins D.I. Trudell J.R. Bertaccini E.J. Davies D.L. Alkana R.L. J. Biol. Chem. 2008; 283: 27698-27706Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar, 40Cheng M.H. Coalson R.D. Cascio M. Proteins. 2007; 71: 972-981Crossref Scopus (21) Google Scholar). GlyRs and GABAARs, which differ significantly in their sensitivities to ethanol, offer a potential method for identifying the structures that control ethanol sensitivity. For example, α1GlyRs do not reliably respond to ethanol concentrations less than 10 mm (32Mascia M.P. Mihic S.J. Valenzuela C.F. Schofield P.R. Harris R.A. Mol. Pharmacol. 1996; 50: 402-406PubMed Google Scholar, 33Davies D.L. Trudell J.R. Mihic S.J. Crawford D.K. Alkana R.L. Alcohol. Clin. Exp. Res. 2003; 27: 743-755Crossref PubMed Scopus (29) Google Scholar, 41Woodward J.J. Nowak M. Davies D.L. Mol. Brain Res. 2004; 125: 86-95Crossref PubMed Scopus (30) Google Scholar). Similarly, γ subunit-containing GABAARs (e.g. α1β2γ2), the most predominantly expressed GABAARs in the central nervous system, are insensitive to ethanol concentrations less than 50 mm (42White G. Lovinger D.M. Weight F.F. Brain Res. 1990; 507: 332-336Crossref PubMed Scopus (174) Google Scholar, 43Weiner J.L. Gu C. Dunwiddie T.V. J. Neurophys. 1997; 77: 1306-1312Crossref PubMed Scopus (94) Google Scholar). In contrast, δ subunit-containing GABAARs (e.g. α4β3δ) have been shown to be sensitive to ethanol concentrations as low as 1–3 mm (44Wei W. Faria L.C. Mody I. J. Neurosci. 2004; 24: 8379-8382Crossref PubMed Scopus (207) Google Scholar, 45Hanchar H.J. Dodson P.D. Olsen R.W. Otis T.S. Wallner M. Nat. Neurosci. 2005; 8: 339-345Crossref PubMed Scopus (245) Google Scholar, 46Hanchar H.J. Chutsrinopkun P. Meera P. Supavilai P. Sieghart W. Wallner M. Olsen R.W. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 8546-8551Crossref PubMed Scopus (110) Google Scholar, 47Liang J. Zhang N. Cagetti E. Houser C.R. Olsen R.W. Spigelman I. J. Neurosci. 2006; 26: 1749-1758Crossref PubMed Scopus (134) Google Scholar, 48Fleming R.L. Wilson W.A. Swartzwelder H.S. J. Neurophysiol. 2007; 97: 3806-3811Crossref PubMed Scopus (70) Google Scholar, 49Glykys J. Peng Z. Chandra D. Homanics G.E. Houser C.R. Mody I. Nat. Neurosci. 2007; 10: 40-48Crossref PubMed Scopus (208) Google Scholar, 50Santhakumar V. Wallner M. Otis T.S. Alcohol. 2007; 41: 211-221Crossref PubMed Scopus (94) Google Scholar, 51Sundstrom-Poromaa I. Smith D.H. Gong Q.H. Sabado T.N. Li X. Light A. Wiedmann M. Williams K. Smith S.S. Nat. Neurosci. 2002; 5: 721-722Crossref PubMed Scopus (295) Google Scholar). Sequence alignment of α1GlyR, γGABAAR, and δGABAAR revealed differences between the Loop 2 regions of these receptor subunits. Since prior studies found that mutations of Loop 2 residues can affect ethanol sensitivity (19Crawford D.K. Trudell J.R. Bertaccini E.J. Li K. Davies D.L. Alkana R.L. J. Neurochem. 2007; 102: 2097-2109Crossref PubMed Scopus (59) Google Scholar, 20Perkins D.I. Trudell J.R. Crawford D.K. Alkana R.L. Davies D.L. J. Neurochem. 