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• W2010046121 abstract "Photoreactive derivatives of the general anesthetic etomidate have been developed to identify their binding sites in γ-aminobutyric acid, type A and nicotinic acetylcholine receptors. One such drug, [3H]TDBzl-etomidate (4-[3-(trifluoromethyl)-3H-diazirin-3-yl]benzyl-[3H]1-(1-phenylethyl)-1H-imidazole-5-carboxylate), acts as a positive allosteric potentiator of Torpedo nACh receptor (nAChR) and binds to a novel site in the transmembrane domain at the γ-α subunit interface. To extend our understanding of the locations of allosteric modulator binding sites in the nAChR, we now characterize the interactions of a second aryl diazirine etomidate derivative, TFD-etomidate (ethyl-1-(1-(4-(3-trifluoromethyl)-3H-diazirin-3-yl)phenylethyl)-1H-imidazole-5-carboxylate). TFD-etomidate inhibited acetylcholine-induced currents with an IC50 = 4 μm, whereas it inhibited the binding of [3H]phencyclidine to the Torpedo nAChR ion channel in the resting and desensitized states with IC50 values of 2.5 and 0.7 mm, respectively. Similar to [3H]TDBzl-etomidate, [3H]TFD-etomidate bound to a site at the γ-α subunit interface, photolabeling αM2-10 (αSer-252) and γMet-295 and γMet-299 within γM3, and to a site in the ion channel, photolabeling amino acids within each subunit M2 helix that line the lumen of the ion channel. In addition, [3H]TFD-etomidate photolabeled in an agonist-dependent manner amino acids within the δ subunit M2-M3 loop (δIle-288) and the δ subunit transmembrane helix bundle (δPhe-232 and δCys-236 within δM1). The fact that TFD-etomidate does not compete with ion channel blockers at concentrations that inhibit acetylcholine responses indicates that binding to sites at the γ-α subunit interface and/or within δ subunit helix bundle mediates the TFD-etomidate inhibitory effect. These results also suggest that the γ-α subunit interface is a binding site for Torpedo nAChR negative allosteric modulators (TFD-etomidate) and for positive modulators (TDBzl-etomidate). Photoreactive derivatives of the general anesthetic etomidate have been developed to identify their binding sites in γ-aminobutyric acid, type A and nicotinic acetylcholine receptors. One such drug, [3H]TDBzl-etomidate (4-[3-(trifluoromethyl)-3H-diazirin-3-yl]benzyl-[3H]1-(1-phenylethyl)-1H-imidazole-5-carboxylate), acts as a positive allosteric potentiator of Torpedo nACh receptor (nAChR) and binds to a novel site in the transmembrane domain at the γ-α subunit interface. To extend our understanding of the locations of allosteric modulator binding sites in the nAChR, we now characterize the interactions of a second aryl diazirine etomidate derivative, TFD-etomidate (ethyl-1-(1-(4-(3-trifluoromethyl)-3H-diazirin-3-yl)phenylethyl)-1H-imidazole-5-carboxylate). TFD-etomidate inhibited acetylcholine-induced currents with an IC50 = 4 μm, whereas it inhibited the binding of [3H]phencyclidine to the Torpedo nAChR ion channel in the resting and desensitized states with IC50 values of 2.5 and 0.7 mm, respectively. Similar to [3H]TDBzl-etomidate, [3H]TFD-etomidate bound to a site at the γ-α subunit interface, photolabeling αM2-10 (αSer-252) and γMet-295 and γMet-299 within γM3, and to a site in the ion channel, photolabeling amino acids within each subunit M2 helix that line the lumen of the ion channel. In addition, [3H]TFD-etomidate photolabeled in an agonist-dependent manner amino acids within the δ subunit M2-M3 loop (δIle-288) and the δ subunit transmembrane helix bundle (δPhe-232 and δCys-236 within δM1). The fact that TFD-etomidate does not compete with ion channel blockers at concentrations