The acetylcholine receptor of the adrenal medulla.

Abstract— The acetylcholine receptor of the bovine adrenal medulla was studied by specific binding of [¹²⁵1]α-bungarotoxin to membrane fractions and by perfusion of the isolated gland. The subcellular distribution of the acetylcholine receptor paralleled the distribution of the plasma membrane markers, acetylcholinesterase and calciumstimulated ATPase. The dissociation constant for the binding of α-bungarotoxin to a purified plasma membrane fraction was calculated from Scatchard plots to be 1.6 nM, with a concentration of 190 fmol of binding sites/mg of membrane protein. Correcting for recovery, this corresponds to 0.9 pmol acetylcholine receptor/g adrenal medulla. In decreasing order of effectiveness, d-tubocurarine, nicotine, acetylcholine, carbamylcholine, acetate plus choline, decamethonium, atropine and hexamethonium inhibited binding of α-bungarotoxin. Perfusion experiments showed the acetylcholine receptor to be entirely nicotinic. Stimulation by nicotine was inhibited by atropine and decamethonium, as well as by hexamethonium. Calculated dissociation constants for these antagonist-receptor interactions were in the range of 1 to 3 × 10^?5 m. α-Bungarotoxin failed to inhibit nicotine-stimulated catecholamine release in the perfused adrenal, most likely because of its limited diffusion into the gland.     

Monoclonal antibodies to Torpedo acetylcholine receptor. Characterisation of antigenic determinants within the cholinergic binding site

Thirteen monoclonal antibodies (mAb) to the acetylcholine receptor (AChR) from Torpedo marmorata showed high avidity for the receptor but none exhibited binding to muscle AChR solubilised from seven other animal species. Five mAb and Fab monomer fragments prepared from two of them, inhibited alpha-bungarotoxin (alpha BuTx) binding to receptor by a maximum of 50%. In the presence of excess mAb the 125I-alpha BuTx bound could be precipitated by anti-IgG indicating that the mAb bound to only one of the two alpha BuTx binding sites on each AChR monomer. This site appeared to have a lower affinity for d-tubocurarine and decamethonium than the non-mAb site. Binding of five anti-site mAb was mutually competitive and four of them (AS2-AS5) were inhibited by other cholinergic ligands and influenced by four non-toxin binding site antibodies. One (AS1) bound within the toxin binding site yet outside the main neurotransmitter binding region. It is concluded that these five mAb distinguish between the two alpha BuTx binding sites on the Torpedo AChR, and bind only to the site which displays lower affinity for d-tubocurarine and other competitive ligands.     

On the mechanism of the acceleration of methanesulfonylation of acetylcholinesterase with cationic accelerators.

1. In order to elucidate some features of the mechanism of the acceleration of methanesulfonylation of acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7) with cationic accelerators, the methanesulfonylation of this enzyme by high concentrations of methanesulfonylfluoride, in the absence and presence of accelerators decamethonium and tetraethylammonium, was studied. 2. The results showed that the accelerator accelerates the reaction by electrostatically improving the binding between acetylcholinesterase and methanesulfonylfluoride without effecting the rate of the decomposition of the enzyme-inhibitor complex into the methanesulfonylated enzyme and product.     

Binding of acetylcholine and related compounds to purified acetylcholine receptor from Torpedo Californica electroplax

The binding properties of the purified acetylcholine receptor from $\text{Torpedo}\text{californica}$ were investigated. One type of binding was observed for acetylcholine (K_D = 2.3 μM), dimethyl tubocurarine (K_D = 6.2 μM), and decamethonium (K_D = 55 μM). No cooperativity was observed in ligand binding. By virtue of its ligand binding properties, the purified receptor is nicotinic in nature.     

Apparent cooperative effects in acetylcholine receptor-mediated ion flux electroplax membrane preparations.

