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• W2027256384 abstract "Etomidate, one of the most potent general anesthetics used clinically, acts at micromolar concentrations as an anesthetic and positive allosteric modulator of γ-aminobutyric acid responses, whereas it inhibits muscle-type nicotinic acetylcholine receptors (nAChRs) at concentrations above 10 μm. We report here that TDBzl-etomidate, a photoreactive etomidate analog, acts as a positive allosteric nAChR modulator rather than an inhibitor, and we identify its binding sites by photoaffinity labeling. TDBzl-etomidate (>10 μm) increased the submaximal response to acetylcholine (10 μm) with a 2.5-fold increase at 60 μm. At higher concentrations, it inhibited the binding of the noncompetitive antagonists [3H]tetracaine and [3H]phencyclidine to Torpedo nAChR-rich membranes (IC50 values of 0. 8 mm). nAChR-rich membranes were photolabeled with [3H]TDBzl-etomidate, and labeled amino acids were identified by Edman degradation. For nAChRs photolabeled in the absence of agonist (resting state), there was tetracaine-inhibitable photolabeling of amino acids in the ion channel at positions M2-9 (δLeu-265) and M2-13 (αVal-255 and δVal-269), whereas labeling of αM2-10 (αSer-252) was not inhibited by tetracaine but was enhanced 10-fold by proadifen or phencyclidine. In addition, there was labeling in γM3 (γMet-299), a residue that contributes to the same pocket in the nAChR structure as αM2-10. The pharmacological specificity of labeling of residues, together with their locations in the nAChR structure, indicate that TDBzl-etomidate binds at two distinct sites: one within the lumen of the ion channel (labeling of M2-9 and -13), an inhibitory site, and another at the interface between the α and γ subunits (labeling of αM2-10 and γMet-299) likely to be a site for positive allosteric modulation. Etomidate, one of the most potent general anesthetics used clinically, acts at micromolar concentrations as an anesthetic and positive allosteric modulator of γ-aminobutyric acid responses, whereas it inhibits muscle-type nicotinic acetylcholine receptors (nAChRs) at concentrations above 10 μm. We report here that TDBzl-etomidate, a photoreactive etomidate analog, acts as a positive allosteric nAChR modulator rather than an inhibitor, and we identify its binding sites by photoaffinity labeling. TDBzl-etomidate (>10 μm) increased the submaximal response to acetylcholine (10 μm) with a 2.5-fold increase at 60 μm. At higher concentrations, it inhibited the binding of the noncompetitive antagonists [3H]tetracaine and [3H]phencyclidine to Torpedo nAChR-rich membranes (IC50 values of 0. 8 mm). nAChR-rich membranes were photolabeled with [3H]TDBzl-etomidate, and labeled amino acids were identified by Edman degradation. For nAChRs photolabeled in the absence of agonist (resting state), there was tetracaine-inhibitable photolabeling of amino acids in the ion channel at positions M2-9 (δLeu-265) and M2-13 (αVal-255 and δVal-269), whereas labeling of αM2-10 (αSer-252) was not inhibited by tetracaine but was enhanced 10-fold by proadifen or phencyclidine. In addition, there was labeling in γM3 (γMet-299), a residue that contributes to the same pocket in the nAChR structure as αM2-10. The pharmacological specificity of labeling of residues, together with their locations in the nAChR structure, indicate that TDBzl-etomidate binds at two distinct sites: one within the lumen of the ion channel (labeling of M2-9 and -13), an inhibitory site, and another at the interface between the α and γ subunits (labeling of αM2-10 and γMet-299) likely to be a site for positive allosteric modulation. The excitatory nicotinic acetylcholine receptor (nAChR) 3The abbreviations used are: nAChRnicotinic