Time-resolved photolabeling by quinacrine azide of a noncompetitive inhibitor site of the nicotinic acetylcholine receptor in a transient, agonist-induced state

Local anesthetics and other noncompetitive inhibitors (NCIs) of the nicotinic acetylcholine receptor, acting at sites other than the acetylcholine-binding sites, block channel opening and/or cation translation through the open channel. In order to characterize the NCI sites and to decide among possible mechanisms of NCI action, we have photolabeled the receptor in membrane from Torpedo electric tissue with the photolyzable NCI [3H]quinacrine azide ([3H]QA), using a continuous-flow, rapid-mixing device and millisecond-duration irradiation. Membrane, [3H]QA, and effectors were mixed, and, after delay times of 20 ms or greater, the mixture was irradiated for 2 ms, quenched, and collected. Brief exposure of the receptor to acetylcholine, but not to hexamethonium or d-tubocurarine, induced a state particularly susceptible to photoincorporation of [3H]QA. This acetylcholine-induced photoincorporation was exclusively into the alpha and beta chains of the receptor, peaked at 100-ms delay time, declined to 15% of maximum after delay times of minutes, and was blocked by the NCIs proadifen and histrionicotoxin. At 20-ms delay, the dependence of labeling by 2 microM [3H]QA on acetylcholine concentration was characterized by an apparent dissociation constant of about 15 microM and a Hill coefficient of 1. The kinetics of the development of susceptibility to photolabeling and the apparent lack of positive cooperativity in the effect of acetylcholine on this development suggest that the preferentially photolabeled state is a transient, rapidly developing, desensitized state, rather than an open-channel state.     

Cembranoid and long-chain alkanol sites on the nicotinic acetylcholine receptor and their allosteric interaction

Long-chain alkanols are general anesthetics which can also act as uncharged noncompetitive inhibitors of the peripheral nicotinic acetylcholine receptor (AChR) by binding to one or more specific sites on the AChR. Cembranoids are naturally occurring, uncharged noncompetitive inhibitors of peripheral and neuronal AChRs, which have no demonstrable general anesthetic activity in vivo. In this study, [3H]tenocyclidine ([3H]TCP), an analogue of the cationic noncompetitive inhibitor phencyclidine (PCP), was used to characterize the cembranoid and long-chain alkanol sites on the desensitized Torpedo californica AChR and to investigate if these sites interact. These studies confirm that there is a single cembranoid site which sterically overlaps the [3H]TCP channel site. This cembranoid site probably also overlaps the sites for the cationic noncompetitive inhibitors, procaine and quinacrine. Evidence is also presented for one or more allosteric cembranoid sites which negatively modulate cembranoid affinity for the inhibitory site. In contrast, long-chain alkanols inhibit [3H]TCP binding through an allosteric mechanism involving two or more alkanol sites which display positive cooperativity toward each other. Double inhibitor studies show that the cembranoid inhibitory site and the alkanol sites are not independent of each other but interfere allosterically with each other's inhibition of [3H]TCP binding. The simplest models consistent with the observed data are presented and discussed.     

Structure of the high-affinity binding site for noncompetitive blockers of the acetylcholine receptor: [3H]chlorpromazine labels homologous residues in the beta and delta chains

The membrane-bound acetylcholine receptor from Torpedo marmorata was photolabeled by the noncompetitive channel blocker [3H]chlorpromazine under equilibrium conditions in the presence of the agonist carbamoylcholine. The amount of radioactivity incorporated into all subunits was reduced by addition of phencyclidine, a specific ligand for the high-affinity site for noncompetitive blockers. The labeled beta chain was purified and digested with trypsin or CNBr, and the resulting fragments were fractionated by high-performance liquid chromatography. Sequence analysis resulted in the identification of Ser-254 and Leu-257 as residues labeled by [3H]chlorpromazine in a phencyclidine-sensitive manner. These residues are located in the hydrophobic and potentially transmembrane segment M II of the beta chain, a region homologous to that containing the chlorpromazine-labeled Ser-262 in the delta chain [Giraudat, J., Dennis, M., Heidmann, T., Chang, J. Y., & Changeux, J.-P. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 2719-2723]. These results show that homologous regions of different receptor subunits contribute to the unique high-affinity site for noncompetitive blockers, a finding consistent with the location of this site on the axis of symmetry of the receptor molecule.     