that inhibit acetylcholine responses indicates that binding to sites at the γ-α subunit interface and/or within δ subunit helix bundle mediates the TFD-etomidate inhibitory effect. These results also suggest that the γ-α subunit interface is a binding site for Torpedo nAChR negative allosteric modulators (TFD-etomidate) and for positive modulators (TDBzl-etomidate). The excitatory nicotinic acetylcholine receptors (nAChRs) 2The abbreviations used are: nAChRnicotinic acetylcholine receptorAChacetylcholineGABAARγ-aminobutyric acid type A receptorTFD-etomidatep-trifluoromethyldiazirinyl etomidate, ethyl-1-(1-(4-(3-trifluoromethyl)-3H-diazirin-3-yl)phenylethyl)-1H-imidazole-5-carboxylateCarbcarbamylcholineTDBzl-etomidate4-[3-(trifluoromethyl)-3H-diazirin-3-yl]benzyl-1-(1-phenylethyl)-1H-imidazole-5-carboxylateTID3-(trifluoromethyl)-3-(m-iodophenyl)diazirinePCPphencyclidineazietomidate2-(3-methyl-3H-diazirin-3-yl)ethyl 1-(phenylethyl)-1H-imidazole-5-carboxylateOPAo-pthalaldehyderpHPLCreversed-phase HPLCV8 proteaseS. aureus endoproteinase Glu-CEndoLys-CLysobacter enzymogenes endoproteinase Lys-CTettetracaineTricineN-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine. and serotonin 5-HT3 receptors and the inhibitory γ-aminobutyric acid type A receptors (GABAARs) and glycine receptors are members of the Cys-loop superfamily of neurotransmitter-gated ion channels (1Wells G.B. Front. Biosci. 2008; 13: 5479-5510Crossref PubMed Scopus (28) Google Scholar, 2Albuquerque E.X. Pereira E.F. Alkondon M. Rogers S.W. Physiol. Rev. 2009; 89: 73-120Crossref PubMed Scopus (1242) Google Scholar, 3Miller P.S. Smart T.G. Trends Pharmacol. Sci. 2010; 31: 161-174Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar). Models of the three-dimensional structures of these receptors can be derived from the cryoelectron microscopy structure of the Torpedo nAChR in the absence of agonist (4Unwin N. J. Mol. Biol. 2005; 346: 967-989Crossref PubMed Scopus (1414) Google Scholar) and from the high resolution crystal structures of distantly related prokaryotic channels (5Hilf R.J. Dutzler R. Nature. 2009; 457: 115-118Crossref PubMed Scopus (469) Google Scholar, 6Bocquet N. Nury H. Baaden M. Le Poupon C. Changeux J.P. Delarue M. Corringer P.J. Nature. 2009; 457: 111-114Crossref PubMed Scopus (590) Google Scholar) and the soluble molluscan acetylcholine-binding proteins (for review, see Ref. 7Rucktooa P. Smit A.B. Sixma T.K. Biochem. Pharmacol. 2009; 78: 777-787Crossref PubMed Scopus (108) Google Scholar). nicotinic acetylcholine receptor acetylcholine γ-aminobutyric acid type A receptor p-trifluoromethyldiazirinyl etomidate, ethyl-1-(1-(4-(3-trifluoromethyl)-3H-diazirin-3-yl)phenylethyl)-1H-imidazole-5-carboxylate carbamylcholine 4-[3-(trifluoromethyl)-3H-diazirin-3-yl]benzyl-1-(1-phenylethyl)-1H-imidazole-5-carboxylate 3-(trifluoromethyl)-3-(m-iodophenyl)diazirine phencyclidine 2-(3-methyl-3H-diazirin-3-yl)ethyl 1-(phenylethyl)-1H-imidazole-5-carboxylate o-pthalaldehyde reversed-phase HPLC S. aureus endoproteinase Glu-C Lysobacter enzymogenes endoproteinase Lys-C tetracaine N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine. The Torpedo nAChR is composed of four homologous subunits (α2βγδ) that assemble around an axis perpendicular to the membrane, forming a cation-conducting channel. Each subunit is made up of three structural domains; that is, an N-terminal extracellular domain, with the two ACh binding sites at the interfaces between the α-γ and α-δ subunits (8Corringer P.J. Le Novère N. Changeux J.P. Annu. Rev. Pharmacol. Toxicol. 