The kinetics of acetylcholine receptor-mediated flux of ²²sodium ions from microsacs has been measured in the presence of activators (carbamylcholine and decamethonium) and an inhibitor (d-tubocurarine) of neural transmission. The dependence of the first-order rate constant, k_obs, for ²²sodium ion efflux on either decamethonium or carbamylcholine concentration does not exhibit cooperativity. The apparent cooperativity observed by Kasai and Changeux in dose-response curves for ²²sodium flux from the same preparation is adequately accounted for by the contribution which efflux from non-excitable microsacs, the main component of the preparation, makes to the measurements. d-Tubocurarine was found to be a non-competitive inhibitor of decamethonium-activated ²²sodium efflux. The results of the kinetic measurements are in agreement with equilibrium measurements of the interaction of decamethonium with the same microsac preparation, i.e. adherence to a classic Langmuir binding isotherm and separate binding sites for activators and inhibitors of neural activity. The results indicate a direct relationship between ligand binding and receptor-mediated ion flux. How these two processes contribute to electrophysiological measurements is not apparent.     

CHARACTERIZATION OF AN α-BUNGAROTOXIN BINDING COMPONENT FROM DROSOPHILA MELANOGASTER

Abstract— We describe an α-bungarotoxin binding component from Dromphila melanoyaster that has the properties expected of an acetylcholine receptor. Toxin binding to a paniculate form of this component has been shown to be proportional to amount of extract, to be saturable and to be destroyed by heat. Localization studies using ¹²⁵I-α-bungarotoxin binding to frozen sections has shown toxin binding to be restricted to synaptic areas of the Drosophila CNS. We have also shown that this toxinbinding component can be treated with Triton X-100 without significantly altering its toxin-binding and pharmacological specificity. The ability of preincubation with cholinergic ligands to block labeled α-bungarotoxin binding to both particulate and detergent treated extracts has been studied. The nicotinic agents nicotine, d-tubocurarine, and acetylcholine are the most effective blocking agents. All of the muscarinic agents tested and the nicotinic agent decamethonium were less effective than acetylcholine in preventing α-bungarotoxin binding.     

Nicotinic Agonists Regulate α-Bungarotoxin Binding Sites of TE671 Human Medulloblastoma Cells

The TE671 human medulloblastoma cell line expresses a variety of characteristics of human neurons. Among these characteristics is the expression of membrane-bound high-affinity binding sites for alpha-bungarotoxin, which is a potent antagonist of functional nicotinic acetylcholine receptors on these cells. These toxin binding sites represent a class of nicotinic receptor isotypes present in mammalian brain. Treatment of TE671 cells during proliferative growth phase with nicotine or carbamylcholine, but not with muscarine or d-tubocurarine, induced up to a five-fold increase in the density of radiolabeled toxin binding sites in crude membrane fractions. This effect was blocked by co-incubation with the nicotinic antagonists d-tubocurarine and decamethonium, but not by mecamylamine or by muscarinic antagonists. Following a 10-13 h lag phase upon removal of agonist, recovery of the up-regulated sites to control values occurred within an additional 10-20 h. These studies indicate that the expression of functional nicotinic acetylcholine receptors on TE671 cells is subject to regulation by nicotinic agonists. Studies of the murine CNS have consistently indicated nicotine-induced up-regulation of nicotinic acetylcholine receptors, thereby supporting the identification of the toxin binding site on these cells as the functional nicotinic receptor. Although a mechanism for this effect is not apparent, nicotine-induced receptor blockade does not appear to be involved.     

Rotifer neuropharmacology--II. Synergistic effect of acetylcholine on local anesthetic activity in Brachionus calyciflorus (Rotifera, Aschelminthes)

A number of compounds showing general anesthetic action in the rotifer Brachionus calyciflorus were investigated in the presence of acetylcholine. Non-ionizing anesthetics, including tricaine, showed no interaction with acetylcholine. However, highly ionized compounds like the local anesthetics procaine and lidocaine, the muscarinic blocker and local anesthetic atropine, and the beta-adrenergic blocker propranolol showed a synergistic effect with acetylcholine. ACh increased the general anesthetic effect of these compounds in a statistically highly significant dose-dependent fashion. To account for the mechanism of this unusual and novel effect it is proposed that these compounds interact with the anesthetic binding site of the rotifer cholinoceptor ionophore in the open state. It is also proposed that non-ionizing compounds have a general membrane effect only. In addition to anesthesia, atropine and propranolol cause foot paralysis in B. calyciflorus. This other novel effect is also enhanced by acetylcholine as well as decamethonium, a neuromuscular blocker.     