acetylcholine receptorAChacetylcholineazietomidate2-(3-methyl-3H-diaziren-3-yl)ethyl 1-(phenylethyl)-1H-imidazole-5-carboxylateTDBzl-etomidate4-[3-(trifluoromethyl)-3H-diazirin-3-yl]benzyl-1-(1-phenylethyl)-1H-imidazole-5-carboxylateTID3-(trifluoromethyl)-3-(m-iodophenyl)diazirineGABAARγ-aminobutyric acid type A receptorCarbcarbamylcholine chlorideV8 proteaseS. aureus endopeptidase Glu-CEndoLys-Cendoproteinase Lys-COPAo-phthalaldehydePCPphencyclidineTMDtransmembrane domainHPLChigh pressure liquid chromatographyTPSTorpedo physiological salineTricineN-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycinePTHphenylthiohydantoin. is a member of a superfamily of neurotransmitter-gated ion channels that also includes the inhibitory GABAARs (1Changeux J.-P. Edelstein S.J. Nicotinic Acetylcholine Receptors: from Molecular Biology to Cognition. Odile Jacob Publishing, New York2005Google Scholar). Information about the three-dimensional structure of these receptors is based upon the crystal structures of homopentameric acetylcholine-binding proteins from molluscs that are homologous to a nAChR extracellular domain (2Brejc K. van Dijk W.J. Klaassen R. Schuurmans M. van der Oost J. Smit A.B. Sixma T.K. Nature. 2001; 411: 269-276Crossref PubMed Scopus (1580) Google Scholar, 3Hansen S.B. Sulzenbacher G. Huxford T. Marchot P. Taylor P. Bourne Y. EMBO J. 2005; 24: 3635-3646Crossref PubMed Scopus (576) Google Scholar, 4Hansen S.B. Taylor P. J. Mol. Biol. 2007; 369: 895-901Crossref PubMed Scopus (104) Google Scholar) and models of the structure of a muscle-type nAChR derived from cryoelectron microscope images of the Torpedo nAChR (5Miyazawa A. Fujiyoshi Y. Unwin N. Nature. 2003; 423: 949-958Crossref PubMed Scopus (1082) Google Scholar, 6Unwin N. J. Mol. Biol. 2005; 346: 967-989Crossref PubMed Scopus (1419) Google Scholar). For each receptor, five homologous subunits associate at a central axis that is the ion channel. The amino-terminal half of each subunit contributes to the extracellular domain where neurotransmitter binding sites are located at subunit interfaces (α-γ and α-δ in the α2βγδ Torpedo nAChR). The transmembrane domain (TMD) of each subunit is made up of a loose bundle of four α helices (M1–M4), with amino acids from each M2 helix contributing to the lumen of the ion channel, that is the binding site for many nAChR inhibitors (7Arias H.R. Bhumireddy P. Bouzat C. Int. J. Biochem. Cell Biol. 2006; 38: 1254-1276Crossref PubMed Scopus (117) Google Scholar). There are also pockets in the TMD within each subunit helix bundle and at subunit interfaces that are potential binding sites for allosteric modulators. Drugs that bind to such sites and act as positive allosteric modulators of agonist binding may represent an important class of therapeutic agents as they will enhance the efficacy of endogenous neurotransmitter signaling while avoiding the prolonged, nonphysiological pattern of receptor activation produced by agonists. nicotinic acetylcholine receptor acetylcholine 2-(3-methyl-3H-diaziren-3-yl)ethyl 1-(phenylethyl)-1H-imidazole-5-carboxylate 4-[3-(trifluoromethyl)-3H-diazirin-3-yl]benzyl-1-(1-phenylethyl)-1H-imidazole-5-carboxylate 3-(trifluoromethyl)-3-(m-iodophenyl)diazirine γ-aminobutyric acid type A receptor carbamylcholine chloride S. aureus endopeptidase Glu-C endoproteinase Lys-C o-phthalaldehyde phencyclidine transmembrane domain high pressure liquid chromatography Torpedo physiological saline N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine phenylthiohydantoin. Drugs that act as positive allosteric modulators of GABAARs include benzodiazepines, which bind in the extracellular domain at a site equivalent to the transmitter binding sites but at a different subunit interface (8Cromer B. Morton C.J. Parker M.W. Trends Biochem. Sci. 2002; 27: 280-287Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar, 9Ernst M. Bruckner S. Boresch S. Sieghart W. Mol. Pharmacol. 