2000; 40: 431-458Crossref PubMed Scopus (706) Google Scholar), a transmembrane domain made up of a loose bundle of four transmembrane helices (M1-M4), and a cytoplasmic domain between the M3 and M4 helices (M3-M4 loop). Amino acids on one face of each M2 helix form the lining of the ion channel, and the M1, M3, and M4 helices form an outer ring that is in contact with membrane lipids (4Unwin N. J. Mol. Biol. 2005; 346: 967-989Crossref PubMed Scopus (1414) Google Scholar, 9Miyazawa A. Fujiyoshi Y. Unwin N. Nature. 2003; 423: 949-955Crossref PubMed Scopus (1078) Google Scholar). Muscle or neuronal nAChRs are targets for drugs potentially useful for treatment of myasthenia gravis and CNS disorders, including nicotine addiction, Alzheimer disease, and schizophrenia (10Jensen A.A. Frølund B. Liljefors T. Krogsgaard-Larsen P. J. Med. Chem. 2005; 48: 4705-4745Crossref PubMed Scopus (477) Google Scholar). In an attempt to develop drugs with strong selectivity for subtypes of muscle or neuronal nAChRs, screens of natural products and synthetic libraries have identified many drugs that act either as positive or negative allosteric modulators (11Romanelli M.N. Gratteri P. Guandalini L. Martini E. Bonaccini C. Gualtieri F. Chem. Med. Chem. 2007; 2: 746-767Crossref Scopus (154) Google Scholar, 12Arias H.R. Adv. Protein Chem. Struct. Biol. 2010; 80: 153-203Crossref PubMed Scopus (59) Google Scholar). However, there is limited information about the diversity of allosteric modulator binding sites or whether positive and negative modulators can bind to the same site, as occurs, for example, at the benzodiazepine site in the GABAAR (13Sigel E. Med. Chem. Rev. 2005; 2: 251-256Google Scholar). In addition to binding sites for non-competitive antagonists within the ion channel (14Arias H.R. Bhumireddy P. Bouzat C. Int. J. Biochem. Cell Biol. 2006; 38: 1254-1276Crossref PubMed Scopus (117) Google Scholar), the transmembrane domain contains potential drug binding sites within each subunit helix bundle and at the interfaces between subunits. Several uncharged, hydrophobic nAChR inhibitors bind in a state-dependent manner within the δ subunit helix bundle (15Arevalo E. Chiara D.C. Forman S.A. Cohen J.B. Miller K.W. J. Biol. Chem. 2005; 280: 13631-13640Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 16Garcia 3rd, G. Chiara D.C. Nirthanan S. Hamouda A.K. Stewart D.S. Cohen J.B. Biochemistry. 2007; 46: 10296-10307Crossref PubMed Scopus (28) Google Scholar). Studies with photoreactive analogs of the general anesthetic etomidate (Fig. 1) led to the identification of binding sites within the transmembrane domain for negative ([3H]azietomidate) and positive ([3H]TDBzl-etomidate) allosteric nAChR modulators (17Husain 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, 18Nirthanan S. Garcia 3rd, G. Chiara D.C. Husain S.S. Cohen J.B. J. Biol. Chem. 2008; 283: 22051-22062Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 19Ziebell 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, 20Chiara D.C. Hong F.H. Arevalo E. Husain S.S. Miller K.W. Forman S.A. Cohen J.B. Mol. Pharmacol. 2009; 75: 1084-1095Crossref PubMed Scopus (27) Google Scholar). Trifluoromethyldiazirinyl-etomidate (TFD-etomidate), another etomidate derivative, has a general anesthetic potency similar to etomidate but lower efficacy as a positive modulator of GABAAR responses while producing, at anesthetic concentrations, significant inhibition of the Torpedo nAChR and the serotonin 5-HT3A receptor (21Husain S.S. Stewart D. Desai R. Hamouda A.K. Li S.G. Kelly E. Dostalova Z. Zhou X. Cotten J.F. Raines D.E. Olsen R.W. Cohen J.B. Forman S.A. Miller K.W. J. Med. Chem. 2010; 53: 6432-6444Crossref PubMed Scopus (22) Google Scholar). TFD-etomidate contains the