2008; 106: 1337-1349Crossref PubMed Scopus (30) Google Scholar, 39Crawford D.K. Perkins D.I. Trudell J.R. Bertaccini E.J. Davies D.L. Alkana R.L. J. Biol. Chem. 2008; 283: 27698-27706Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar), the non-conserved residues in Loop 2 of GlyR and GABAAR subunits could provide the physical-chemical and structural bases underlying the differences in ethanol sensitivity between these receptors. The present study tested the hypothesis that the structure of Loop 2 can markedly affect the ethanol sensitivity of GlyRs and GABAARs. To accomplish this, we performed multiple mutations that replaced the Loop 2 region of the α1 subunit in α1GlyRs and the Loop 2 region of the γ subunit of α1β2γ2 GABAARs with corresponding non-conserved residues from the δ subunit of GABAAR and tested the sensitivity of these receptors to ethanol. As predicted, replacing Loop 2 of WT α1GlyRs with the homologous residues from the δGABAAR subunit (δL2), but not the γGABAAR subunit (γL2), markedly increased the sensitivity of the receptor to ethanol. Similarly, replacing the non-conserved residues of the γ subunit of α1β2γ2 GABAARs with δL2 also markedly increased ethanol sensitivity of GABAARs. These findings support the hypothesis and suggest that Loop 2 may play a role in controlling ethanol sensitivity across the Cys-loop superfamily of receptors. The findings also provide the basis for suggesting structure-function relationships in a new molecular model of the GlyR based on the bacterial Gloeobacter violaceus pentameric LGIC homologue (GLIC). Adult female Xenopus laevis frogs were purchased from Nasco (Fort Atkinson, WI). Gentamicin, 3-aminobenzoic acid ethyl ester, glycine, GABA, ethanol, zinc chloride, strychnine, picrotoxin, diazepam, and collagenase were purchased from Sigma. All other chemicals used were of reagent grade. Glycine, GABA, and strychnine stock solutions were prepared from powder. Stock solutions of picrotoxin and diazepam were prepared in DMSO and then diluted to an appropriate concentration with the extracellular solution just before use. To avoid adverse effects from DMSO exposure, the final concentration (v/v) of DMSO was not higher than 0.5%. Picrotoxin stocks and solutions were wrapped in foil to avoid UV exposure. The amino acid sequences for α1GlyR and δ- and γGABAAR subunits were aligned, and the Loop 2 regions were compared (Table 1). Individual point mutations in the α1GlyR or γGABAAR subunit cDNA were created so that the resulting Loop 2 region matched that of the δGABAAR or the γGABAAR subunits. Xenopus oocytes were isolated and injected with human GlyR cDNAs (1 ng/32 nl) or GABAAR cDNAs (1:1:10 ratio for a total volume of 1 ng of α1β2γ2) cloned into the mammalian expression vector pCIS2 or pBKCMV, as described previously (33Davies D.L. Trudell J.R. Mihic S.J. Crawford D.K. Alkana R.L. Alcohol. Clin. Exp. Res. 2003; 27: 743-755Crossref PubMed Scopus (29) Google Scholar), and verified by partial sequencing (DNA Core Facility, University of Southern California). After injection, oocytes were stored in incubation medium (modified Barth's saline supplemented with 2 mm sodium pyruvate, 0.5 mm theophylline, and 50 mg/liter gentamycin) in Petri dishes (VWR, San Dimas, CA). All solutions were sterilized by passage through 0.22-μm filters. Oocytes, stored at 18 °C, usually expressed GlyRs the day after injection and GABAARs 3–4 days after injection. Oocytes were used in experiments for up to 7 days after injection.TABLE 1Loop 2 sequence alignment for the α1GlyR subunit, δ− and γGABAAR subunits, α1nAChR subunit, and GLICSubunitPositionSequenceHuman GlyR α150SIAETTMDYRHuman GABAAR δ43HISEANMEYTHuman GABAAR γ264PVNAINMEYTHuman nAChR α142NVDEVNQIVEGLIC29SLDDKAETFK Open table in a new tab Native δ-containing GABAARs (α4β2/3δ and α6β2/3δ) have been shown to be sensitive to low ethanol concentrations (1–3 mm) in a variety of preparations (44Wei W. Faria L.C. Mody I. J. Neurosci. 