nAChR-rich Membranes Research Articles

Identifying Barbiturate Binding Sites in a Nicotinic Acetylcholine Receptor with [3H]Allyl m-Trifluoromethyldiazirine Mephobarbital, a Photoreactive Barbiturate

At concentrations that produce anesthesia, many barbituric acid derivatives act as positive allosteric modulators of inhibitory GABAA receptors (GABAARs) and inhibitors of excitatory nicotinic acetylcholine receptors (nAChRs). Recent research on [(3)H]R-mTFD-MPAB ([(3)H]R-5-allyl-1-methyl-5-(m-trifluoromethyldiazirinylphenyl)barbituric acid), a photoreactive barbiturate that is a potent and stereoselective anesthetic and GABAAR potentiator, has identified a second class of intersubunit binding sites for general anesthetics in the α1β3γ2 GABAAR transmembrane domain. We now characterize mTFD-MPAB interactions with the Torpedo (muscle-type) nAChR. For nAChRs expressed in Xenopus oocytes, S- and R-mTFD-MPAB inhibited ACh-induced currents with IC50 values of 5 and 10 µM, respectively. Racemic mTFD-MPAB enhanced the equilibrium binding of [(3)H]ACh to nAChR-rich membranes (EC50 = 9 µM) and inhibited binding of the ion channel blocker [(3)H]tenocyclidine in the nAChR desensitized and resting states with IC50 values of 2 and 170 µM, respectively. Photoaffinity labeling identified two binding sites for [(3)H]R-mTFD-MPAB in the nAChR transmembrane domain: 1) a site within the ion channel, identified by photolabeling in the nAChR desensitized state of amino acids within the M2 helices of each nAChR subunit; and 2) a site at the γ-α subunit interface, identified by photolabeling of γMet299 within the γM3 helix at similar efficiency in the resting and desensitized states. These results establish that mTFD-MPAB is a potent nAChR inhibitor that binds in the ion channel preferentially in the desensitized state and binds with lower affinity to a site at the γ-α subunit interface where etomidate analogs bind that act as positive and negative nAChR modulators.

Identification of Propofol Binding Sites in a Nicotinic Acetylcholine Receptor with a Photoreactive Propofol Analog*

Propofol, a widely used intravenous general anesthetic, acts at anesthetic concentrations as a positive allosteric modulator of γ-aminobutyric acid type A receptors and at higher concentration as an inhibitor of nicotinic acetylcholine receptors (nAChRs). Here, we characterize propofol binding sites in a muscle-type nAChR by use of a photoreactive analog of propofol, 2-isopropyl-5-[3-(trifluoromethyl)-3H-diazirin-3-yl]phenol (AziPm). Based upon radioligand binding assays, AziPm stabilized the Torpedo nAChR in the resting state, whereas propofol stabilized the desensitized state. nAChR-rich membranes were photolabeled with [(3)H]AziPm, and labeled amino acids were identified by Edman degradation. [(3)H]AziPm binds at three sites within the nAChR transmembrane domain: (i) an intrasubunit site in the δ subunit helix bundle, photolabeling in the nAChR desensitized state (+agonist) δM2-18' and two residues in δM1 (δPhe-232 and δCys-236); (ii) in the ion channel, photolabeling in the nAChR resting, closed channel state (-agonist) amino acids in the M2 helices (αM2-6', βM2-6' and -13', and δM2-13') that line the channel lumen (with photolabeling reduced by >90% in the desensitized state); and (iii) at the γ-α interface, photolabeling αM2-10'. Propofol enhanced [(3)H]AziPm photolabeling at αM2-10'. Propofol inhibited [(3)H]AziPm photolabeling within the δ subunit helix bundle at lower concentrations (IC50 = 40 μm) than it inhibited ion channel photolabeling (IC50 = 125 μm). These results identify for the first time a single intrasubunit propofol binding site in the nAChR transmembrane domain and suggest that this is the functionally relevant inhibitory binding site.

