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PubMed1998

Functional characterization of mongoose nicotinic acetylcholine receptor alpha-subunit: resistance to alpha-bungarotoxin and high sensitivity to acetylcholine.

The mongoose is resistant to snake neurotoxins. The mongoose muscle nicotinic acetylcholine receptor (AChR) alpha-subunit contains a number of mutations in the ligand-binding domain and exhibits poor binding of alpha-bungarotoxin (alpha-BTX). We characterized the functional properties of a hybrid (alpha-mongoose/beta gamma delta-rat) AChR. Hybrid AChRs, expressed in Xenopus oocytes, respond to acetylcholine with depolarizing current, the mean maximal amplitude of which was greater than that mediated by the rat AChR. The IC50 of alpha-BTX to the hybrid AChR was 200-fold greater than that of the rat, suggesting much lower affinity for the toxin. Hybrid AChRs exhibited an apparent higher rate of desensitization and higher affinity for ACh (EC50 1.3 vs. 23.3 microM for the rat AChR). Hence, changes in the ligand-binding domain of AChR not only affect the binding properties of the receptor, but also result in marked changes in the characteristics of the current.

Acetylcholine↗
PubMed1997

Effects of lobeline and dimethylphenylpiperazinium iodide (DMPP) on N-methyl-D-aspartate (NMDA)-evoked acetylcholine release in vitro: evidence for a lack of involvement of classical neuronal nicotinic acetylcholine receptors.

Biochemical, behavioral and electrophysiological evidence suggests interactions between pathways containing neuronal nicotinic acetylcholine receptors (NAChRs) and excitatory amino acid receptors. Recently, protective effects of nicotine against N-methyl-D-aspartate (NMDA)-induced toxicity in primary cortical cultures were reported. To address possible interactions between NAChR and NMDA receptor containing pathways, several NAChR agonists were evaluated for their effects on NMDA-evoked [3H]acetylcholine ([3H]ACh) release from slices of rat striatum. Nicotine, cytisine and epibatidine had no effect on NMDA-evoked release or basal release of [3H]ACh over a wide range of concentrations. Lobeline and dimethylphenylpiperazinium iodide (DMPP), however, decreased basal [3H]ACh release and attenuated NMDA-evoked [3H]ACh release with EC50 values of 35 and 155 microM, respectively. The NAChR antagonists, dihydro-beta-erythroidine (DH beta E) and d-tubocurarine had no effect on NMDA-evoked [3H]ACh release, whereas mecamylamine attenuated the NMDA-evoked [3H]ACh evoked release with an EC50 value of 144 microM. Methyllycaconitine (MLA), a highly selective and potent antagonist of the alpha-bungarotoxin-sensitive alpha 7 NAChR subtype, also had no effect on NMDA-evoked [3H]ACh release at concentrations upto 10 microM. The inhibitory effects of DMPP and lobeline on NMDA-evoked [3H[ACh release were relatively insensitive to mecamylamine, d-tubocurarine, MLA and DH beta E. In addition, DMPP or lobeline-induced attenuation of basal [3H]ACh release was insensitive to blockade by sulpiride, a dopamine (D2) receptor antagonist. In contrast to their effects on NMDA-evoked striatal [3H]ACh release, both DMPP and lobeline increased basal release of striatal [3H]DA and hippocampal [3H]norepinephrine ([3H]NE) and did not attenuate NMDA-evoked release of these two transmitters. Instead, DMPP and lobeline appeared to have an additive effect on both NMDA-evoked hippocampal [3H]NE release and striatal [3H]DA release. These pharmacological results suggest that the inhibitory effects on lobeline and DMPP on striatal [3H]ACh release are independent of their interactions with classical NAChRs or the NMDA receptor complex itself.

Acetylcholine↗
PubMed1988

Liquid chromatography with electrochemical detection for the determination of choline and acetylcholine in plasma and red blood cells. Failure to detect acetylcholine in blood of humans and mice.

An assay is described for the measurement of choline in plasma and red blood cells using liquid chromatography, an enzyme reactor and electrochemical detection after a simple sample pretreatment. The intra-assay coefficient of variation for choline was 6.2 and 3.8% in plasma and in red blood cells, respectively. Using this method we have re-investigated the presence of acetylcholine in blood constituents. We were not able to demonstrate acetylcholine with a limit of detection of 10 pmol per ml of plasma or per ml of red blood cells.