same photoreactive 3-trifluoromethyl-3- phenyl diazirine group as TDBzl-etomidate, which can react with most amino acid side chains (22Sigrist H. Mühlemann M. Dolder M. J. Photochem. Photobiol. B. 1990; 7: 277-287Crossref Scopus (33) Google Scholar). In this report we characterize the interactions of TFD-etomidate with the Torpedo nAChR by use of radioligand binding assays and by direct identification of the nAChR amino acids photolabeled by [3H]TFD-etomidate. Torpedo nAChR-rich membranes were isolated from Torpedo californica electric organs (Aquatic Research Consultants, San Pedro, CA) as described previously (23Middleton R.E. Cohen J.B. Biochemistry. 1991; 30: 6987-6997Crossref PubMed Scopus (177) Google Scholar). Racemic ethyl 1-(1-(4-(3-((trifluoromethyl)-3H-diazirin-3-yl)phenyl)ethyl)-1H-imidazole-5-carboxylate (p-trifluoromethyldiazirinyl-etomidate (TFD-etomidate)) and [3H]TFD-etomidate (40 Ci/mmol) were synthesized as described (21Husain S.S. Stewart D. Desai R. Hamouda A.K. Li S.G. Kelly E. Dostalova Z. Zhou X. Cotten J.F. Raines D.E. Olsen R.W. Cohen J.B. Forman S.A. Miller K.W. J. Med. Chem. 2010; 53: 6432-6444Crossref PubMed Scopus (22) Google Scholar). [3H]Phencyclidine ([3H]PCP; 27 Ci/mmol) was from PerkinElmer Life Sciences, and [3H]tetracaine (30 Ci/mmol) was from Sibtech (Newington, CT). [3H]ACh (1.9 Ci/mmol) was synthesized from choline and [3H]acetic anhydride. Staphylococcus aureus glutamyl endopeptidase Glu-C (V8 protease) was from MP Biomedical (Solon, OH), and endoproteinase Lys-C (EndoLys-C) was from Roche Diagnostics. l-1-tosylamido-2-phenylethyl chloromethyl ketone-treated trypsin was from Worthington Biomedical (Freehold, NJ). Effects of TFD-etomidate on the equilibrium binding of [3H]PCP, [3H]tetracaine, and [3H]ACh to Torpedo nAChR-rich membranes were determined using a centrifugation assay. TFD-etomidate was prepared as a 50 mm stock solution in methanol and diluted to final concentrations in Torpedo physiological saline (250 mm NaCl, 5 mm KCl, 3 mm CaCl2, 2 mm MgCl2, and 5 mm sodium phosphate, pH 7.0). For [3H]PCP and [3H]tetracaine binding, nAChR-rich membranes at 0.7 mg of protein/ml (0.8 μm ACh binding sites) in 200 μl of Torpedo physiological saline were incubated for 1 h at room temperature with [3H]PCP (6 nm) or [3H]tetracaine (9 nm) in the absence or presence of the agonist carbamylcholine (Carb) and in the absence or presence of TFD-etomidate. For [3H]ACh, membrane suspensions (1 ml, 0.25 mg of protein/ml, 40 nm ACh binding sites) were pretreated with 0.5 mm diisopropyl-phosphofluoridate to inhibit acetylcholinesterase, then incubated for 1 h with [3H]ACh (15 nm) in the absence or presence of TFD-etomidate. Bound and free 3H were separated by centrifugation (18,000 × g for 1 h) and then quantified by liquid scintillation counting. The nonspecific binding of [3H]ACh, [3H]tetracaine, or [3H]PCP to Torpedo nAChR-rich membranes was determined in the presence of 1 mm Carb, tetracaine, or proadifen, respectively. The concentration-dependent inhibition of [3H]PCP or [3H]tetracaine binding by TFD-etomidate was fit using a single site model f(x) = {f0/(1 + (x/IC50))} + fns, where f(x) is binding in the presence of TFD-etomidate at concentration x, f0 is specific binding in the absence of TFD-etomidate, fns is nonspecific binding, and IC50 is the concentration of competitor that inhibits the specific binding by 50%. Standard two-electrode voltage clamp techniques were used to characterize the effects of TFD-etomidate and etomidate on wild type Torpedo nAChR expressed in Xenopus oocytes. Stage V and VI oocytes were obtained from anesthetized adult Xenopus frogs in accordance with