2005; 68: 1291-1300Crossref PubMed Scopus (121) Google Scholar), and general anesthetics of diverse chemical structure, including volatiles, neurosteroids, and intravenous agents such as etomidate and barbiturates (10Franks N.P. Lieb W.R. Nature. 1994; 367: 607-614Crossref PubMed Scopus (1633) Google Scholar, 11Hemmings H.C. Akabas M.H. Goldstein P.A. Trudell J.R. Orser B.A. Harrison N.L. Trends Pharmacol. Sci. 2005; 26: 503-510Abstract Full Text Full Text PDF PubMed Scopus (405) Google Scholar). General anesthetic binding sites, distinct from the transmitter and benzodiazepine sites, are in the GABAAR TMD in the pockets within subunits or at subunit interfaces (11Hemmings H.C. Akabas M.H. Goldstein P.A. Trudell J.R. Orser B.A. Harrison N.L. Trends Pharmacol. Sci. 2005; 26: 503-510Abstract Full Text Full Text PDF PubMed Scopus (405) Google Scholar, 12Mihic S.J. Ye Q. Wick M.J. Koltchines 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, 13Belelli D. Lambert J.J. Peters J.A. Wafford K. Whiting P.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11031-11036Crossref PubMed Scopus (350) Google Scholar, 14Yamakura T. Bertaccini E. Trudell J.R. Harris R.A. Annu. Rev. Pharmacol. Toxicol. 2001; 41: 23-51Crossref PubMed Scopus (238) Google Scholar, 15Hosie A.M. Wilkins M.E. Da Silva H.M.A. Smart T.G. Nature. 2006; 444: 486-489Crossref PubMed Scopus (595) Google Scholar, 16Li G.-D. Chiara D.C. Sawyer G.W. Husain S.S. Olsen R.W. Cohen J.B. J. Neurosci. 2006; 26: 11599-11605Crossref PubMed Scopus (251) Google Scholar). In contrast, most general anesthetics act as negative allosteric modulators of nAChRs (14Yamakura T. Bertaccini E. Trudell J.R. Harris R.A. Annu. Rev. Pharmacol. Toxicol. 2001; 41: 23-51Crossref PubMed Scopus (238) Google Scholar). Positive allosteric nAChR modulators have been identified, including natural products such as physostigmine and galantamine, which are active on muscle and neuronal nAChRs (17Okonjo K.O. Kuhlmann J. Maelicke A. Eur. J. Biochem. 1991; 200: 671-677Crossref PubMed Scopus (62) Google Scholar, 18Samochocki M. Hoffle A. Fehrenbacher A. Jostock R. Ludwig J. Christner C. Radina M. Zerlin M. Ullmer C. Pereira E.F.R. Lubbert H. Albuquerque E.X. Maelicke A. J. Pharmacol. Exp. Ther. 2003; 305: 1024-1036Crossref PubMed Scopus (301) Google Scholar, 19Akk G. Steinbach J.H. J. Neurosci. 2005; 25: 1992-2001Crossref PubMed Scopus (47) Google Scholar), and compounds identified recently through high throughput drug screens that have selectivity for one or more neuronal nAChR subtypes (for reviews, see Refs. 20Bertrand D. Gopalakrishnan M. Biochem. Pharmacol. 2007; 74: 1155-1163Crossref PubMed Scopus (206) Google Scholar and 21Romanelli M.N. Gratteri P. Guandalini L. Martini E. Bonaccini C. Gualtieri F. ChemMedChem. 2007; 2: 746-767Crossref PubMed Scopus (155) Google Scholar). Although physostigmine and galantamine probably bind in the nAChR extracellular domain (4Hansen S.B. Taylor P. J. Mol. Biol. 2007; 369: 895-901Crossref PubMed Scopus (104) Google Scholar, 22Schrattenholz A. Godovac-Zimmermann J. Schafer H.J. Albuquerque E.X. Maelicke A. Eur. J. Biochem. 1993; 216: 671-677Crossref PubMed Scopus (74) Google Scholar), the locations of the binding sites for the other modulators are unknown. We report here that TDBzl-etomidate, a photoreactive general anesthetic developed to provide an improved definition of etomidate binding sites in GABAARs (23Husain S.S. Nirthanan S. Ruesch D. Solt K. Cheng Q. Li G.D. Arevalo E. Olsen R.W. Raines D.E. Forman S.A. Cohen J.B. Miller K.W. J. Med. Chem. 2006; 49: 4818-4825Crossref PubMed Scopus (39) Google Scholar) (Fig. 1), acts as a novel positive allosteric modulator of muscle-type Torpedo nAChR. We used photoaffinity labeling, an experimental approach that directly identifies amino acids contributing to a drug binding site without assumptions about the protein points of contact (24Kotzyba-Hibert F. Kapfer I. Goeldner M. Angew. Chem. Int. Ed. Engl. 1995; 34: 1296-1312Crossref Scopus (387) Google Scholar, 25Vodovozova E.L. Biochemistry (Mosc.). 