Acetylcholine↗
PubMed1991

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.

Acetylcholine↗
PubMedDate not supplied

Acetylcholine release from growth cones detected with patches of acetylcholine receptor-rich membranes.

Synaptic potentials can be evoked at nerve-muscle junctions in vitro within minutes after an exploring growth cone contacts a receptive myotube. Functional transmission is also evident in vivo on the time scale of minutes after motor axons enter adjacent myotomes. The ability to release acetylcholine (ACh) may be induced in motor nerve terminals after they contact receptive target cells. Alternately, growth cones may be capable of releasing ACh before contact. To examine the development of ACh release we have used isolated patches of acetylcholine receptor(AChR)-rich membrane as sensitive detectors of ACh. We report here that the growth cones of embryonic chick ciliary ganglion neurones can release ACh, even when the cells are grown in the absence of target myotubes.

Acetylcholine↗
PubMed1990

Chemical transduction with fluorescent lipid membranes using selective interactions of acetylcholine receptor with agonist/antagonist and acetylcholine with substrate.

Alterations in the physical structure of vesicles and monolayers of phospholipids and soybean lecithin were monitored by measurement on the average fluorescence intensity changes from N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)dipalmitoyl-L-a-phosphatidyl ethanolamine (NBD-PE) located in the lipid matrices. This probe was intimately dispersed at a concentration of 1-2 mol-% in lipid membranes and had an emission sensitive to local environmental structure. Alterations in the structure of soybean lecithin vesicles were induced by the selective interaction of acetylcholine receptor with the agonist carbamylcholine and the antagonist alpha-bungarotoxin. Structural changes in vesicles with a 7:3 mole ratio of dipalmitoylphosphatidyl choline to dipalmitoylphosphatidic acid were observed for selective interactions between acetylcholinesterase and acetylcholine. Enhancement of fluorescence emission from the lipid membranes provided transduction of the selective binding events of the receptor and enzyme. A maximum sensitivity of about a 30% enhancement per micromole of carbamylcholine and a detection limit for the toxin of 10 nM were observed for the receptor. Fluorescence microscopy was used to establish that protein could be incorporated in monolayer lipid membranes and to provide information about potential mechanisms of fluorescence enhancement. These studies show that lipid membranes containing NBD-PE can be used as generic transducers of protein-ligand interactions.

Acetylcholine↗
PubMed1976

Dynamic properties of isolated acetylcholine receptor proteins: release of calcium ions caused by acetylcholine binding.

Interaction of Ca and acetylcholine (AcCh) ions with purified acetylcholine receptor (AcChR) from Torpedo californica and Electrophorus electricus has been investigated in view of these ions' role proposed in bioelectricity. Spectrophotometric Ca titration using murexide as an indicator and an ultrafiltration method with 45Ca show that AcChR proteins have a high binding capacity for Ca ions. Per macromolecule of 260,000 daltons, up to 60 Ca ions can be bound with at least three Ca dissociation constants. A linear inhibition of AcCh binding to AcChR by Ca was observed in the 0.1-1 mM Ca range, indicating competition of AcCh and Ca for AcChR. The addition of AcCh to a Ca-AcChR solution at 1.2 mM Ca causes release of four to six bound Ca ions from AcChR when a maximum of two AcCh ions are bound per 260,000 dalton macromolecule. The subsequent addition of alpha-bungarotoxin causes reuptake of up to six Ca ions by AcChR. These results suggest that the neural activator AcCh and the inhibitor alpha-bungarotoxin induce opposing shifts between different conformational states of isolated AcChR.

Acetylcholine↗
PubMed1999

Acetylcholine and epibatidine binding to muscle acetylcholine receptors distinguish between concerted and uncoupled models.