local and federal guidelines for animal care. Torpedo nAChR was expressed as described (24Sullivan D.A. Cohen J.B. J. Biol. Chem. 2000; 275: 12651-12660Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). Oocytes were injected with ∼25 ng of total subunit mRNA at a ratio of 2α:1β:1γ:1δ and used 24–72 h after injection. Oocytes were voltage-clamped at −50 mV (Warner Instrument Oocyte Clamp OC-725C) and perfused continually at ∼2 ml/min with ND96 (100 mm NaCl, 2 mm KCl, 10 mm Hepes, 1 mm EGTA, 1 mm CaCl2, 0.8 mm MgCl2, pH 7.5). Oocytes were exposed to ACh alone or to ACh and inhibitor for 20 s, and oocytes were washed for ∼3 min between each application. Data were collected using a Digidata 1322A (Axon Instruments, Foster City, CA) and analyzed using Clampex/Clampfit 8.2 (Axon Instruments). TFD-etomidate was dissolved in DMSO at a concentration of 10 mm, with the final concentration of DMSO at <0.05% at the highest TFD-etomidate concentration used. Etomidate (IV grade) at a concentration of 2 mg/ml was obtained from Ben Venue Laboratories, Bedford, OH. Torpedo nAChR-rich membranes at 2 mg of protein/ml in Torpedo physiological saline supplemented with 1 mm oxidized glutathione were incubated at room temperature for 40 min with 1 μm [3H]TFD-etomidate in the absence or presence of other drugs. For photolabeling on an analytical or preparative scale, 75-μl (150 pmol of ACh binding sites in a 96-well polyvinyl chloride microtiter plate) or 5-ml aliquots (15 nmol of ACh sites in 5-cm plastic Petri dishes) were irradiated on ice with a 365-nm UV lamp (Model EN-16, Spectronics Corp., Westbury, NJ) for 30 min at a distance of less than 2 cm. Electrophoresis sample buffer (12.5 mm Tris-HCL, 2% SDS, 8% sucrose, 1% glycerol, 0.01% bromphenol blue, pH 6.8) was added to samples photolabeled on an analytical scale, and the polypeptides were resolved on 8% polyacrylamide, 0.33% bisacrylamide gels. [3H]TFD-etomidate photoincorporation into the membrane polypeptides was visualized by fluorography (Amplify, Amersham Biosciences GE Healthcare) using Eastman Kodak Co. BIOMAX XAR film (exposure for 4–6 weeks at −80 °C), and 3H incorporation into individual polypeptide bands was quantified by liquid scintillation counting of excised gel bands (23Middleton R.E. Cohen J.B. Biochemistry. 1991; 30: 6987-6997Crossref PubMed Scopus (177) Google Scholar). For experiments on the preparative scale, the photolabeled membranes were pelleted by centrifugation (18,000 × g for 1 h) and then resuspended in sample buffer for electrophoresis. The β, γ, and δ subunits were excised from the Coomassie Blue-stained gels and recovered by passive elution, concentrated to a final volume of 300 μl by centrifugal filtration (Vivaspin 15 mr 5000 concentrators; Vivascience, Stonehouse, UK), and then acetone-precipitated (75% acetone at −20 °C overnight) and resuspended in digestion buffer (15 mm Tris, 0.5 mm EDTA, 0.1% SDS, pH 8.1). The band containing the nAChR α subunit was excised from the stained gel, soaked in overlay buffer (5% sucrose, 125 mm Tris-HCL, 0.1% SDS, pH 6.8) for 30 min, and then transferred to the wells of a 15% acrylamide mapping gel and subjected to in-gel digestion with 100 μg of V8 protease to generate fragments of 20 kDa (αV8-20), 18 kDa (αV8-18), and 10 kDa (αV8-10) (25White B.H. Cohen J.B. Biochemistry. 