On Some Structural Analogies between Acetylcholinesterase and the Macromolecular Receptor of Acetylcholine

Several properties of the enzyme acetylcholinesterase (AChE) isolated in vitro are compared with those of the membrane receptor(s) of acetylcholine expressed by the in vivo electrical response of the electroplax membrane. AChE strongly binds in vitro effectors of the electroplax: agonists e.g., decamethonium or antagonists, e.g., d-tubocurarine and flaxedil. It also reacts covalently with an affinity labeling reagent of the acetylcholine receptor site(s) in vivo (TDF). Two classes of sites on AChE molecule account for the binding of these quaternary nitrogen containing compounds: (1) the anionic site of the active center and (2) noncatalytic "peripheral anionic centers" located outside the active center. A disulfide bond breaking agent, dithiothreitol (DTT) alters in a parallel manner the reaction of AChE and the excitable membrane of the electroplax to TDF. The irreversibility of TDF action is lost in both cases, after exposure to DTT. Both AChE and the acetylcholine receptor thus contain disulfide bonds—they are closely related but not necessarily identical proteins.     

Uses of fluorescent cholinergic analogues to study binding sites for cholinergic ligands in Torpedo californica acetylcholine receptor.

A series of synthetic 1,n-bis(3-aminopyridinio)-alkane fluorescent probes have been used to determine the ligand binding properties of the acetylcholine receptor purified from Torpedo californica electroplax. At equilibrium, the probes bound to a single class of sites. The binding affinity of the fluorescent decamethonium analogues increased progressively as the number of methylene groups (n) increased from 4 to 12 and decreased in the range of 16--18 such groups. The receptor bound 1,12-bis(3-aminopyridinio)dodecane and 1,14-bis(3-aminopyridinio)tetradecane with the highest affinity while related monofunctional probes such as 1-(3-amino-pyridinio)propane were bound with a substantially lower affinity. The data indicate that the receptor interacts strongly with both ends of a bifunctional probe such as 1,14-bis(3-aminopyridinio)tetradecane. Also, competition between bifunctional fluorescent probe binding and the binding of conventional cholinergic ligands, was investigated and led to the conclusion that the probes, which are antagonists, form ternary complexes in the presence of acetylcholine.     

Purification and molecular properties of the acetylcholine receptor from Torpedo electroplax

The acetylcholine receptor of Torpedo electroplax is purified by affinity adsorption using cobra toxin (Naja naja siamensis) covalently attached to Sepharose 4B. Desorption by 10 mm benzoquinonium produces a protein that binds α-[¹²⁵I]bungarotoxin but not [³H]acetylcholine or other reversible cholinergic ligands. On the other hand, desorption by 1 m carbamylcholine produces an acetylcholine receptor protein that binds [³H]acetylcholine, [³H]decamethonium, [³H]nicotine, [¹⁴C]dimethyl-d-tubocurarine, and α-[¹²⁵I]bungarotoxin. The batch method of affinity adsorption employed gives recoveries of acetylcholine receptor (as measured by acetylcholine binding) averaging 69.2 ± 14.6%. The purity of the isolated acetylcholine receptor protein is estimated to be at best 87% as judged by disc gel electrophoresis and electrofocusing.The purified acetylcholine receptor binds 7.8 nmoles acetylcholine/mg protein based on estimation of protein concentration by a spectrophotometric method. Of these, 2.7 nmoles exhibit high affinity (K_D = 0.02 μM) and 5.1 nmoles a lower affinity (K_D = 1.97 μM. If the protein concentration used is that obtained by amino acid analysis, the total specific activity would be 10.4 nmoles acetylcholine bound per milligram protein. The subunit carrying one acetylcholine binding site is estimated to range between 83,000 and 112,000 daltons. In contrast to the membrane-bound or Lubrol-solubilized acetylcholine receptor, the purified acetylcholine receptor shows no autoinhibition with acetylcholine concentrations up to 10 μm. Binding of acetylcholine was totally inhibited by α-bungarotoxin or cobra toxin and was partially blocked by four nicotinic drugs, but not by two muscarinic ones. The amino acids of the acetylcholine receptor are analyzed and compared to those of acetylcholinesterase.     