The muscle acetylcholine receptor (AChR) has served as a prototype for understanding allosteric mechanisms of neurotransmitter-gated ion channels. The phenomenon of cooperative agonist binding is described by the model of Monod et al. (Monod, J., Wyman, J., and Changeux, J. P. (1965) J. Mol. Biol. 12, 88-118; MWC model), which requires concerted switching of the two binding sites between low and high affinity states. The present study examines binding of acetylcholine (ACh) and epibatidine, agonists with opposite selectivity for the two binding sites of mouse muscle AChRs. We expressed either fetal or adult AChRs in 293 HEK cells and measured agonist binding by competition against the initial rate of 125I-alpha-bungarotoxin binding. We fit predictions of the MWC model to epibatidine and ACh binding data simultaneously, taking as constants previously determined parameters for agonist binding and channel gating steps, and varying the agonist-independent parameters. We find that the MWC model describes the apparent dissociation constants for both agonists but predicts Hill coefficients that are far too steep. An Uncoupled model, which relaxes the requirement of concerted state transitions, accurately describes binding of both ACh and epibatidine and provides parameters for agonist-independent steps consistent with known aspects of AChR function.

Acetylcholine↗
PubMed2005

The Drosophila acetylcholine receptor subunit D alpha5 is part of an alpha-bungarotoxin binding acetylcholine receptor.

The central nervous system of Drosophila melanogaster contains an alpha-bungarotoxin-binding protein with the properties expected of a nicotinic acetylcholine receptor. This protein was purified 5800-fold from membranes prepared from Drosophila heads. The protein was solubilized with 1% Triton X-100 and 0.5 M sodium chloride and then purified using an alpha-cobratoxin column followed by a lentil lectin affinity column. The purified protein had a specific activity of 3.9 micromol of 125I-alpha-bungarotoxin binding sites/g of protein. The subunit composition of the purified receptor was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. This subunit profile was identical with that revealed by in situ labeling of the membrane-bound protein using the photolyzable methyl-4-azidobenzoimidate derivative of 125I-alpha-bungarotoxin. The purified receptor reveals two different protein bands with molecular masses of 42 and 57 kDa. From sedimentation analysis of the purified protein complex in H2O and D2O and gel filtration, a mass of 270 kDa was calculated. The receptor has a s(20,w) of 9.4 and a Stoke's radius of 7.4 nm. The frictional coefficient was calculated to be 1.7 indicating a highly asymmetric protein complex compatible with a transmembrane protein forming an ion channel. The sequence of a peptide obtained after tryptic digestion of the 42-kDa protein allowed the specific identification of the Drosophila D alpha5 subunit by sequence comparison. A peptide-specific antibody raised against the D alpha5 subunit provides further evidence that this subunit is a component of an alpha-bungarotoxin binding nicotinic acetylcholine receptor from the central nervous system of Drosophila.

Acetylcholine↗
PubMed2006

Alpha-conotoxin OmIA is a potent ligand for the acetylcholine-binding protein as well as alpha3beta2 and alpha7 nicotinic acetylcholine receptors.

The molluskan acetylcholine-binding protein (AChBP) is a homolog of the extracellular binding domain of the pentameric ligand-gated ion channel family. AChBP most closely resembles the alpha-subunit of nicotinic acetylcholine receptors and in particular the homomeric alpha7 nicotinic receptor. We report the isolation and characterization of an alpha-conotoxin that has the highest known affinity for the Lymnaea AChBP and also potently blocks the alpha7 nAChR subtype when expressed in Xenopus oocytes. Remarkably, the peptide also has high affinity for the alpha3beta2 nAChR indicating that alpha-conotoxin OmIA in combination with the AChBP may serve as a model system for understanding the binding determinants of alpha3beta2 nAChRs. alpha-Conotoxin OmIA was purified from the venom of Conus omaria. It is a 17-amino-acid, two-disulfide bridge peptide. The ligand is the first alpha-conotoxin with higher affinity for the closely related receptor subtypes, alpha3beta2 versus alpha6beta2, and selectively blocks these two subtypes when compared with alpha2beta2, alpha4beta2, and alpha1beta1deltaepsilon nAChRs.

Amino Acid Sequence↗
PubMed2002

Acetylcholine and alcohol sensitivity of neuronal nicotinic acetylcholine receptors: mutations in transmembrane domains.