3H]Chlorpromazine Photolabeling of the Torpedo Nicotinic Acetylcholine Receptor Identifies Two State-Dependent Binding Sites in the Ion Channel

Chlorpromazine (CPZ), a potent nicotinic acetylcholine receptor (nAChR) noncompetitive antagonist, binds with higher affinity in the ion channel in the desensitized state than in the closed channel state and with low affinity to additional sites in nAChR-rich membranes. For nAChR equilibrated with agonist, we confirm previous reports that [(3)H]CPZ occupies a site near the cytoplasmic end of the M2 ion channel domain, photolabeling positions M2-2, M2-6, and/or M2-9 in each subunit. We find that [(3)H]CPZ also binds at the extracellular end of the channel, photolabeling amino acids at positions M2-16 (alpha,gamma), M2-17 (alpha,beta,delta), and M2-20 (alpha,beta,delta). The photolabeling at the cytoplasmic end of the channel is fully inhibitable by phencyclidine or proadifen, whereas neither drug inhibits [(3)H]CPZ photolabeling at the extracellular end, establishing that positively charged drugs can bind simultaneously at the cytoplasmic and extracellular ends of the ion channel. [(3)H]CPZ photolabeling is not detected in the transmembrane domain outside the ion channel, but it photolabels alphaMet-386 and alphaSer-393 in the cytoplasmic alphaMA helix. In the nAChR equilibrated with alpha-bungarotoxin to stabilize the nAChR in a closed state, [(3)H]CPZ photolabels amino acids at M2-5 (alpha), M2-6 (alpha,beta,delta), and M2-9 (beta,delta), with no labeling at M2-2. These results provide novel information about the modes of drug binding within the nAChR ion channel and indicate that within the nAChR transmembrane domain, the binding of cationic aromatic amine antagonists can be restricted to the ion channel domain, in contrast to the uncharged, allosteric potentiators and inhibitors that also bind within the delta subunit helix bundle and at subunit interfaces.

3H]Epibatidine Photolabels Non-equivalent Amino Acids in the Agonist Binding Site of Torpedo and α4β2 Nicotinic Acetylcholine Receptors

Nicotinic acetylcholine receptor (nAChR) agonists, such as epibatidine and its molecular derivatives, are potential therapeutic agents for a variety of neurological disorders. In order to identify determinants for subtype-selective agonist binding, it is important to determine whether an agonist binds in a common orientation in different nAChR subtypes. To compare the mode of binding of epibatidine in a muscle and a neuronal nAChR, we photolabeled Torpedo alpha(2)betagammadelta and expressed human alpha4beta2 nAChRs with [(3)H]epibatidine and identified by Edman degradation the photolabeled amino acids. Irradiation at 254 nm resulted in photolabeling of alphaTyr(198) in agonist binding site Segment C of the principal (+) face in both alpha subunits and of gammaLeu(109) and gammaTyr(117) in Segment E of the complementary (-) face, with no labeling detected in the delta subunit. For affinity-purified alpha4beta2 nAChRs, [(3)H]epibatidine photolabeled alpha4Tyr(195) (equivalent to Torpedo alphaTyr(190)) in Segment C as well as beta2Val(111) and beta2Ser(113) in Segment E (equivalent to Torpedo gammaLeu(109) and gammaTyr(111), respectively). Consideration of the location of the photolabeled amino acids in homology models of the nAChRs based upon the acetylcholine-binding protein structure and the results of ligand docking simulations suggests that epibatidine binds in a single preferred orientation within the alpha-gamma transmitter binding site, whereas it binds in two distinct orientations in the alpha4beta2 nAChR.

Identification of Binding Sites in the Nicotinic Acetylcholine Receptor for TDBzl-etomidate, a Photoreactive Positive Allosteric Effector

Etomidate, one of the most potent general anesthetics used clinically, acts at micromolar concentrations as an anesthetic and positive allosteric modulator of gamma-aminobutyric acid responses, whereas it inhibits muscle-type nicotinic acetylcholine receptors (nAChRs) at concentrations above 10 microm. 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 microm) increased the submaximal response to acetylcholine (10 microm) with a 2.5-fold increase at 60 microm. At higher concentrations, it inhibited the binding of the noncompetitive antagonists [(3)H]tetracaine and [(3)H]phencyclidine to Torpedo nAChR-rich membranes (IC(50) values of 0. 8 mm). nAChR-rich membranes were photolabeled with [(3)H]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 (deltaLeu-265) and M2-13 (alphaVal-255 and deltaVal-269), whereas labeling of alphaM2-10 (alphaSer-252) was not inhibited by tetracaine but was enhanced 10-fold by proadifen or phencyclidine. In addition, there was labeling in gammaM3 (gammaMet-299), a residue that contributes to the same pocket in the nAChR structure as alphaM2-10. 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 alpha and gamma subunits (labeling of alphaM2-10 and gammaMet-299) likely to be a site for positive allosteric modulation.