Characterization of the transient agonist-triggered state of the acetylcholine receptor rapidly labeled by the noncompetitive blocker [3H]chlorpromazine: additional evidence for the open channel conformation

The kinetics of covalent labeling of the alpha, beta, gamma, and delta chains of the acetylcholine receptor (AcChR) from Torpedo marmorata by the noncompetitive blocker [3H]chlorpromazine ([3H]CPZ) are investigated by using rapid mixing photolabeling techniques. In an initial study [Heidmann, T., & Changeux, J. P. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 1897-1901], it was shown that the rate of [3H]CPZ labeling increases 100-1000-fold upon simultaneous addition of nicotinic agonists to the AcChR and that prior addition of these agonists abolishes the effect. The data were interpreted in terms of the rapid labeling of the transient active state of the AcChR where the ion channel is in its open configuration. This interpretation was recently challenged [Cox, R. N., Kaldany, R. R. J., Di Paola, M., & Karlin, A. (1985) J. Biol. Chem. 260, 7186-7193] on the ground of studies with a different noncompetitive blocker, [3H]quinacrine azide, and the suggestion was made that this compound labels the rapidly desensitized closed channel conformation of the AcChR. In this paper it is shown that the rate of rapid labeling of the AcChR by [3H]CPZ decreases to negligible values upon exposure of the AcChR to nicotinic agonists, in the 100-500-ms time range. The absolute values of the rate constants of this decrease (10-15 s-1 for saturating concentrations of acetylcholine and carbamoylcholine) and their variation with agonist concentration (apparent dissociation constants of 40 microM and 0.4 mM for acetylcholine and carbamoylcholine, respectively) are those expected for the rapid desensitization of the AcChR.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structure of the agonist-binding site of the nicotinic acetylcholine receptor. [3H]acetylcholine mustard identifies residues in the cation-binding subsite.

To characterize the structure of the agonist-binding site of the Torpedo nicotinic acetylcholine receptor (AChR), we have used [3H]acetylcholine mustard [( 3H]AChM), a reactive analog of acetylcholine, to identify residues contributing to the cation-binding subsite. Reaction of [3H]AChM, in its aziridinium form, with AChR-rich membrane suspensions, resulted initially in reversible, high affinity binding (K approximately 0.3 microM) followed by slow alkylation of the acetylcholine-binding site. Incorporation of label into AChR alpha-subunit was inhibited by agonists and competitive antagonists, but not by noncompetitive antagonists, and reaction with 3 microM [3H]AChM for 2 h resulted in specific alkylation of 0.6% of alpha-subunits. Within the alpha-subunit, greater than 90% of specific incorporation was contained within an 18-kDa Staphylococcus aureus V8 proteolytic fragment beginning at Val-46 and containing N-linked carbohydrate. To identify sites of specific alkylation, [3H]AChM-labeled alpha-subunit was digested with trypsin, and the digests were fractionated by reverse phase high pressure liquid chromatography. Specifically labeled material was recovered within a single peak containing a peptide extending from Leu-80 to Lys-107. NH2-terminal amino acid sequencing revealed specific release of 3H in cycle 14 corresponding to alpha-subunit Tyr-93. Identification of Tyr-93 as the site of alkylation was confirmed by radiosequence analysis utilizing o-phthalaldehyde to establish that the released 3H originated from a peptide containing prolines at residues 2 and 9. Because [3H]AChM contains as its reactive group a positively charged quaternary aziridinium, alpha-subunit Tyr-93 is identified as contributing to the cation-binding domain of the AChR agonist-binding site. The selective reaction of [3H]AChM with tyrosyl rather than acidic side chains indicates the importance of aromatic interactions for the binding of the quaternary ammonium group, and the lack of reaction with the tyrosyl or acidic side chains within alpha 190-200 emphasizes the selective orientation of acetylcholine within its binding site.     