BACKGROUND: The effect of n-alcohols on glycine and gamma-aminobutyric acid type A receptors depends on two specific amino acids (AAs) located in the transmembrane domains (TM) 2 and 3. Our aim was to assess whether the corresponding AAs in the neuronal nicotinic acetylcholine receptor (nAChR) also formed a binding pocket for alcohols. METHODS: We made single AA substitutions in the homologous sites in rat neuronal nAChR alpha2 and alpha4 (alphaL261 and alphaL283) and expressed them in Xenopus laevis oocytes in combination with beta4 wild type. The effect of different n-alcohols was studied in alpha4(L261A)beta4 and alpha4(L283A)beta4 nAChRs. The effect of ethanol, propanol, and octanol on acetylcholine (ACh) responses was studied in alpha2(L261X)beta4 and alpha2(L283X)beta4 nAChRs. RESULTS: Most of the mutations in the alpha2 subunit, in either the 261 or the 283 position, induced changes in ACh sensitivity and increased alcohol action, but none was able to reduce ethanol potentiation. In alpha4(L283A)beta4, enhancement of potentiation by short-chain alcohols was observed, as well as a change from inhibition to potentiation for long-chain alcohols. The exposure of the AAs was assessed through the action of a charged thiol-specific reagent on alpha2(L261C)beta4 and alpha2(L283C)beta4, and these experiments suggest that the AA in TM2 is located in a water-accessible position, whereas the AA in TM3 is inaccessible. However, a noncharged thiol-specific reagent did not affect either ACh responses or ethanol effect on alpha2(L261C)beta4. CONCLUSIONS: The AAs located at positions 261 and 283 of the alpha2 and alpha4 nAChR subunits do not seem to form a binding pocket for alcohols. Additional studies are required to determine whether alcohols act on a site near these AAs or on sites unrelated to the TM2-TM3 site found in glycine and gamma-aminobutyric acid type A receptors.

Acetylcholine↗
PubMed1980

Reconstitution of a functional acetylcholine receptor. Polypeptide chains, ultrastructure, and binding sites for acetylcholine and local anesthetics.

The 'reconstitution cycle' is composed of the following sequence of operations. Highly purified receptor-rich membranes prepared from Torpedo marmorata electric organ are exposed to pH 11 to remove the 43,000-Mr protein and dispersed into solution by sodium cholate under conditions where more than 85% of the receptor protein is in its 9-S form. Elimination of the detergent by filtration on a Sephadex column (or dialysis) yields a 'reconstituted receptor' fraction, under conditions which conserve part of the endogenous lipids, or 'reconstituted vesicles' in the presence of an excess of exogenous lipids. The polypeptide composition of these fractions was analysed by sodium dodecylsulfate gel electrophoresis. Conditions are defined for quantitative measurements of the various polypeptide chains. The 40,000-Mr chain, which is labelled by the affinity reagent 4-(N-maleimido)phenyl [3H]trimethylammonium and therefore carries the acetylcholine receptor site, is the dominant polypeptide in the alkaline-treated membranes and the reconstituted acetylcholine receptor. Electron microscopy discloses that many of the alkaline-treated membranes no longer form closed vesicles and do not show the transverse asymmetry of the native membranes observed after tannic acid fixation. In the reconstituted receptor fractions, the receptor molecules reaggregate into discs and may be exposed on both faces of the discs. In the reconstituted vesicles, receptor rosettes are integrated to the lipid vesicles. With native membranes, the radioactive local anesthetic [3H]trimethisoquin binds to three classes of sites: non-specific, low-affinity and high-affinity. Carbamylcholine causes an increase in the number of high-affinity sites up to approximately 0.7 times the number of alpha-125I-bungarotoxin sites. This ratio, the three classes of binding sites, and their regulation by carbamylcholine are conserved through the reconstitution cycle.

Acetylcholine↗
PubMed1995

Activation of recombinant mouse acetylcholine receptors by acetylcholine, carbamylcholine and tetramethylammonium.