2004; 24: 8379-8382Crossref PubMed Scopus (207) Google Scholar, 45Hanchar H.J. Dodson P.D. Olsen R.W. Otis T.S. Wallner M. Nat. Neurosci. 2005; 8: 339-345Crossref PubMed Scopus (245) Google Scholar, 46Hanchar H.J. Chutsrinopkun P. Meera P. Supavilai P. Sieghart W. Wallner M. Olsen R.W. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 8546-8551Crossref PubMed Scopus (110) Google Scholar, 47Liang J. Zhang N. Cagetti E. Houser C.R. Olsen R.W. Spigelman I. J. Neurosci. 2006; 26: 1749-1758Crossref PubMed Scopus (134) Google Scholar, 48Fleming R.L. Wilson W.A. Swartzwelder H.S. J. Neurophysiol. 2007; 97: 3806-3811Crossref PubMed Scopus (70) Google Scholar, 49Glykys J. Peng Z. Chandra D. Homanics G.E. Houser C.R. Mody I. Nat. Neurosci. 2007; 10: 40-48Crossref PubMed Scopus (208) Google Scholar, 50Santhakumar V. Wallner M. Otis T.S. Alcohol. 2007; 41: 211-221Crossref PubMed Scopus (94) Google Scholar, 51Sundstrom-Poromaa I. Smith D.H. Gong Q.H. Sabado T.N. Li X. Light A. Wiedmann M. Williams K. Smith S.S. Nat. Neurosci. 2002; 5: 721-722Crossref PubMed Scopus (295) Google Scholar) However, these receptors are difficult to express in oocytes. This topic has been the subject of several reviews (52Olsen R.W. Hanchar H.J. Meera P. Wallner M. Alcohol. 2007; 41: 201-209Crossref PubMed Scopus (110) Google Scholar, 53Borghese C.M. Harris R.A. Alcohol. 2007; 41: 155-162Crossref PubMed Scopus (59) Google Scholar, 54Mody I. Glykys J. Wei W. Alcohol. 2007; 41: 145-153Crossref PubMed Scopus (51) Google Scholar). The goal of the present study was to test the hypothesis that the structure of Loop 2 can markedly affect the ethanol sensitivity of GlyRs and GABAARs. We used the δ Loop 2 as a vehicle for testing this hypothesis. In this context, and given the difficulties described above, we did not include WT δ-containing GABAARs in the current paper. Two-electrode voltage clamp recording was performed using techniques similar to those previously reported (33Davies D.L. Trudell J.R. Mihic S.J. Crawford D.K. Alkana R.L. Alcohol. Clin. Exp. Res. 2003; 27: 743-755Crossref PubMed Scopus (29) Google Scholar). Briefly, electrodes pulled (P-30; Sutter Instruments, Novato, CA) from borosilicate glass (1.2-mm thick walled filamented glass capillaries (WPI, Sarasota, FL)) were back-filled with 3 m KCl to yield resistances of 0.5–3 megaohms. All electrophysiological recordings were conducted within a chamber that contains a vibration-resistant platform that supports an oocyte bath, two micro positioners (WPI (Sarasota, FL) or Narishige International USA, Inc. (East Meadow, NY)), and bath clamp (33Davies D.L. Trudell J.R. Mihic S.J. Crawford D.K. Alkana R.L. Alcohol. Clin. Exp. Res. 2003; 27: 743-755Crossref PubMed Scopus (29) Google Scholar). Oocytes were perfused in a 100-μl oocyte bath with modified Barth's saline with or without drugs via a custom high pressure drug delivery system (Alcott Chromatography, Norcross, GA) at 2 ml/min using 116 OD high pressure PEEK tubing (Upchurch Scientific, Oak Harbor, WA). Oocytes were voltage-clamped at a membrane potential of −70 mV using a Warner Instruments model OC-725C (Hamden, CT) oocyte clamp. A chart recorder (Barnstead/Thermolyne, Dubuque, IA) continuously plotted the clamped currents. The peak currents were measured and used in data analysis. All experiments were performed