Naturally Occurring Disulfide-bound Dimers of Three-fingered Toxins

Disulfide-bound dimers of three-fingered toxins have been discovered in the Naja kaouthia cobra venom; that is, the homodimer of alpha-cobratoxin (a long-chain alpha-neurotoxin) and heterodimers formed by alpha-cobratoxin with different cytotoxins. According to circular dichroism measurements, toxins in dimers retain in general their three-fingered folding. The functionally important disulfide 26-30 in polypeptide loop II of alpha-cobratoxin moiety remains intact in both types of dimers. Biological activity studies showed that cytotoxins within dimers completely lose their cytotoxicity. However, the dimers retain most of the alpha-cobratoxin capacity to compete with alpha-bungarotoxin for binding to Torpedo and alpha7 nicotinic acetylcholine receptors (nAChRs) as well as to Lymnea stagnalis acetylcholine-binding protein. Electrophysiological experiments on neuronal nAChRs expressed in Xenopus oocytes have shown that alpha-cobratoxin dimer not only interacts with alpha7 nAChR but, in contrast to alpha-cobratoxin monomer, also blocks alpha3beta2 nAChR. In the latter activity it resembles kappa-bungarotoxin, a dimer with no disulfides between monomers. These results demonstrate that dimerization is essential for the interaction of three-fingered neurotoxins with heteromeric alpha3beta2 nAChRs.

3H]Benzophenone Photolabeling Identifies State-Dependent Changes in Nicotinic Acetylcholine Receptor Structure

Interactions of benzophenone (BP) with the Torpedo nicotinic acetylcholine receptor (nAChR) were characterized by electrophysiological analyses, radioligand binding assays, and photolabeling of nAChR-rich membranes with [3H]BP to identify the amino acids contributing to its binding sites. BP acted as a low potency noncompetitive antagonist, reversibly inhibiting the ACh responses of nAChRs expressed in Xenopus oocytes (IC50 = 600 microM) and the binding of the noncompetitive antagonist [3H]tetracaine to nAChR-rich membranes (IC50 = 150 microM). UV irradiation at 365 nm resulted in covalent incorporation of [3H]BP into the nAChR subunits (delta > alpha approximately beta > gamma), with photoincorporation limited to the nAChR transmembrane domain. Comparison of nAChR photolabeling in the closed state (absence of agonist) and desensitized state (equilibrated with agonist) revealed selective desensitized state labeling in the delta subunit of deltaPhe-232 in deltaM1 and deltaPro-286/deltaIle-288 near the beginning of deltaM3 that are within a pocket at the interface between the transmembrane and extracellular domains. There was labeling in the closed state within the ion channel at position M2-13 (alphaVal-255, betaVal-261, and deltaVal-269) that was reduced by 90% upon desensitization and labeling in the transmembrane M3 helices of the beta and gamma subunits (betaMet-285, betaMet-288, and gammaMet-291) that was reduced by 50-80% in the desensitized state. Labeling at the lipid interface (alphaMet-415 in alphaM4) was unaffected by agonist. These results provide a further definition of the regions in the nAChR transmembrane domain that differ in structure between the closed and desensitized states.

Photolabeling the Torpedo Nicotinic Acetylcholine Receptor with 4-Azido-2,3,5,6-tetrafluorobenzoylcholine, a Partial Agonist