Decidium. A novel fluorescent probe of the agonist/antagonist and noncompetitive inhibitor sites on the nicotinic acetylcholine receptor

We have examined the interaction of the nicotinic acetylcholine receptor with decidium diiodide, a bisquaternary analogue of ethidium containing 10 methylene groups between the endocyclic and trimethylamino quaternary nitrogens. Decidium inhibits mono-[125I]iodo-alpha-toxin binding, inhibits agonist-elicited 22Na+ influx in intact cells, augments agonist competition with mono-[125I]iodo-alpha-toxin binding, and enhances [3H]phencyclidine (PCP) binding to a noncompetitive inhibitor site. These effects occur over similar concentration ranges (half-maximum effects between 0.1 and 0.4 microM). Thus, decidium binds to the agonist site and converts the receptor to a desensitized state exhibiting increased affinity for agonist and heterotropic inhibitors. These properties are similar to metaphilic antagonists characterized in classical pharmacology. At higher concentrations decidium associates directly with the noncompetitive inhibitor site identified by [3H]phencyclidine binding. Dissociation constants of decidium at this site in the resting and desensitized states are determined to be 29 and 1.2 microM, respectively. Analysis of fluorescence excitation and emission maxima reveal that binding to both the agonist and noncompetitive inhibitor sites is associated with approximately 2-fold enhancement of fluorescence. The excitation maximum for decidium bound at the agonist site appears at 490 nm while that for decidium bound at the noncompetitive inhibitor site appears at 530 compared to 480 nm in buffer. These results suggest that decidium experiences a more hydrophobic environment upon binding to the nicotinic acetylcholine receptor sites, particularly to the noncompetitive inhibitor site. Fluorescence energy transfer between N'-fluorescein isothiocyanate-lysine-23 alpha-toxin (FITC-toxin), and decidium is not detected when each is bound to one of the two agonist sites on the receptor. This allows a minimal distance to be estimated between fluorophores. In contrast, energy transfer is observed between decidium nonspecifically associated with the membrane or with nonspecific sites and the FITC-toxin at the agonist sites.     

The noncompetitive blocker [(3)H]chlorpromazine labels segment M2 but not segment M1 of the nicotinic acetylcholine receptor alpha-subunit

The membrane bound acetylcholine receptor from Torpedo marmorata was photolabeled by the noncompetitive channel blocker ]3H]chlorpromazine under equilibrium conditions in the presence of the agonist carbamoylcholine. The radioactivity incorporated into the AChR subunits was reduced by addition of phencyclidine, a specific ligand for the high-affinity side for noncompetitive blockers. The alpha-subunit was purified and digested with trypsin and/or CNBr and the resulting fragments fractionated by HPLC. Sequence analysis resulted in the identification of Ser-248 as a major residue labeled by [3H]chlorpromazine in a phencyclidine-sensitive manner. This residue is located in the hydrophobic and putative transmembrane segment M2 of the alpha-subunit, a region homologous to that containing the chlorpromazine-labeled Ser-262 in the delta-chain [1] and Ser-254 and Leu-257 in the beta-chain [2]. Extended sequence analysis of the hydrophobic segment M1 further showed that no labeling-occurred in this region.     

Binding sites for alpha-bungarotoxin and the noncompetitive inhibitor phencyclidine on a synthetic peptide comprising residues 172-227 of the alpha-subunit of the nicotinic acetylcholine receptor.