2007; 72: 1-20Crossref PubMed Scopus (106) Google Scholar), to identify its binding sites in the nAChR TMD. Azietomidate, another photoreactive etomidate analog that is a general anesthetic and positive modulator of GABAARs, inhibits nAChRs similarly to etomidate (26Husain S.S. Ziebell M.R. Ruesch D. Hong F. Arevalo E. Kosterlitz J.A. Olsen R.W. Forman S.A. Cohen J.B. Miller K.W. J. Med. Chem. 2003; 46: 1257-1265Crossref PubMed Scopus (81) Google Scholar). Photoaffinity labeling established that [3H]azietomidate binds in the nAChR ion channel (27Ziebell M.R. Nirthanan S. Husain S.S. Miller K.W. Cohen J.B. J. Biol. Chem. 2004; 279: 17640-17649Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar), whereas in GABAARs it binds in the TMD at the interface between β and α subunits that contains the γ-aminobutyric acid binding sites in the extracellular domain (16Li G.-D. Chiara D.C. Sawyer G.W. Husain S.S. Olsen R.W. Cohen J.B. J. Neurosci. 2006; 26: 11599-11605Crossref PubMed Scopus (251) Google Scholar). Upon photoactivation, azietomidate, an aliphatic diazirine, forms a carbonium ion that reacts preferentially with acidic side chains and with nucleophilic residues (tyrosine and methionine) but not with aliphatic side chains. In contrast, TDBzl-etomidate, an aryl diazirine, forms a carbene intermediate that reacts efficiently with aliphatic and most other side chains (24Kotzyba-Hibert F. Kapfer I. Goeldner M. Angew. Chem. Int. Ed. Engl. 1995; 34: 1296-1312Crossref Scopus (387) Google Scholar). Based upon the pharmacological specificity of residue labeling and residue location in the nAChR structure, TDBzl-etomidate binds at two distinct sites in the transmembrane domain, one located at the interface between the α and γ subunits that is likely to be the site for positive allosteric modulation and the other within the lumen of the ion channel that is an inhibitory site. Materials—Membranes rich in nAChRs, containing 1–2 nmol of [3H]acetylcholine (ACh) binding sites/mg of protein, were isolated from Torpedo californica electric organs (Aquatic Research Consultants, San Pedro, CA) as described previously (28Middleton R.E. Cohen J.B. Biochemistry. 1991; 30: 6987-6997Crossref PubMed Scopus (177) Google Scholar). Nonradioactive TDBzl-etomidate and [3H]TDBzl-etomidate (16 Ci/mmol) were synthesized as described previously (23Husain S.S. Nirthanan S. Ruesch D. Solt K. Cheng Q. Li G.D. Arevalo E. Olsen R.W. Raines D.E. Forman S.A. Cohen J.B. Miller K.W. J. Med. Chem. 2006; 49: 4818-4825Crossref PubMed Scopus (39) Google Scholar). (R)(+)-etomidate was a gift from Dr. David Gemmell (Organon Laboratories Ltd., Cambridge, UK). [3H]Phencyclidine (PCP; 27 Ci/mmol) was from PerkinElmer Life Sciences, and [3H]tetracaine (30 Ci/mmol) was from Sibtech. [3H]ACh (1.9 Ci/mmol) was synthesized from choline and [3H]acetic anhydride. Staphylococcus aureus glutamyl endopeptidase Glu-C (V8 protease) was from ICN Biomedical, and endoproteinase Lys-C (EndoLys-C) was from Roche Applied Sciences. l-1-Tosylamido-2-phenylethyl chloromethyl ketone-treated trypsin was from Worthington. Electrophysiology—Standard two-electrode voltage clamp (oocyte clamp OC-725B, Warner Instruments) techniques were used to study the effects of etomidate and TDBzl-etomidate on wild-type Torpedo α2βγδ nAChRs expressed in Xenopus oocytes as described previously (29Blanton M.P. Xie Y. Dangott L.J. Cohen J.B. Mol. Pharmacol. 