The interactions of a photoreactive analogue of benzoylcholine, 4-azido-2,3,5,6-tetrafluorobenzoylcholine (APFBzcholine), with nicotinic acetylcholine receptors (nAChRs) were studied using electrophysiology and photolabeling. APFBzcholine acted as a low-efficacy partial agonist, eliciting maximal responses that were 0.3 and 0.1% of that of acetylcholine for embryonic mouse and Torpedo nAChRs expressed in Xenopus oocytes, respectively. Equilibrium binding studies of [3H]APFBzcholine with nAChR-rich membranes from Torpedo electric organ revealed equal affinities (K(eq) = 12 microM) for the two agonist binding sites. Upon UV irradiation at 254 nm, [3H]APFBzcholine was photoincorporated into the nAChR alpha, gamma, and delta subunits in an agonist-inhibitable manner. Photolabeled amino acids in the agonist binding sites were identified by Edman degradation of isolated, labeled subunit fragments. [3H]APFBzcholine photolabeled gammaLeu-109/deltaLeu-111, gammaTyr-111, and gammaTyr-117 in binding site segment E as well as alphaTyr-198 in alpha subunit binding site segment C. The observed pattern of photolabeling is examined in relation to the predicted orientation of the azide when APFBzcholine is docked in the agonist binding site of a homology model of the nAChR extracellular domain based upon the structure of the snail acetylcholine binding protein.

Identification of amino acids in the nicotinic acetylcholine receptor agonist binding site and ion channel photolabeled by 4-[(3-trifluoromethyl)-3H-diazirin-3-yl]benzoylcholine, a novel photoaffinity antagonist.

[(3)H]4-[(3-trifluoromethyl)-3H-diazirin-3-yl]benzoylcholine (TDBzcholine) was synthesized and used as a photoaffinity probe to map the orientation of an aromatic choline ester within the agonist binding sites of the Torpedo nicotinic acetylcholine receptor (nAChR). TDBzcholine acts as a nAChR competitive antagonist that binds at equilibrium with equal affinity to both agonist sites (K(D) approximately 10 microM). Upon UV irradiation (350 nm), nAChR-rich membranes equilibrated with [(3)H]TDBzcholine incorporate (3)H into the alpha, gamma, and delta subunits in an agonist-inhibitable manner. The specific residues labeled by [(3)H]TDBzcholine were determined by N-terminal sequence analysis of subunit fragments produced by enzymatic cleavage and purified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and/or reversed-phase high-performance liquid chromatography. For the alpha subunit, [(3)H]TDBzcholine photoincorporated into alphaCys-192, alphaCys-193, and alphaPro-194. For the gamma and delta subunits, [(3)H]TDBzcholine incorporated into homologous leucine residues, gammaLeu-109 and deltaLeu-111. The photolabeling of these amino acids suggests that when the antagonist TDBzcholine occupies the agonist binding sites, the Cys-192-193 disulfide and Pro-194 from the alpha subunit Segment C are oriented toward the agonist site and are in proximity to gammaLeu-109/deltaLeu-111 in Segment E, a conclusion consistent with the structure of the binding site in the molluscan acetylcholine binding protein, a soluble protein that is homologous to the nAChR extracellular domain.

Mass Spectrometry of Nicotinic Acetylcholine Receptors and Associated Proteins as Models for Complex Transmembrane Proteins

Studies were conducted to optimize matrix-assisted laser desorption/ionization, time-of-flight mass spectrometry (MALDI TOF MS) in analyzing the composition of nicotinic acetylcholine receptors (nAChR) from Torpedo californica electric tissue in their membrane-bound, detergent-solubilized, and affinity-purified states. Mass spectra obtained from nAChR-rich membrane fractions gave reasonably good representations of protein compositions indicated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis of those same samples. Efficiency of extraction of nAChR from membranes was not markedly different for most detergents, but quality and signal size of mass spectra were clearly influenced by detergent composition and concentration, protein concentration, and MALDI matrix composition. The best spectra, allowing detection and accurate size determinations for samples containing as little as 10 fmol of pure nAChR, were obtained for samples solubilized in Triton X-100 and assayed by use of a sinapinic acid matrix. Although informative spectra could be obtained for nAChR affinity purified on α-cobratoxin (Naja naja siamensis) columns and extracted using sinapinic acid, superior spectra with much higher signal:noise were obtained if extraction media contained Triton X-100 or sodium dodecyl sulfate. nAChR subunit masses determined were similar regardless of the membrane-associated, detergent-solubilized, or affinity-purified state of the preparation. These studies illustrate how masses can be determined for nAChR subunits and for other protein components in Torpedo membrane preparations, such as RAPsyn and Na+-K+-ATPase α and β subunits. They also provide an underpinning for streamlined analysis of the composition of complex transmembrane proteins using MALDI TOF MS.