The binding of the competitive antagonist alpha-bungarotoxin (alpha-Btx) and the noncompetitive inhibitor phencyclidine (PCP) to a synthetic peptide comprising residues 172-227 of the alpha-subunit of the Torpedo acetylcholine receptor has been characterized. 125I-alpha-Btx bound to the 172-227 peptide in a solid-phase assay and was competed by alpha-Btx (IC50 = 5.0 x 10(-8) M), d-tubocurarine (IC50 = 5.9 X 10(-5)M), and NaCl (IC50 = 7.9 x 10(-2)M). In the presence of 0.02% sodium dodecyl sulfate, 125I-alpha-Btx bound to the 56-residue peptide with a KD of 3.5 nM, as determined by equilibrium saturation binding studies. Because alpha-Btx binds to a peptide comprising residues 173-204 with the same affinity and does not bind to a peptide comprising residues 205-227, the competitive antagonist and hence agonist binding site lies between residues 173 and 204. After photoaffinity labeling, [3H]PCP was bound to the 172-227 peptide. [3H]PCP binding was inhibited by chlorpromazine (IC50 = 6.3 x 10(-5)M), tetracaine (IC50 = 4.2 x 10(-6)M), and dibucaine (IC50 = 2.7 x 10(-4)M). Equilibrium saturation binding studies in the presence of 0.02% sodium dodecyl sulfate showed that [3H]PCP bound at two sites, a major site of high affinity with an apparent KD of 0.4 microM and a minor low-affinity site with an apparent KD of 4.6 microM. High -affinity binding occurred at a single site on peptide 205-227 (KD = 0.27 microM) and was competed by chlorpromazine but not by alpha-Btx.(ABSTRACT TRUNCATED AT 250 WORDS)     

Photoaffinity labeling of Torpedo nicotinic receptor with the agonist [3H]DCTA: identification of amino acid residues which contribute to the binding of the ester moiety of acetylcholine

Torpedo marmorata acetylcholine binding sites were photolabeled using 360 nm light, at equilibrium in the desensitized state, with the agonist [3H]DCTA utilizing the CeIV/glutathione procedure described previously (Grutter, et al. (1999) Biochemistry 38, 7476-7484). Photoincorporation of [3H]DCTA was concentration-dependent with a maximum of 7.5% specific labeling on the alpha-subunit and 1.2% on the gamma-subunit. The apparent dissociation constants for labeling of the alpha- and gamma-subunits were 2.2 +/- 1.1 and 3.6 +/- 2.8 microM, respectively. The alpha-chains isolated from receptor-rich membranes photolabeled in the absence or in the presence of carbamylcholine were cleaved with CNBr using an efficient "in gel" procedure. The resulting peptide fragments were purified by HPLC and further submitted to trypsinolysis. The digest was analyzed by HPLC leading to a single radioactive peak which, by microsequencing, revealed two sequences extending from alpha Lys-179 and from alpha His-186, respectively. Radioactive signals could be unambiguously attributed to positions corresponding to residues alpha Tyr-190, alpha Cys-192, alpha Cys-193, and alpha Tyr-198. These four identified [3H]DCTA-labeled residues, which have been also labeled with other affinity and photoaffinity probes including the agonist [3H]nicotine, belong to loop C of the ACh binding site. The chemical structure of [3H]DCTA, together with its well-defined and powerful photochemical reactivity, provides convincing evidence that loop C-labeled residues are primarily involved in the interaction with the ester moiety of acetylcholine.     

Mapping of the acetylcholine binding site of the nicotinic acetylcholine receptor: [3H]nicotine as an agonist photoaffinity label

The agonist [3H]nicotine was used as a photoaffinity label for the acetylcholine binding sites on the Torpedo nicotinic acetylcholine receptor (AChR). [3H]nicotine binds at equilibrium with Keq = 0.6 microM to the agonist binding sites. Irradiation with 254-nm light of AChR-rich membranes equilibrated with [3H]nicotine resulted in covalent incorporation into the alpha- and gamma-subunits, which was inhibited by agonists and competitive antagonists but not by noncompetitive antagonists. Inhibition of labeling by d-tubocurarine demonstrated that the alpha-subunit was labeled via both agonist sites but the gamma-subunit was labeled only via the site that binds d-tubocurarine with high affinity. Within the alpha-subunit, 93% of the labeling was contained within a 20-kDa Staphylococcus aureus V8 proteolytic fragment beginning at Ser-173. Sequence analysis of this peptide indicated that approximately 80% of the incorporation was into Tyr-198, approximately 13% was into Cys-192, and approximately 7% was into Tyr-190. Chymotryptic digestion of the alpha-subunit confirmed that Tyr-198 was the principal amino acid labeled by [3H]nicotine. This confirmation required a novel radio-sequencing strategy employing omicron-phthalaldehyde, since the efficiency of photolabeling was low (approximately 1.0%) and the labeled chymotryptic peptide was not isolated in sufficient quantity to be identified by mass. [3H]Nicotine, which is the first photoaffinity agonist used, labels primarily Tyr-198 in contrast to competitive antagonist affinity labels, which label primarily Tyr-190 and Cys-192/Cys-193.     

Quinacrine binds to the lipid-protein interface of the Torpedo acetylcholine receptor: a fluorescence study.

It has been argued both that there is a high affinity noncompetitive inhibitor binding site in the lumen of the acetylcholine receptor and that this lumen exists on the central axis of the receptor. Such a site would be expected to be 20-40 A from the membrane lipids. We tested whether, in fact, quinacrine, a potent fluorescent noncompetitive inhibitor, binds to such a site. We measured quenching of receptor-bound quinacrine fluorescence by fluorescence dipolar energy transfer to lipid probes, 5-(N-dodecanoylamino)eosin and N-(3-sulfopropyl)-4-(p-didecylaminostyryl)pyridinium, or by collision with paramagnetic lipid probes 2,2,6,6-tetramethylpiperidine-1-oxyl and 3-doxyl-17 beta-hydroxy-5 alpha-androstane (spin-labeled androstane). Initial control experiments established that in the presence of carbamylcholine, quinacrine binds to a phencyclidine-sensitive site on the Torpedo receptor with a Kd equal to 0.14 microM and with a quantum yield of 0.18. Fluorescence energy transfer from receptor-bound quinacrine had a magnitude consistent with quinacrine being less than 10 A from the lipid fluorescent probes. 2,2,6,6-Tetramethylpiperidine-1-oxyl and spin-labeled androstane were two to five times more effective at quenching receptor-bound quinacrine fluorescence than the fluorescence from membrane-partitioned 5-(dodecanoylamino)fluorescein. These results suggest that the quinacrine binding site is too close to the lipid domain to be in the lumen of the receptor, and therefore it is probably located on the outer surface of the membrane-spanning domain of the acetylcholine receptor.     

Thiol-group modification of Torpedo californica acetylcholine receptor: subunit localization and effects on function

The effects of thiol-group modifications on acetylcholine receptor (ACHR) function were measured with purified ACHR reconstituted into asolectin vesicles. N-Phenylmaleimide (NPM) was used to modify sulfhydryl groups on ACHR in the absence of any prior reduction of dithiothreitol, so that only the functional relevance of free sulfhydryls was examined. Modification by NPM led to the inhibition of ion-channel activity without a detectable effect on ligand binding. The ion flux inhibition by NPM primarily affected channel activation, since the initial rates of activation were decreased over a wide range of carbamylcholine concentrations. The [3H]NPM subunit labeling pattern of ACHR (a multisubunit membrane protein with alpha 2 beta gamma delta stoichiometry) revealed that there was preferential labeling of the gamma subunit. At high NPM concentrations, the number of sulfhydryl groups on the gamma subunit that could be modified with NPM was approximately two. Detergent was required during labeling for functionally relevant thiol-group modifications, and most of the label was protected from protease digestion in the reconstituted membranes. These results are consistent with the presence of the NPM modification in a bilayer and/or cytoplasmic domain.     