1999; 55: 269-278Crossref PubMed Scopus (55) Google Scholar). TDBzl-etomidate was prepared at 0.1 m in methanol and diluted into low calcium ND96 recording solution (96 mm NaCl, 2 mm KCl, 0.3 mm CaCl2, 1 mm MgCl2, 5 mm HEPES, pH 7.6) containing 1 μm atropine. Radioligand Binding Assays—The concentration-dependent effects of TDBzl-etomidate on the equilibrium binding of [3H]ACh, [3H]tetracaine, or [3H]PCP to Torpedo nAChR-rich membranes in Torpedo physiological saline (TPS; 250 mm NaCl, 5 mm KCl, 3 mm CaCl2, 2 mm MgCl2, 5 mm NaPO4, pH 7.0) were studied by centrifugation binding assays in a TOMY TX201 centrifuge as described previously (30White B.H. Howard S. Cohen S.G. Cohen J.B. J. Biol. Chem. 1991; 266: 21595-21607Abstract Full Text PDF PubMed Google Scholar). TDBzl-etomidate was prepared at 50 mm in methanol and then diluted into TPS. For [3H]tetracaine (19 nm) or [3H]PCP (6 nm; ±1 mm carbamylcholine (Carb)), membrane suspensions (0.2 ml, 750 μg of protein/ml) were equilibrated with the drugs for 2 h prior to centrifugation. For the [3H]ACh binding assay, membrane suspensions were pretreated with diisopropylphosphofluoridate (∼0.5 mm) to inhibit acetylcholinesterase activity, then [3H]ACh (11 nm) and drugs were equilibrated with dilute membrane suspensions (1 ml, 80 μg of protein/ml, 40 nm ACh binding sites) for 45 min before centrifugation. Nonspecific binding was determined in the presence of 1 mm Carb for [3H]ACh, in the presence of 0.2 mm tetracaine for [3H]tetracaine, and in the presence of 1 mm proadifen (+Carb) or 1 mm tetracaine (-Carb) for [3H]PCP. At 1 mm TDBzl-etomidate, the highest concentration tested, the methanol concentration was 2%, which in control experiments altered [3H]ACh and [3H]PCP binding by <5% and reduced [3H]tetracaine binding by 20%. Data Analysis—The concentration dependence of etomidate inhibition of ACh-induced currents or TDBzl-etomidate inhibition of radioligand binding was fit to the single site binding equation f(x) = f0/[1 + (x/IC50)] + fns where f(x) is the current or total radioligand bound in the presence of inhibitor concentration x, f0 is the current or specific radioligand bound in the absence of inhibitor, fns is the leak current in the absence of ACh or nonspecific radioligand binding, and IC50 is the concentration of inhibitor associated with the inhibition of 50% of ACh-induced currents or radioligand binding. Photolabeling of nAChR-rich Membranes with [3H]TDBzl-etomidate—Frozen nAChR-rich Torpedo membranes were thawed, diluted 3-fold with TPS, and pelleted by centrifugation. The membrane pellets were then resuspended at 2 mg of protein/ml (∼2.4 μm ACh binding sites) in TPS with oxidized glutathione (1 mm) added to serve as an aqueous scavenger. [3H]TDBzl-etomidate was added to the membrane suspension and mixed by gentle agitation for about 10 min prior to the addition of other ligands. For photolabeling on an analytical scale, the membrane samples in polypropylene microcentrifuge tubes were equilibrated with 0.8 μm [3H]TDBzl-etomidate and additional ligands for 90 min at 4 °C. Following this, aliquots of 130 μl from each sample were placed in a 96-well microtiter plate. For photolabeling on a preparative scale, the membrane suspensions (10 mg of protein/condition) at 2 mg/ml concentration were incubated in 6-cm plastic Petri dishes for 45 min at 4 °C with 1.25 μm [3H]TDBzl-etomidate in the presence or absence of additional ligands. Membrane suspensions were then irradiated for 30 min on ice at 360 nm in a horizontal photochemical chamber reactor (Rayonet RPR-200, Southern New England Ultraviolet Co.) using RPR-3500 bulbs. Following irradiation, the samples were dissolved in sample buffer for SDS-PAGE. SDS-Polyacrylamide Gel Electrophoresis and Proteolytic Digestions—Photolabeled membranes, solubilized in sample buffer, were separated on 1.5-mm-thick acrylamide gels (31White B.H. Cohen J.B. Biochemistry. 