Site of resting state inhibition of the nicotinic acetylcholine receptor by a hydrophobic inhibitor.

The lipophilic photoactivatable probe 3-(trifluoromethyl)-3-(m-iodophenyl) diazirine (TID) is a noncompetitive, resting-state inhibitor of the nicotinic acetylcholine receptor (nAChR) that requires tens of milliseconds of preincubation to inhibit agonist-induced cation efflux. At equilibrium, [(125)I]TID photoincorporates into both the ion channel and the lipid-protein interface of the Torpedo nAChR. To determine which of these regions is responsible for resting-state inhibition, we characterized the interactions between [(125)I]TID and nAChR-rich membranes milliseconds after mixing, by use of time-resolved photolabeling. Photolabeling was performed after preincubation times of 2 ms or 600 s (equilibrium), and the efficiencies of incorporation at specific residues were determined by amino-terminal sequence analysis of nAChR-subunit proteolytic fragments isolated by SDS-PAGE and/or reversed-phase HPLC. Equilibration of TID with lipid was complete within a millisecond as determined by both stopped-flow fluorescence quenching of diphenylhexatriene in lipid bilayers and photoincorporation into nAChR-rich membrane phospholipids. Equilibration with the lipid-protein interface (alphaM4) was slightly slower, reaching approximately 50% that at equilibrium after 2 ms preincubation. In contrast, equilibration with the channel region (alpha 2 and deltaM2) was much slower, reaching only 10% that at equilibrium after 2 ms preincubation. Within the ion channel, the ratio of [(125)I]TID incorporation between M2 residues 9', 13', and 16' was independent of preincubation time. We conclude that TID's access to the ion channel is more restricted than to the lipid-protein interface and that TID bound within the ion channel is responsible for flux inhibition upon activation of the nAChR.

Probing the Structure of the Nicotinic Acetylcholine Receptor with 4-Benzoylbenzoylcholine, a Novel Photoaffinity Competitive Antagonist

[(3)H]4-Benzoylbenzoylcholine (Bz(2)choline) was synthesized as a photoaffinity probe for the Torpedo nicotinic acetylcholine receptor (nAChR). [(3)H]Bz(2)choline acts as an nAChR competitive antagonist and binds at equilibrium with the same affinity (K(D) = 1.4 microm) to both agonist sites. Irradiation at 320 nm of nAChR-rich membranes equilibrated with [(3)H]Bz(2)choline results in the covalent incorporation of [(3)H]Bz(2)choline into the nAChR gamma- and delta-subunits that is inhibitable by agonist, with little specific incorporation in the alpha-subunits. To identify the sites of photoincorporation, gamma- and delta-subunits, isolated from nAChR-rich membranes photolabeled with [(3)H]Bz(2)choline, were digested enzymatically, and the labeled fragments were isolated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and/or reversed-phase high performance liquid chromatography. For the gamma-subunit, Staphylococcus aureus V8 protease produced a specifically labeled peptide beginning at gammaVal-102, whereas for the delta-subunit, endoproteinase Asp-N produced a labeled peptide beginning at deltaAsp-99. Amino-terminal sequence analysis identified the homologous residues gammaLeu-109 and deltaLeu-111 as the primary sites of [(3)H]Bz(2)choline photoincorporation. This is the first identification by affinity labeling of non-reactive amino acids within the acetylcholine-binding sites, and these results establish that when choline esters of benzoic acid are bound to the nAChR agonist sites, the para substituent is selectively oriented toward and in proximity to amino acids gammaLeu-109/deltaLeu-111.

Specific phosphorylation of Torpedo 43K rapsyn by endogenous kinase(s) with thiamine triphosphate as the phosphate donor.