Location of ligand-binding sites on the nicotinic acetylcholine receptor alpha-subunit

The portions of the Torpedo californica nicotinic acetylcholine receptor (AChR) alpha-subunit that contribute to the allosteric antagonist-binding site and to the agonist-binding site have been localized by affinity labeling and proteolytic mapping. [3H]Meproadifen mustard was employed as an affinity label for the allosteric antagonist-binding site and [3H]tubocurare as a photoaffinity label for the agonist-binding site. Both labels were found in a 20-kDa proteolytic fragment generated from the AChR alpha-subunit by Staphylococcus aureus V8 protease. This 20-kDa peptide also contains the 3H-labeled 4-(N-maleimido)-alpha-benzyltrimethylammonium iodide-reactive site and binds 125I-alpha-bungarotoxin. N-terminal sequencing established that the 20-kDa fragment began at Ser-173 of the alpha-subunit. Fluorescein isothiocyanate-conjugated concanavalin A could be bound to the second of the two major V8 cleavage products, an 18-kDa peptide. This peptide was also sensitive to treatment with endo-beta-N-acetyl-glucosaminidase H, consistent with the presence of N-linked carbohydrate on this fragment. The N terminus of this peptide was found to be Val-46 of the alpha-subunit sequence. Experiments designed to map disulfide bonds within the AChR alpha-subunit indicate that no bonds exist between the 18-kDa fragment (containing Cys-128 and Cys-142) and the 20-kDa fragment (containing Cys-192, Cys-193, and Cys-222). These results establish that the 20-kDa fragment contributes to both the acetylcholine and the allosteric antagonist-binding sites, whereas there is no evidence that the 18-kDa fragment is part of either site.     

Photolabeling of membrane-bound Torpedo nicotinic acetylcholine receptor with the hydrophobic probe 3-trifluoromethyl-3-(m-[125I]iodophenyl)diazirine

The hydrophobic, photoactivatable probe 3-trifluoromethyl-3-(m-[125I]iodophenyl)diazirine ([125I]TID) was used to label acetylcholine receptor rich membranes purified from Torpedo californica electric organ. All four subunits of the acetylcholine receptor (AChR) were found to incorporate label, with the gamma-subunit incorporating approximately 4 times as much as each of the other subunits. Carbamylcholine, an agonist, and histrionicotoxin, a noncompetitive antagonist, both strongly inhibited labeling of all AChR subunits in a specific and dose-dependent manner. In contrast, the competitive antagonist alpha-bungarotoxin and the noncompetitive antagonist phencyclidine had only modest effects on [125I]TID labeling of the AChR. The regions of the AChR alpha-subunit that incorporate [125I]TID were mapped by Staphylococcus aureus V8 protease digestion. The carbamylcholine-sensitive site of labeling was localized to a 20-kDa V8 cleavage fragment that begins at Ser-173 and is of sufficient length to contain the three hydrophobic regions M1, M2, and M3. A 10-kDa fragment beginning at Asn-339 and containing the hydrophobic region M4 also incorporated [125I]TID but in a carbamylcholine-insensitive manner. Two further cleavage fragments, which together span about one-third of the alpha-subunit amino terminus, incorporated no detectable [125I]TID. The mapping results place constraints on suggested models of AChR subunit topology.     

Biosynthesis of bacterial glycogen. Isolation and characterization of the pyridoxal-P allosteric activator site and the ADP-glucose-protected pyridoxal-P binding site of Escherichia coli B ADP-glucose synthase.

[3H]Pyridoxal-P can be covalently incorporated into Escherichia coli B mutant strain AC70R1 ADP-glucose synthase by reduction with NaBH4. Two distinct lysine residues can be modified by the allosteric activator pyridoxal-P. Incorporation of [3H]pyridoxal-P in the presence of substrate ADP-glucose + MgCl2 prevents pyridoxylation of an ADP-glucose-protected site and allows modification of the allosteric activator site. Incorporation of [3H]pyridoxal-P in the presence of the allosteric effector, 1,6-hexanediol-P2, protects against pyridoxylation of the allosteric activator site and allows modification of the ADP-glucose-protected site. The activator site CNBr [3H]pyridoxyl-P peptide was purified to homogeneity in the presence of urea by Sephadex G-50 and CM-cellulose chromatography. The peptide consists of 59 residues, with a molecular weight of 6750. The NH2-terminal of the peptide has a 16-residue sequence overlap with the previously determined NH2-terminal sequence of the native enzyme. The activator site pyridoxyl-P lysine is identified as residue 38 of the native enzyme's NH2 terminus. The ADP-glucose-protected site CNBr [3H]pyridoxyl peptide was purified to homogeneity by Sephadex G-50 and DEAE-cellulose chromatography. The peptide consists of 21 residues, with a molecular weight of 2460. The sequence of this peptide has been elucidated.     