1988; 27: 8741-8751Crossref PubMed Scopus (85) Google Scholar). The resolved polypeptides were visualized by staining with Coomassie Blue R-250 stain (analytical scale experiments) or with GelCode® Blue stain reagent (Pierce) (preparative scale experiments). The stained analytical gels were prepared for fluorography using Amplify (Amersham Biosciences), and the dried gels were exposed to film (Eastman Kodak Co. X-Omat) at -80 °C for various times (4–8 weeks). In parallel experiments, the incorporation of 3H into individual nAChR subunits was quantified by liquid scintillation counting of excised gel slices containing the polypeptide bands of interest (28Middleton R.E. Cohen J.B. Biochemistry. 1991; 30: 6987-6997Crossref PubMed Scopus (177) Google Scholar). For preparative scale photolabeling, the gel bands containing the nAChR β, γ, and δ subunits were excised and passively eluted for 3 days into 12 ml of elution buffer (100 mm NH4HCO3, 0.1% SDS, 2.5 mm dithiothreitol). The α subunit bands were excised and placed in the wells of 15-cm-long, 15% acrylamide “mapping” gels with a 5-cm-long 4.5% acrylamide stacker for limited “in-gel” digestion with S. aureus V8 protease (31White B.H. Cohen J.B. Biochemistry. 1988; 27: 8741-8751Crossref PubMed Scopus (85) Google Scholar). After electrophoresis, the mapping gel was stained with Coomassie Blue R-250, and the proteolytic fragments of 20 kDa (αV8-20), 18 kDa (αV8-18), and 10 kDa (αV8-10) were excised and eluted. The eluates were then filtered and concentrated to <400 μl by centrifugal filtration (Vivaspin 15 Mr 5,000 concentrators, Vivascience Inc., Edgewood, NY). nAChR subunits or subunit fragments were precipitated with 75% acetone (12 h at -20 °C) and then resuspended in 200 μl of resuspension buffer (12 mm Tris, 0.5 mm EDTA, 0.1% SDS, pH 8.6). Based upon the MicroBCA Protein Assay (Pierce), 200–400 μg of nAChR β, γ, and δ subunits and 30–50 μg of αV8-20, αV8-18, and αV8-10 were isolated from 10 mg of membranes. The αV8-20 fragment (∼40 μg) and δ subunit (∼200 μg) were digested with EndoLys-C (0.5 units/sample) for 14 days. Aliquots of the γ and δ subunits in resuspension buffer were digested for 3 days with V8 protease (100%, w/w). Aliquots of the β subunit or αV8-10 in resuspension buffer supplemented with Genapol C-100 (0.5%) were digested with trypsin (10 units/sample) for 2 days. All digests were carried out at room temperature. The digests of αV8-20 and αV8-10 as well as the V8 protease digests of the γ and δ subunits were fractionated directly by reversed-phase HPLC, whereas the trypsin digests of β subunits and the EndoLys-C digests of δ subunits were further separated on a 1.5-mm-thick, 16.5% T, 6% C Tricine SDS-PAGE gel (32Schagger H. von Jagow G. Anal. Biochem. 1987; 166: 368-379Crossref PubMed Scopus (10480) Google Scholar, 33Blanton M.P. Cohen J.B. Biochemistry. 1994; 33: 2859-2872Crossref PubMed Scopus (212) Google Scholar). The Tricine gels were sectioned into 5-mm slices, the polypeptides in each slice were eluted, and the eluates containing the peaks of 3H were concentrated and fractionated by reversed-phase HPLC. Reversed-phase HPLC—Samples were fractionated by reversed-phase HPLC on an Agilent 1100 binary HPLC system at 40 °C using a Brownlee C4-Aquapore 7-μm (100 × 2.1 mm) column with an upstream C2 guard column. The elution of peptides was monitored by absorbance at 214 nm. Solvent A was 0.08% trifluoroacetic acid; solvent B consisted of 60% acetonitrile, 40% 2-propanol, and 