43K rapsyn is a peripheral protein specifically associated with the nicotinic acetylcholine receptor (nAChR) present in the postsynaptic membrane of the neuromuscular junction and of the electrocyte, and is essential for its clustering. Here, we demonstrate a novel specific phosphorylation of 43K rapsyn by endogenous protein kinase(s) present in Torpedo electrocyte nAChR-rich membranes and identify thiamine triphosphate (TTP) as the phosphate donor. In the presence of Mg(2+) and [gamma-(32)P]-TTP, 43K rapsyn is specifically phosphorylated with a (32)P-half-maximal incorporation at approximately 5-25 microM TTP. The presence of TTP in the cytosol and of 43K rapsyn at the cytoplasmic face of the postsynaptic membrane, together with TTP-dependent phosphorylation of 43K rapsyn without added exokinases, suggests that TTP-dependent-43K-rapsyn phosphorylation may occur in vivo. In addition, phosphoamino acid and chemical stability analysis suggests that the residues phosphorylated are predominantly histidines. Inhibition of phosphorylation by Zn(2+) suggests a possible control of 43K rapsyn phosphorylation state by its zinc finger domain. Endogenous kinase(s) present in rodent brain membranes can also use [gamma-(32)P]-TTP as a phosphodonor. The use of a phosphodonor (TTP) belonging to the thiamine family but not to the classical (ATP, GTP) purine triphosphate family represents a novel phosphorylation pathway possibly important for synaptic proteins.

Identification of amino acids of the torpedo nicotinic acetylcholine receptor contributing to the binding site for the noncompetitive antagonist [(3)H]tetracaine.

[(3)H]Tetracaine is a noncompetitive antagonist of the Torpedo nicotinic acetylcholine receptor (nAChR) that binds with high affinity in the absence of cholinergic agonist (K(eq) = 0.5 microM) and weakly (K(eq) = 30 microM) in the presence of agonist (i.e., to nAChR in the desensitized state). In the absence of agonist, irradiation at 302 nm of nAChR-rich membranes equilibrated with [(3)H]tetracaine results in specific photoincorporation of [(3)H]tetracaine into each nAChR subunit. In this report, we identify the amino acids of each nAChR subunit specifically photolabeled by [(3)H]tetracaine that contribute to the high-affinity binding site. Subunits isolated from nAChR-rich membranes photolabeled with [(3)H]tetracaine were subjected to enzymatic digestion, and peptides containing (3)H were purified by SDS-polyacrylamide gel electrophoresis followed by reversed phase HPLC. N-terminal sequence analysis of the isolated peptides demonstrated that [(3)H]tetracaine specifically labeled two sets of homologous hydrophobic residues (alphaLeu(251), betaLeu(257), gammaLeu(260), and deltaLeu(265); alphaVal(255) and deltaVal(269)) as well as alphaIle(247) and deltaAla(268) within the M2 hydrophobic segments of each subunit. The labeling of these residues establishes that the high-affinity [(3)H]tetracaine-binding site is located within the lumen of the closed ion channel and provides a definition of the surface of the M2 helices facing the channel lumen.

Photoaffinity labeling the torpedo nicotinic acetylcholine receptor with [(3)H]tetracaine, a nondesensitizing noncompetitive antagonist.

Tetracaine (N,N-dimethylaminoethyl-4-butylaminobenzoate) and related N,N-dialkylaminoethyl substituted benzoic acid esters have been used to characterize the high-affinity binding site for aromatic amine noncompetitive antagonists in the Torpedo nicotinic acetylcholine receptor (nAChR). [(3)H]Tetracaine binds at equilibrium to a single site with a K(eq) value of 0.5 microM in the absence of agonist or presence of alpha-bungarotoxin and with a K(eq) value of 30 microM in the presence of agonist (i.e., for nAChR in the desensitized state). Preferential binding to nAChR in the absence of agonist is also seen for N,N-DEAE and N,N-diethylaminopropyl esters, both binding with 10-fold higher affinity in the absence of agonist than in the presence, and for the 4-ethoxybenzoic acid ester of N, N-diethylaminoethanol, but not for the 4-amino benzoate ester (procaine). Irradiation at 302 nm of nAChR-rich membranes equilibrated with [(3)H]tetracaine resulted in covalent incorporation with similar efficiency into nAChR alpha, beta, gamma, and delta subunits. The pharmacological specificity of nAChR subunit photolabeling as well as its dependence on [(3)H]tetracaine concentration establish that the observed photolabeling is at the high-affinity [(3)H]tetracaine-binding site. Within alpha subunit, >/=95% of specific photolabeling was contained within a 20-kilodalton proteolytic fragment beginning at Ser(173) that contains the M1 to M3 hydrophobic segments. With all four subunits contributing to [(3)H]tetracaine site, the site in the closed channel state of the nAChR is most likely within the central ion channel domain.