Photoaffinity labeling of the acetylcholine transporter.

The acetylcholine (AcCh) binding site in the AcCh transporter-vesamicol receptor (AcChT-VR) present in synaptic vesicles isolated from the electric organ of Torpedo was characterized. A high-affinity analogue of AcCh containing an aryl azido group, namely, cyclohexylmethyl cis-N-(4-azidophenacyl)-N-methylisonipecotate bromide (AzidoAcCh), was synthesized in nonradioactive and highly tritiated forms. AzidoAcCh was shown to be a competitive inhibitor of [3H]AcCh active transport and binding of [3H]-vesamicol to the allosteric site. The [3H]AzidoAcCh saturation curve was determined. In all cases the AcChT.AzidoAcCh complex exhibited an inhibition or dissociation constant of about 0.3 microM. Binding of [3H]AzidoAcCh was inhibited by vesamicol and AcCh. AzidoAcCh irreversibly blocked greater than 90% of the [3H]vesamicol binding sites after multiple rounds of photolysis and reequilibration with fresh ligand. Autofluorographs of synaptic vesicles photoaffinity-labeled with [3H]AzidoAcCh showed specific labeling of material exhibiting a continuous distribution from 50 to 250 kDa after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The result demonstrates that the AcChT has an unexpected structure highly suggestive of the synaptic vesicle proteoglycan.     

Reaction of quinacrine mustard with the acetylcholine receptor from Torpedo californica

Amines with local anesthetic activity are typically also noncompetitive inhibitors of the agonist-induced increase in cation permeability mediated by the nicotinic acetylcholine receptor. Quinacrine is such an agent, and we have synthesized tritiated quinacrine mustard, a derivative capable of reacting with nucleophiles. Quinacrine mustard was reacted with receptor-rich membrane from torpedo electric tissue, excess reagent was removed by partition into liposomes, and the modified receptor was extracted and reconstituted with exogenous phospholipid. After reaction of the native membrane with 10 microM quinacrine mustard for 5 min, binding of cobratoxin to the acetylcholine binding sites is inhibited 15%; in contrast, receptor-mediated 86Rb uptake in the reconstituted vesicles is inhibited 70%. When the reaction with quinacrine mustard is carried out in the presence of 10 microM carbamylcholine or 10 microM d-tubocurarine, there is no block of the acetylcholine binding sites; nevertheless, the inhibition of Rb uptake is greater than that resulting from reaction in the absence of acetylcholine binding site ligands. Conversely, when the reaction is carried out in the presence of either 100 microM quinacrine or 100 microM proadifen (also a potent noncompetitive inhibitor), either with or without carbamylcholine or d-tubocurarine, the inhibition of 86Rb uptake is about 70% smaller. Under the same conditions that we used in the functional studies, quinacrine mustard reacts with the four types of chains that constitute the receptor complex, alpha 2 beta gamma delta. The presence of the acetylcholine binding site ligands, however, results in increased reaction with the alpha and beta chains, while the presence of the noncompetitive inhibitors, with or without the acetylcholine binding site ligands, results in decreased reaction with the alpha and beta chains. We conclude that the alpha and beta chains contribute to one or more functionally significant binding sites for noncompetitively inhibiting amines.     

Structure of the noncompetitive antagonist-binding site of the Torpedo nicotinic acetylcholine receptor. [3H]meproadifen mustard reacts selectively with alpha-subunit Glu-262.