1988; 27: 8741-8751Crossref PubMed Scopus (85) Google Scholar). After electrophoresis, the α subunit fragments, visualized by staining with GelCode Blue (Pierce), were recovered from the gel bands and resuspended in digestion buffer as described above. αV8-20 (∼30 μg) and the δ subunits (∼150 μg) were digested with EndoLys-C (0.5–1 units) for 2 weeks at room temperature. Aliquots of αV8-10 (30 μg) and β subunit (∼150 μg) were diluted with 0.5% Genapol in 50 mm NH4HCO3 buffer, pH 8.1, to reduce SDS concentration (<0.02%) and then digested with trypsin (1:1 protein to enzyme ratio) in the presence of 0.4 mm CaCl2 for 12 h (β subunit) or for 2 days (αV8-10). The proteolytic digests of αV8-20 and αV8-10 as well as intact αV8-18 were then fractionated by reversed-phase HPLC (rpHPLC). The trypsin digest of β subunit and the EndoLys-C digest of δ subunit were fractionated on small pore (16.5% T, 6% C) Tricine SDS-PAGE gels (26Schägger H. von Jagow G. Anal. Biochem. 1987; 166: 368-379Crossref PubMed Scopus (10470) Google Scholar). For the β subunit digest, fragments beginning near the N termini of βM2 and βM4 were isolated for sequence analysis by rpHPLC from material eluted from gel bands with apparent molecular masses of ∼10 and ∼7 kDa, respectively (27White B.H. Cohen J.B. J. Biol. Chem. 1992; 267: 15770-15783Abstract Full Text PDF PubMed Google Scholar). For the δ subunit digests, fragments beginning near the N termini of δM1 and δM2 were isolated by rpHPLC from material migrating with a apparent molecular mass of 10–14 kDa (16Garcia 3rd, G. Chiara D.C. Nirthanan S. Hamouda A.K. Stewart D.S. Cohen J.B. Biochemistry. 2007; 46: 10296-10307Crossref PubMed Scopus (28) Google Scholar). To examine [3H]TFD-etomidate photoincorporation within subunit M2-M3 loops and M3 segments, aliquots (∼150 μg) of β, γ, and δ subunits were digested overnight with V8 protease (200 μg). The digests were fractionated by rpHPLC, and the hydrophobic peaks of 3H were pooled and sequenced as described below. rpHPLC was performed on an HP 1100 binary system using a Brownlee Aquapore BU-300 column (70 μm, 100 × 2.1 mm; PerkinElmer #0711-0064) and Brownlee Newguard RP-2 guard column at 40 °C. Solvent A was composed of 0.08% trifluoroacetic acid (TFA) in water, and solvent B was 0.05% TFA in 60% acetonitrile, 40% 2-propanol. A nonlinear elution gradient at 0.2 ml/min was employed (25% to 100% solvent B in 100 min), and fractions were collected every 2.5 min. The elution of peptides was monitored by the absorbance at 210 nm, and the elution of 3H associated was monitored by liquid scintillation counting of a 10% aliquot. For sequence analysis, rpHPLC fractions containing the αM4 and δM1 segments were loaded onto PVDF filters using Prosorb® sample preparation cartridges (Applied Biosystems #401950) as recommended by the manufacturer. All other samples were drop-loaded onto Micro TFA filters (Applied Biosystems #401111) at 45 °C, with the filters treated with Biobrene after sample loading. Sequencing was performed on an Applied Biosystems PROCISETM 492 protein sequencer. For each cycle of Edman degradation, ⅙ was used for amino acid identification/quantification, and ⅚ was used for 3H counting. The mass of amino acid detected was quantified from its peak height then fit (shown as a dotted line in FIGURE 5, FIGURE 6, FIGURE 7, FIGURE 8, FIGURE 9) using SigmaPlot 11 to the equation f(x) = I0Rx, where I0 was the initial amount of the peptide sequenced (in pmol), R was the repetitive yield, and f(x) was the pmol detected in cycle x. Amino acids Ser, His, Trp, and Cys were excluded from the fits due to known inaccuracies in their quantification. Photolabeling efficiency in cpm/pmol at a specific residue was calculated by (cpmx − cpm(x − 1)/5IoRx.FIGURE 6[3H]TFD-etomidate photolabels amino acids in γM3 (γMet-295 and γMet-299) but not in βM3 or δM3. Shown are 3H (○,▿) and PTH-amino acids (□, ◊) released during sequencing through the M2-M3 loop and M3 helix of the nAChR γ (A), δ (B), and β (C) subunits isolated from nAChRs from the photolabeling of Fig. 5 in the absence (○, □) or presence (▿, ◊) of tetracaine. The major 3H peaks from rpHPLC fractionations of V8 protease digests of γ, δ, and β subunits (supplemental Fig. S4) were sequenced with OPA treatment at cycle 6 of Edman degradation (indicated by an arrow), which prevents further sequencing of peptides not containing a proline at this cycle and chemically isolates the subunit fragments beginning at γThr-276, δThr-281, and βThr-273, respectively. A, after treatment with OPA, sequencing continued for the fragment beginning at γThr-276 (I0 = 26 and 20 pmol; -and +Tet) and for the equivalent, contaminating δ subunit fragment, (δThr-281; I0 ∼3 pmol). In the cycles before OPA treatment, sequences were also present beginning at γIle-209 and γAsn-439 (each at ∼ 20 pmol), with OPA reducing those levels by >95%. The peaks of 3H release in cycles 20 and 24 indicated photolabeling of γMet-295 (7 cpm/pmol) and γMet-299 (5 cpm/pmol) in the absence and presence of tetracaine. B, after OPA treatment in cycle 6, the primary sequence began at δThr-281 (−Tet, I0 = 30 pmol) is shown. Based upon the levels of 3H release, photolabeling of any amino acid, if it occurred, was at <0.5 cpm/pmol. C, after OPA treatment, sequencing continued for fragments beginning at βThr-273 (□) and the contaminating fragment beginning at δ-Thr281 (8 pmol each). Photolabeling of any amino acids in βM3, if it occurred, would be at <0.5 cpm/pmol.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 7Agonist-enhanced [3H]TFD-etomidate photolabeling within αM2, βM2, and δM2. 3H (○, ●) and PTH-amino acids (□, ■) released during sequence analysis of nAChR subunit fragments beginning at the N termini of αM2 (A), βM2 (B), and δM2 (C) isolated from nAChRs photolabeled on a preparative scale with 0. 8 μm [3H]TFD-etomidate in the absence (○, □) or presence (●, ■) of 1 mm Carb. Tricine-PAGE and rpHPLC fractionations of the subunit digests are shown in supplemental Fig. S1B, S2, B and D, and S3, B and D. A, the primary sequence began at αMet243 (I0 = 7 (□) and 5 (■) pmol), with a secondary sequence beginning at βMet-249 (<2 pmol). The peaks of 3H release in cycles 6, 9, 10, 13, 16, and 20 of Edman degradation indicate photolabeling (−Carb/+Carb, in cpm/pmol) of αSer-248 (1.4/7.8), αLeu-251 (3.2/15), αSer-252 (2.8/27), αVal-255 (1.4/13), αLeu-258 (2/2), and αGlu-262 (1/4). B, the only sequence detected began at βMet-249 (I0 = 17 (□) and 18 (■) pmol). The peaks of 3H release in cycles 6, 9, 13, and 16 indicated labeling (−Carb/+Carb, in cpm/pmol) of βSer-254 (0.6/3.5), βLeu-257 (5/12), βVal-261 (2/11), and βLeu-265 (<0.3/3). C, the only sequence detected began at δMet-257 (I0 = 13 (□) and 19 (■) pmol). The peaks of 3H release in cycles 6, 9, 13, 16, and 20 indicated photolabeling (−Carb/+Carb, in cpm/pmol) of δLeu-262 (0.3/2.2), δLeu-265 (2.4/15), δVal-269 (2/5), δLeu-272 (<0.3/2.7), δGln-276 (<0.5/3.5).View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 8Effect of agonist on [3H]TFD-etomidate photolabeling within γM3 (A), δM3 (B), and δM1. Shown are 3H (○, ●) and PTH-amino acids (□, ■) released during sequencing of subunit fragments from the photolabeling experiment of Fig. 7 of nAChRs in the absence (○, □) or presence (●, ■) of 1 mm Carb. A and B, photoincorporation within the M2-M3 loop and M3 helix was determined, as in Fig. 6, by sequence analysis of the major peaks of 3H recovered when V8 protease digests of γ (A) and δ (B) subunits were fractionated by rpHPLC with OPA treatment at cycle 6 of Edman degradation (indicated by arrow). A, after treatment with OPA in