Structure of the Agonist-Binding Sites of the Torpedo Nicotinic Acetylcholine Receptor: Affinity-Labeling and Mutational Analyses Identify γTyr-111/δArg-113 as Antagonist Affinity Determinants

Photoaffinity labeling of the Torpedo nicotinic acetylcholine receptor (nAChR) with [3H]d-tubocurarine (dTC) has identified a residue within the gamma-subunit which, along with the analogous residue in delta-subunit, confers selectivity in binding affinities between the two agonist sites for dTC and alpha-conotoxin (alpha Ctx) MI. nAChR gamma-subunit, isolated from nAChR-rich membranes photolabeled with [3H]dTC, was digested with Staphylococcus aureus V8 protease, and a 3H-labeled fragment was purified by reversed-phase high-performance liquid chromatography. Amino-terminal sequence analysis of this fragment identified 3H incorporation in gamma Tyr-111 and gamma Tyr-117 at about 5% and 1% of the efficiency of [3H]dTC photoincorporation at gamma Trp-55, the primary site of [3H]dTC photoincorporation within gamma-subunit [Chiara, D. C., and Cohen, J. B. (1997) J. Biol. Chem 272, 32940-32950]. The Torpedo nAChR delta-subunit residue corresponding to gamma Tyr-111 (delta Arg-113) contains a positive charge which could confer the lower binding affinity seen for some competitive antagonists at the alpha-delta agonist site. To test this hypothesis, we examined by voltage-clamp analysis and/or by [125I]alpha-bungarotoxin competition binding assays the interactions of acetylcholine (ACh), dTC, and alpha Ctx MI with nAChRs containing gamma Y111R or delta R113Y mutant subunits expressed in Xenopus oocytes. While these mutations affected neither ACh equilibrium binding affinity nor the concentration dependence of channel activation, the gamma Y111R mutation decreased by 10-fold dTC affinity and inhibition potency. Additionally, each mutation conferred a 1000-fold change in the equilibrium binding of alpha Ctx MI, with delta R113Y enhancing and gamma Y111R weakening affinity. Comparison of these results with previous results for mouse nAChR reveals that, while the same regions of gamma- (or delta-) subunit primary structure contribute to the agonist-binding sites, the particular amino acids that serve as antagonist affinity determinants are species-dependent.

Identification of tryptophan 55 as the primary site of [ 3H]nicotine photoincorporation in the γ-subunit of the Torpedo nicotinic acetylcholine receptor

[ 3H]nicotine has been used as a photoaffinity agonist to identify amino acids within the Torpedo nicotinic acetylcholine receptor (nAChR) γ-subunit that contributes to the structure of the agonist binding site. UV irradiation (254 nm) of nAChR-rich membranes equilibrated with [ 3H]nicotine results in covalent incorporation into α- and γ-subunits that is inhibitable by agonists and competitive antagonists, but not by non-competitive antagonists (Middleton, R.E. and Cohen, J.B. (1991) Biochemistry 30, 6887–6897). To identify sites of specific incorporation, SDS-PAGE and reversed-phase HPLC were used to isolate proteolytic fragments of [ 3H]nicotine-labeled γ-subunit. Amino-terminal sequence analysis identified γTrp-55 as the major site of [ 3H]nicotine photoincorporation in γ-subunit. Thus γTrp-55 is the first amino acid within a non-α-subunit to be identified by affinity labeling in direct contact with a bound agonist.

Identification of Amino Acids Contributing to High and Low Affinity d-Tubocurarine Sites in the TorpedoNicotinic Acetylcholine Receptor