[3H]Meproadifen mustard, an affinity label for the noncompetitive antagonist site of the nicotinic acetylcholine receptor (AChR), specifically alkylates the AChR alpha-subunit when the acetylcholine-binding sites are occupied by agonist (Dreyer, E. B., Hasan, F., Cohen, S. G., and Cohen, J. B. (1986) J. Biol. Chem. 261, 13727-13734). In this report, we identify the site of alkylation within the alpha-subunit as Glu-262. AChR-rich membranes from Torpedo californica electric organ were reacted with [3H]meproadifen mustard in the presence of carbamylcholine and in the absence or presence of nonradioactive meproadifen to define specific alkylation of the noncompetitive antagonist site. Alkylated alpha-subunits were isolated and subjected to chemical or enzymatic cleavage. When digests with CNBr in 70% trifluoroacetic acid or 70% formic acid were fractionated by gel filtration high performance liquid chromatography (HPLC), specifically labeled material was recovered in the void volume fractions. Based upon NH2-terminal sequence analysis, for both digests, the void volume fractions contained a fragment beginning at Gln-208 before the M1 hydrophobic sequence, whereas the sample from the digest in trifluoroacetic acid also contained as a primary sequence a fragment beginning at Thr-244 and extending through the M2 hydrophobic sequence. Sequence analysis revealed no release of 3H for the sample from digestion in formic acid, whereas for the trifluoroacetic acid digest, there was specific release of 3H in cycle 19, which would correspond to Glu-262. This site of alkylation was confirmed by isolation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and reversed-phase HPLC of a specifically labeled fragment from an endoproteinase Lys-C digest of the alkylated alpha-subunit. NH2-terminal amino acid sequencing revealed release of 3H at cycle 20 from a fragment beginning at Met-243 and extending into the M3 hydrophobic sequence. Because [3H]meproadifen mustard contains, as its reactive group, a positively charged quaternary aziridinium ion, Glu-262 of the alpha-subunit is identified as a contributor to the cation-binding domain of the noncompetitive antagonist-binding site and thus of the ion channel.     

Interaction of noncompetitive inhibitors with the acetylcholine receptor. The site specificity and spectroscopic properties of ethidium binding

The spectroscopic properties and specificity of binding of a fluorescent quaternary amine, ethidium, with acetylcholine receptor-enriched membranes from Torpedo californica have been examined. Competition binding with [3H]phencyclidine in the presence of carbamylcholine showed that ethidium binds with high affinity to a noncompetitive inhibitor site (KD = 3.6 X 10(-7) M). However, in the presence of alpha-toxin, ethidium's affinity is substantially lower (KD approximately 1 X 10(-3) M). Ethidium was also found to enhance [3H]acetylcholine binding with a KD characteristic of ethidium binding to a high-affinity noncompetitive inhibitor site. These findings indicate that ethidium binds to an allosteric site which is regulated by agonist binding and can convert the agonist sites from low to high affinity. Fluorescence titrations of ethidium in the presence of carbamylcholine yielded a similar KD (2.5 X 10(-7) M) and showed an ethidium stoichiometry of one site/acetylcholine receptor monomer. Ethidium was completely displaced by noncompetitive inhibitors such as phencyclidine, histrionicotoxin, and dibucaine. The enhanced fluorescence lifetime of the bound species showed that the increased fluorescence intensity reflects a 13-fold increase in quantum yield for the complex compared to ethidium in buffer. Fractional dissociation of ethidium with phencyclidine produced a double-exponential fluorescence decay rate with lifetime components characteristic of ethidium free in solution and bound to the receptor. These data argue that the alterations in ethidium fluorescence elicited by other ligands is due to a change in the fraction of specifically bound ethidium rather than a change in quantum yield of a pre-existing ethidium-acetylcholine receptor complex. The extent of polarization indicates that bound ethidium is strongly immobilized. The magnitude of the quantum yield enhancement and the shifts of excitation and emission maxima of bound ethidium suggest that its binding site is within a hydrophobic domain with limited accessibility to the aqueous phase.