0.05% trifluoroacetic acid. A stepwise linear gradient was used: 0 min, 25% solvent B; 15 min, 28% solvent B; 30 min, 37% solvent B; 45 min, 52% solvent B; 60 min, 73% solvent B; 75 min, 100% solvent B; 80 min, 100% solvent B; 85 min 25% solvent B; and 90 min, 25% solvent B. The flow rate was 200 μl/min with 0.5-ml fractions collected. Sequence Analysis—Material for sequence analyses was isolated from four independent preparative photolabelings: Ia/b and IIa/b (control versus tetracaine), IIIa/b/c (control, +Carb, +proadifen), and IVa/b/c (+Carb, +PCP, +Carb+PCP). Amino-terminal sequencing was performed on an Applied Biosystems Procise 492 protein sequencer that was modified so that ⅙ of each cycle was analyzed for PTH derivative quantitation and ⅚ was collected for scintillation counting. HPLC fractions were usually drop-loaded at 45 °C onto BiobrenePlus®-treated glass fiber filters (Applied Biosystems catalog number 601111). Fractions containing αM4 or δM1, which are not sequenced efficiently on glass fiber supports, were immobilized on polyvinylidene filters using Prosorb® sample preparation cartridges (Applied Biosystems catalog number 401959). In some instances, the sequencing run was interrupted, and the sample on the filter was treated with o-phthalaldehyde (OPA), which reacts preferentially with primary amines over secondary amines (i.e. proline) and hence can be used to block Edman degradation of any peptide not containing an amino-terminal proline at that cycle (28Middleton R.E. Cohen J.B. Biochemistry. 1991; 30: 6987-6997Crossref PubMed Scopus (177) Google Scholar, 34Brauer A.W. Oman C.L. Margolies M.N. Anal. Biochem. 1984; 137: 134-142Crossref PubMed Scopus (106) Google Scholar). The quantity of amino acids released was determined by peak heights, and the amount of each peptide was obtained from a nonlinear least squares fit (Sigma Plot, SPSS) of the equation f(x) = I0 × Rx where f(x) is pmol of the peptide residue in cycle x, I0 is the initial amount of peptide (in pmol), and R is the average repetitive yield. PTH derivatives known to have poor recovery (Ser, Cys, His, and Trp) were omitted from the fit because of known problems with their quantitations. The efficiency of photolabeling of amino acid residues (cpm/pmol) was calculated as (cpmx - cpm(x - 1))/(5 × I0 × Rx). Molecular Modeling—A homology model of the T. californica nAChR, constructed using the Discovery Studio (Accelerys, Inc.) software package from the structural model of the Torpedo marmorata nAChR (Protein Data Bank code 2BG9), is essentially that of T. marmorata nAChR because the sequences of the four subunits differ between species only by 38 amino acid substitutions, including 16 in the transmembrane helices, without any insertions or deletions. CDOCKER was used to dock 50 energy-minimized structures of TDBzl-etomidate (volume, 284 Å3) into potential binding sites in the nAChR TMD defined by a sphere of 15-Å radius centered within the ion channel or of 12-Å radius centered at each of the five subunit interfaces or within the helical bundle of the δ subunit. The spheres within the ion channel and at the subunit interfaces extended from the level of M2-6 to M2-23, whereas for the δ subunit intrahelical bundle the sphere extended approximately from slightly below M2-13 and above M2-23. Because of the atom limitations of CDOCKER, the nAChR model was “trimmed” by removal of the M4 helices, the cytoplasmic extensions, and extracellular regions >14 Å above the membrane. Each set of 50 solutions was evaluated using CalculateBindingEnergies, and for visualization, each TDBzl-etomidate binding pocket was represented by the Connolly surface of the ensemble of the 10 docking solutions with the most favorable binding energies (Fig. 8 and supplemental