d-Tubocurarine (dTC) is a potent competitive antagonist of the Torpedo nicotinic acetylcholine receptor (nAChR) that binds non-equivalently to the two agonist sites (Kd values of 30 nM and 8 microM). When nAChR-rich membranes equilibrated with [3H]dTC are irradiated with 254 nm UV light, [3H]dTC is covalently incorporated into the alpha-, gamma-, and delta-subunits in a concentration-dependent and agonist-inhibitable manner, consistent with the localization of the high and low affinity dTC binding sites at the alpha-gamma- and alpha-delta-subunit interfaces, respectively (Pedersen, S. E. and Cohen, J. B. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 2785-2789). We report on the amino acids within alpha-, gamma-, and delta-subunits that are the sites of specific photoincorporation of [3H]dTC. Subunits isolated from nAChR-rich membranes photolabeled with [3H]dTC were subjected to enzymatic digestion, and peptides containing 3H were isolated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and/or reversed-phase high performance liquid chromatography. Isolated peptides were then subjected to NH2-terminal sequence analysis to identify specifically labeled residues. Within the alpha-subunit, 95% of specific incorporation was contained within a 20-kDa proteolytic fragment beginning at Ser-173, with alphaTyr-190 the primary site of [3H]dTC photoincorporation and alphaCys-192 and alphaTyr-198 labeled at lower efficiency. Within gamma- and delta-subunits, specific labeling was contained within proteolytic fragments of 14 and 21 kDa, respectively, beginning at gammaAla-49 and deltaThr-51. gammaTrp-55 and deltaTrp-57 were identified as the sites of specific [3H]dTC photoincorporation. Sequence alignment studies reveal gammaTrp-55 and deltaTrp-57 to be homologous residues at whose position in receptor subunit primary structure a unique pattern of conservation exists in all nAChR (neuronal and muscle). Specifically, all subunits that associate with an alpha-subunit to form an agonist site contain a tryptophan homologous to gammaTrp-55/deltaTrp-57. This pattern of conservation may indicate a functional significance for tryptophan at that location in all nAChR agonist sites.

Barbiturate Action Is Dependent on the Conformational State of the Acetylcholine Receptor

Barbiturates act on many neuronal ion channels by poorly understood mechanisms. The authors investigated the hypothesis that barbiturates inhibit the transient open-channel conformation of the nicotinic acetylcholine receptor (nAchR) by binding to a discrete site. Inhibition curves of the agonist-stimulated efflux of 8bRb+ from nAchR-rich membrane vesicles prepared from the electric tissue of Torpedo nobiliana were obtained for 14 barbiturates using a filter assay. When added simultaneously with agonist, all agents inhibited the ion efflux with half-inhibitory concentrations (IC50), varying from 23 microM for pentobarbital to 880 microM for barbital, and with Hill coefficients of one. The effect of several barbiturates on the agonist concentration-response curve for carbachol-stimulated efflux indicated that this inhibitory action was not competitive. The IC50s of these agents did not correlate with their octanol/water partition coefficients, nor with general anesthetic potency, although a degree of channel inhibition occurred with many agents at general anesthetic concentrations. The existence of a barbiturate-inhibitory site of action was indicated by the structural specificity. This conclusion was supported by the Hill coefficient of one, and by the high inhibitory potencies, which ruled out membrane perturbations as a mechanism. This site on the transient open-channel conformation exhibits different structure-activity relationships than an allosteric site established by equilibrium barbiturate binding on the resting conformation of the AchR. Thus, barbiturate action depends on the nAchR's conformational state.

Photoaffinity labeling of muscle-type nicotinic acetylcholine receptors and neuronal/nicotinic α-bungarotoxin binding sites with a derivative of α-bungarotoxin

Neuronal/nicotinic α-bungarotoxin binding sites (nBgtS) found in the nervous system are not well characterized. In this study, photolabile toxin derivatives have been used in affinity labeling protocols to investigate the subunit composition of nBgtS expressed by different neuron-like cell lines. Data obtained was compared to the known subunit composition of toxin-binding muscle-type nicotinic acetylcholine receptors (nAChR). Muscle-type nAChR-rich membranes prepared from Torpedo electroplax contain components with corrected apparent molecular sizes of 41, 46, 50, 62 and 66 kDa that are reactive with toxin. The photoaffinity labeling patterns for preparations derived from cells of the TE671 clone, which express muscle-type nAChR, are very similar to that of cells of the IMR-32 or SH-SY5Y clonal lines, which express nBgtS. There is consistent labeling of four polypeptides with corrected apparent molecular weights of 40, 43, 47 and 56 kDa. These results suggest that both mammalian muscle-type nAChR and mammalian nBgtS are similarly composed of at least four kinds of subunits.