at room temperature (20–23 °C). For agonist concentration response experiments, WT or mutant GlyRs or GABAARs were exposed to 1 μm to 3 mm glycine or 1 μm to 10 mm GABA for 60 s, using 5–15-min washouts between applications to ensure complete receptor resensitization. We used a concentration of glycine or GABA producing 10 ± 2% of the maximal effect (EC10). Agonist EC10 was applied as a control pre- and post-ethanol treatment. When testing ethanol potentiation, the oocytes were preincubated with ethanol for 60 s prior to co-application of ethanol and agonist for 60 s (18Davies D.L. Crawford D.K. Trudell J.R. Mihic S.J. Alkana R.L. J. Neurochem. 2004; 89: 1175-1185Crossref PubMed Scopus (27) Google Scholar). Washout periods (5–15 min) were allowed between agonist and drug applications to ensure complete resensitization of receptors. WT and mutant α1GlyR responses were measured across an ethanol concentration range of 1–30 mm. GABAAR responses were measured across an ethanol concentration range of 1–50 mm. Ethanol, in the absence of glycine or GABA, did not significantly affect the holding currents of the GlyRs and GABAARs tested. Oocytes expressing WT, δL2, and γL2 GlyRs were tested for response to low (10 μm) and high (100 μm) concentrations of zinc chloride (ZnCl2), a bimodal allosteric modulator of the GlyR. Glycine EC10 was applied for 60 s. Oocytes were preincubated with ZnCl2 for 60 s, followed by co-application with glycine EC10 for 60 s. Wash-out periods (5–15 min) were allowed between drug applications to ensure complete resensitization of receptors. Oocytes expressing WT, δL2, and γL2 GlyRs were tested for response to the competitive GlyR antagonist strychnine or the noncompetitive GlyR antagonist picrotoxin. Glycine EC10 was applied for 60 s. Oocytes were preincubated with strychnine (50 nm) or picrotoxin (100 μm) for 60 s, followed by co-application with glycine EC10 for 60 s. Washout periods (5–15 min) were allowed between drug applications to ensure complete resensitization of receptors. Oocytes expressing WT and δL2 GABAARs were tested for response to the benzodiazepine agonist diazepam. GABA EC10 was applied for 60 s. Oocytes were preincubated with diazepam (1 μm) for 60 s, followed by co-application with GABA EC10 for 60 s. Washout periods (5–15 min) were allowed between drug applications to ensure complete resensitization of receptors. Biotinylation of surface-expressed proteins was modified from a previous protocol published by Chen et al. (55Chen Z.W. Chang C.S. Leil T.A. Olcese R. Olsen R.W. Mol. Pharmacol. 2005; 68: 152-159Crossref PubMed Scopus (40) Google Scholar). Four days after cDNA injections, oocytes (15 oocytes/group) were incubated with 1.5 mg/ml membrane-impermeable sulfosuccinimidyl 2-(biotinamido)-ethyl-1,3-dithiopropionate (Pierce) for 30 min at room temperature. After washing once with 25 mm Tris (pH 8.0) and twice with phosphate-buffered saline, oocytes were homogenized in 500 μl of lysis buffer (40 mm Tris (pH 7.5), 110 mm NaCl, 4 mm EDTA, 0.08% Triton X-100, 1% protease inhibitor mixture (Vector Laboratories, Burlingame, CA)). The yolk and cellular debris were removed by centrifugation at 3600 × g for 10 min. Aliquots of the supernatant were mixed with 2× SDS loading buffer and stored at −20 °C to assess total receptor fraction. The remaining supernatant was incubated with streptavidin beads (Pierce
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