1. The kinetic properties of cloned mouse embryonic nicotinic acetylcholine receptors (AChRs) expressed in HEK 293 cells or Xenopus oocytes were examined using high concentrations of acetylcholine (ACh), carbamylcholine (CCh), or tetramethylammonium (TMA). The rate constants of agonist binding and channel gating were estimated by fitting kinetic models to idealized open and closed intervals over a range of agonist concentrations. 2. Once doubly liganded, TMA-activated receptors open at approximately 3000 s-1. The equilibrium binding constants for TMA are 525 and 12,800 microM. Doubly liganded CCh-activated receptors open at approximately 11,500 s-1; the equilibrium binding constants for this agonist are 14 and 570 microM. If we assume that doubly liganded, ACh-activated receptors open at 60,000 s-1, then the equilibrium binding constants for ACh are 20 and > 650 microM, similar to those for CCh. For all three agonists the higher affinity site both binds and releases agonists more slowly than does the lower affinity site. 3. ACh and CCh bind to the two sites equally rapidly, at approximately 2 x 10(7) and 4 x 10(7) M-1 s-1 at the first and second binding sites, respectively. Compared with ACh, the TMA association rate is approximately 100 times slower at the first binding site, and approximately 30 times slower at the second binding site. These results indicate that at both binding sites the association rate of TMA is not limited by diffusional or steric factors. 4. All three agonists dissociate from the receptor binding sites at similar rates. The dissociation rate for all agonists was approximately 40 times slower at the first binding site than at the second. These results suggest that the interaction of the quarternary amine moiety with the receptor determines the rate of release of the agonist, and that the nature of this interaction is quite different at the two binding sites. 5. Although the channel opening rates for the three agonists varied approximately 20-fold, the channel closing rates were not strongly agonist dependent, and varied less than 3-fold. We speculate that the ester moiety of the agonist promotes both rapid binding and fast opening of the ligand receptors, and that interactions of the quarternary amine moiety of the agonist with the receptor determine the channel closing rate constant.

Acetylcholine↗
PubMed1997

Positive cooperativity of acetylcholine and other agonists with allosteric ligands on muscarinic acetylcholine receptors.

It is well known that allosteric modulators of muscarinic acetylcholine receptors can both diminish and increase the affinity of receptors for their antagonists. We investigated whether the allosteric modulators can also increase the affinity of receptors for their agonists. Twelve agonists and five allosteric modulators were tested in experiments on membranes of CHO cells that had been stably transfected with genes for the M1-M4 receptor subtypes. Allosterically induced changes in the affinities for agonists were computed from changes in the ability of a fixed concentration of each agonist to compete with [3H]N-methylscopolamine for the binding to the receptors in the absence and the presence of varying concentrations of allosteric modulators. The effects of allosteric modulators varied greatly depending on the agonists and the subtypes of receptors. The affinity for acetylcholine was augmented by (-)-eburnamonine on the M2 and M4 receptors and by brucine on the M1 and M3 receptors. Brucine also enhanced the affinities for carbachol, bethanechol, furmethide, methylfurmethide, pilocarpine, 3-(3-pentylthio-1,2,5-thiadiazol-4-yl)-1,2,5,6-tetrahydro-1- methylpyridine (pentylthio-TZTP), oxotremorine-M, and McN-A-343 on the M1, M3, and M4 receptors, for pentylthio-TZTP on the M2 receptors, and for arecoline on the M3 receptors. (-)-Eburnamonine enhanced the affinities for carbachol, bethanechol, furmethide, methylfurmethide, pentylthio-TZTP, pilocarpine, oxotremorine and oxotremorine-M on the M2 receptors and for pilocarpine on the M4 receptors. Vincamine, strychnine, and alcuronium displayed fewer positive allosteric interactions with the agonists, but each allosteric modulator displayed positive cooperativity with at least one agonist on at least one muscarinic receptor subtype. The highest degrees of positive cooperativity were observed between (-)-eburnamonine and pilocarpine and (-)-eburnamonine and oxotremorine-M on the M2 receptors (25- and 7-fold increases in affinity, respectively) and between brucine and pentylthio-TZTP on the M2 and brucine and carbachol on the M1 receptors (8-fold increases in affinity). The discovery that it is possible to increase the affinity of muscarinic receptors for their agonists by allosteric modulators offers a new way to subtype-specific pharmacological enhancement of transmission at cholinergic (muscarinic) synapses.