Figs. S5 and S6). For the binding site in the channel, the 10 orientations were contained within a pocket of ∼900 Å3, extending from M2-9 to M2-23. For the binding site at the interface between γ and α subunits, the pocket had a volume of ∼580 Å3 and did not extend below M2-9. TDBzl-etomidate binding was also predicted within less constrained pockets at the β-α (∼1380 Å3), α-γ (∼890 Å3), and α-δ (1120 Å3) interfaces that were less well defined and extended into the ion channel and the lipid interface. No stable binding was predicted at the δ-β interface or within the δ subunit helix bundle. TDBzl-etomidate, a Positive Modulator of ACh Responses—We used two-electrode voltage clamp to compare the effects of TDBzl-etomidate and etomidate on the ACh current responses for Torpedo nAChRs expressed in Xenopus oocytes (Fig. 2, A and B). Neither etomidate nor TDBzl-etomidate, when applied in the absence of agonist, produced any detectable current response (i.e. <0.1% of the maximal response for ACh). As reported previously (26Husain S.S. Ziebell M.R. Ruesch D. Hong F. Arevalo E. Kosterlitz J.A. Olsen R.W. Forman S.A. Cohen J.B. Miller K.W. J. Med. Chem. 2003; 46: 1257-1265Crossref PubMed Scopus (81) Google Scholar), when coapplied in the presence of 10 μm ACh, a concentration producing ∼20% of the maximal response, etomidate produced a dose-dependent inhibition characterized by an IC50 of 20 μm. In contrast, TDBzl-etomidate at concentrations above 10 μm increased the ACh response with a 2.5-fold increase seen at the highest concentration studied (60 μm). At that concentration the methanol concentration was 0.05%, but in control experiments we established that methanol at 0.5% altered the ACh response by less than 5%. Consistent enhancement of the ACh response was seen when TDBzl-etomidate was applied in either increasing or decreasing concentrations, and the enhancement was fully reversible when the ACh response was measured minutes after removal of TDBzl-etomidate. We also measured the effects of TDBzl-etomidate on the equilibrium binding of [3H]ACh and drugs that bind in the ion channel preferentially in the resting state ([3H]tetracaine (35Middleton R.E. Strnad N.P. Cohen J.B. Mol. Pharmacol. 1999; 56: 290-299Crossref PubMed Scopus (50) Google Scholar)) or desensitized state ([3H]PCP (36Oswald R.E. Heidmann T. Changeux J.-P. Biochemistry. 1983; 22: 3128-3136Crossref PubMed Scopus (67) Google Scholar)) (Fig. 2C). When [3H]ACh equilibrium binding was measured at a concentration sufficient to occupy ∼20% of sites, TDBzl-etomidate increased binding by ∼20% at the highest concentration studied (300 μm). In the absence of agonist it inhibited [3H]tetracaine or [3H]PCP binding with an IC50 of 0.9 mm, and in the presence of agonist it inhibited [3H]PCP binding with an IC50 of 0.5 mm. Photoincorporation of [3H]TDBzl-etomidate into nAChR-rich Membranes—We used SDS-PAGE followed by fluorography (Fig. 3A) or liquid scintillation counting of excised gel bands (Fig. 3, B and C) to provide an initial characterization of the pattern and pharmacological specificity of nAChR subunit photolabeling. Membranes were photolabeled after equilibration with [3H]TDBzl-etomidate (0.8 μm) in the absence of other drugs or in the presence of the agonist Carb and/or drugs that bind in the ion channel preferentially in the desensitized state (proadifen (37Boyd N.D. Cohen J.B. Biochemistry. 1984; 23: 4023-4033Crossref PubMed Scopus (102) Google Scholar) or PCP)" @default.
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• W2027256384 title "Identification of Binding Sites in the Nicotinic Acetylcholine Receptor for TDBzl-etomidate, a Photoreactive Positive Allosteric Effector" @default.
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