cycle 6, sequencing continued for the fragment beginning at γThr-276 (I0 = 12 (□) and 16 (■) pmol), with the equivalent, contaminating δ subunit fragment (δThr-281, I0 ∼ 4 pmol, both conditions) as a secondary sequence. The peaks of 3H release in cycles 20 and 24 indicated photolabeling (−Carb/+Carb, in cpm/pmol) of γMet-295 (11/6) and γMet-299 (7/4). B, after treatment with OPA in cycle 6, sequencing continued for the fragment beginning at δThr-281 (I0 = 17 (□) and 23 (■) pmol). The major peak of 3H release in cycle 8, seen only in the +Carb sample, indicated agonist-induced labeling of δIle-288 (+Carb, 3.7 cpm/pmol) in the δM2-M3 loop. C, the fragment beginning at δPhe-206 (□, -Carb, 25 pmol; ■, +Carb, 23 pmol), containing the δM1 helix was isolated by Tricine SDS-PAGE and rpHPLC from EndoLys-C digests of δ subunits (from an experiment with nAChRs photolabeled at 0.4 μm [3H]TFD-etomidate). The fragment beginning at δPhe-206 was recovered by rpHPLC from the same 10/14-kDa gel band as the δM2 fragment, with the δPhe-206 and δMet-257 fragments eluting at ∼50 and 70% organic, respectively (supplemental Fig. 3, C and D). Because the fragment beginning at δPhe-206 contains a proline at cycle 20, the sequencing filter was treated with OPA at cycle 20 to ensure that any 3H release after this cycle originated from the δM1 helix. The peaks of 3H release in cycles 27 and 31 in the +Carb sample indicated agonist-induced labeling of δPhe-232 and δCys-236 at 0.5 cpm/pmol.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 9Agonist-insensitive [3H]TFD-etomidate photolabeling in αM4 (top) and βM4 (bottom). Shown are 3H (○, ●) and PTH-amino acids (□, ■) released during sequencing of subunit fragments from the experiment of Fig. 7 of nAChR photolabeled by [3H]TFD-etomidate in the absence (○, □) or presence (●, ■) of 1 mm Carb. Top, the α subunit fragment beginning at αTyr-401 (7 pmol for each condition) containing the αM4 helix (indicated by the bar) was isolated along with an overlapping fragment beginning at αSer-388 (2 pmol) by rpHPLC fractionation of trypsin digests of the αV8-10 fragment supplemental Fig. S1C). The peaks of 3H release in cycles 13 and 15 indicated photolabeling labeling (−Carb/+Carb, in cpm/pmol) of αCys-412 (35/30) and αMet-415 (11/6) at the lipid-exposed face of αM4. Bottom, the fragment beginning at βAsp-427 (□, −Carb, 3.5 pmol; ■, +Carb, 1.8 pmol) containing the βM4 helix (indicated by the bar), was isolated by rpHPLC fractionation of polypeptides eluted from a gel band with apparent molecular mass of 7 kDa from a Tricine SDS-PAGE fractionation of trypsin digests of the β subunit. Each sample also contained the fragment beginning at βLys-216 before βM1 (3 pmol each condition). The 3H release at cycle 15 indicated photolabeling of βTyr-441(−Carb/+Carb, 17/16 cpm/pmol), as no 3H release was seen in cycle 15 when other fractions more enriched in the βLys-216 fragment were sequenced.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To chemically isolate during sequence analysis the nAChR subunit fragments produced by V8 protease that begin at βThr-273, γThr-276, or δThr-281 within the M2-M3 loops (16Garcia 3rd, G. Chiara D.C. Nirthanan S. Hamouda A.K. Stewart D.S. Cohen J.B. Biochemistry. 2007; 46: 10296-10307Crossref PubMed Scopus (28) Google Scholar), sequencing filters were treated with o-ph" @default.
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• W2010046121 title "Multiple Transmembrane Binding Sites for p-Trifluoromethyldiazirinyl-etomidate, a Photoreactive Torpedo Nicotinic Acetylcholine Receptor Allosteric Inhibitor" @default.
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