Acetylcholine↗
PubMed1979

Acetylcholine receptor degradation in adult rat diaphragms in organ culture and the effect of anti-acetylcholine receptor antibodies.

Acetylcholine receptor located at the neuromuscular synapse of normal innervated adult muscle fibers is extremely stable metabolically. We have studied the kinetics of receptor degradation in both normal innervated and denervated rat diaphragms in organ culture. These studies show that degradation of receptor-bound 125I-alpha-bungarotoxin is a valid measure of junctional receptor degradation. Degradation of junctional receptor is similar or identical to degradation of extrajunctional receptor in many ways: 1) both require energy, 2) both are inhibited by specific lysosomal protease inhibitors, 3) both are inhibited by treatment with colchicine, and 4) both are stimulated by treatment with anti-acetylcholine receptor antibodies. The one important distinction between degradation of junctional and extrajunctional receptor is a 10-fold difference in rate constant for the process.

Acetylcholine↗
PubMed1984

[Determination of acetylcholine by inhibition of the specific binding of tritiated cis-methyldioxolane: application to the study of the effects of acetylcholine accumulation in a murine neuroblastoma culture].

The radioreceptor assay for acetylcholine (ACh) is based on the ability of the ACh to compete with the specific binding of [3H] cis-methyldioxolane to muscarinic receptors of rat cerebral cortex membranes. The technique described was used to measure ACh levels in NS 20 cells treated with an organophosphorus compound. The down regulation of muscarinic acetylcholine receptors of neuroblastoma cells observed is probably related to ACh accumulation.

Acetylcholine↗
PubMed1982

Inelastic electron tunneling spectroscopic study of interaction of acetylcholine and beta-methyl acetylcholine with alumina surface.

The conformation of acetylcholine (Ach) and its muscarinic analogue beta-methyl acetylcholine (beta-MeAch) on an alumina surface was analyzed by inelastic electron tunneling spectroscopy (IETS). This method detects vibrational modes of organic molecules that are active in both Raman (R) and IR spectroscopies. By using previously recorded and interpreted R and IR spectra of Ach and beta-MeAch in solid-state and aqueous solutions we studied the perturbations due to adsorption. The results were used to interpret the interaction of both molecules with the alumina surface, and a comparison to that with receptors or with acetylcholinesterase was attempted. In the case of nonhydrolytic interaction, the positive trimethylammonium groups of both molecules seemed to be attracted by the negative oxygen ions of the surface. There was evidence that the O--C--C--N skeleton of Ach changed its conformation in aqueous solution and adopted the solid-state conformation, which is very similar to that of beta-MeAch. This conformation once established, Ach appeared to interact with the alumina surface in the same way as did beta-MeAch: both tunneling spectra were very similar. There was also evidence that in the acetyl part of both molecules the C=O double bond was broken and that the oxygen atom coordinated with an Al+ cation. The acetyl skeleton did not show important conformational changes for either molecule. In the case of hydrolytic interaction of Ach or beta-MeAch, the products of the hydrolysis, acetate ion and choline--the latter also adsorbed in ionic form--were found on the alumina surface. In both cases the conformation of the lateral groups bonded to the choline and acetyl skeletons was also analyzed.

Acetylcholine↗
PubMed1994

Acetylcholine mustard labels the binding site aspartate in muscarinic acetylcholine receptors.

Acetylcholine mustard (AChM) is an analogue of acetylcholine (ACh) in which the onium headgroup is replaced by a chemically reactive aziridinium moiety. AChM aziridinium has agonist activity, but, having bound, reacts with and blocks the muscarinic receptor (mAChR) binding site. Purified mAChRs from rat forebrain have been specifically labeled with [3H]AChM. The linkage formed is cleaved by hydroxylamine, is found within cyanogen bromide (CNBr) peptides with molecular masses of approximately 2.4 and 3.9 kDa, and is close to a disulfide-bonded cysteine. Edman degradation reveals a site of label attachment 26 residues C-terminal to a CNBr cleavage site. As in the case of the alkylating antagonist analogue [3H]propylbenzilylcholine mustard, these findings indicate that a conserved aspartic acid residue in transmembrane helix 3 of the mAChRs, corresponding to Asp-105 (m1 sequence), is the site of label attachment.

Acetylcholine↗