Evidence that the acetylcholine binding site is not formed by the sequence alpha 127-143 of the acetylcholine receptor

The sequence alpha 127-143 of the alpha subunit of the acetylcholine receptor has been proposed to contain several important features: (1) the acetylcholine binding site, (2) the only N-glycosylation site of the alpha subunit, at asparagine-alpha 141, and (3) two cysteine residues, at alpha 128 and alpha 142, that may participate in a disulfide bond known to be near the binding site. We tested these hypotheses by using antisera to receptor and its subunits and monoclonal antibodies to the synthetic peptide alpha 127-143 cyclized by a disulfide bond between alpha 128 and alpha 142. Antisera to receptor and its alpha subunit were able to immunoprecipitate the iodinated peptide, and this reaction was inhibited by soluble receptor, but not by membrane-bound receptor. alpha-Bungarotoxin did not inhibit antiserum binding to solubilized receptor. Similarly, cholinergic ligands had little or no effect on binding to immobilized receptors of anti-peptide monoclonal antibodies. In addition, these monoclonal antibodies, when bound to the receptor, did not affect toxin binding kinetics. By contrast, preincubation with concanavalin A did inhibit monoclonal antibody binding. Reduction of the receptor significantly decreased the binding of three of the monoclonal antibodies, but subsequent alkylation with N-ethylmaleimide or the affinity labeling reagent bromoacetylcholine had no additional effect on binding. A dithiothreitol concentration about 100-fold higher that the one needed to reduce the disulfide near the acetylcholine binding site was necessary to inhibit monoclonal antibody binding. We conclude that the sequence alpha 127-143 is not fully exposed on the surface when the receptor is in the membrane.(ABSTRACT TRUNCATED AT 250 WORDS)     

Tryptophan and cystein residues of the acetylcholine receptors of Torpedo species. Relationship to binding of cholinergic ligands.

Several methods were used to analyze for tryptophan in the acetylcholine (ACh) receptors purified from the electric organs of the electric rays, Torpedo californica and Torpedo marmorata. The best value of tryptophan was 2.4 mol %. When excited at 290 nm, both receptors fluoresced with a maximum at 336, but there was no change in the fluorescence emission spectra upon binding of carbamylcholine, d-tubocurarine, ACh, or decamethonium. The free SH content of the Torpedo receptors varied in different preparations, and was highest in that purified from fresh T. californica using deaerated solutions and dialysis under nitrogen, and lowest in that prepared from the aged lyophilized membranes of T. marmorata. The maximum free SH content was 20 nmol/mg of protein or 0.22 mol %, equal to at most 18% of the total cysteic acid residues. Reaction of either 33% or of all the SH residues with p-chloromercuribenzoate reduced maximum ACh binding to the pure receptor prepared from fresh T. californica by only 23%.     

A monoclonal antibody to a synthetic fragment of rabies virus glycoprotein binds ligands of the nicotinic cholinergic receptor

Rabies virus glycoprotein and snake venom curaremimetic neurotoxins share a region of high homology (30-45 for neurotoxins and 190-203 for the glycoprotein) in the regions that are believed to be responsible for binding the nicotinic acetylcholine receptor. Monoclonal antibodies raised to the 190-203 synthetic fragment of rabies virus glycoprotein were immobilized on a high performance affinity chromatography column and were able to bind neurotoxins. Toxins were displaced from the affinity column by elution at acidic pH and by affinity competition with acetylcholine at neutral pH. Furthermore, the affinity column proved to be useful for the purification of cholinergic ligands. Overall, these results indicate that the paratope of our monoclonal antibodies could behave as an 'internal image' of the nicotinic cholinergic receptor acetylcholine binding site.     

The interaction of cyproheptadine with agents affecting neuromuscular transmission.

Using the rat phrenic nerve diaphragm, cyproheptadine at concentrations of 1 to 8 mug/ml did not affect or slightly augmented indirect muscle twitches, but potentiated blockade by tubocurarine, decamethonium and succinylcholine, and antagonized the augmentation of twitches by neostigmine. Ketamine, choline and tetraethylammonium at concentrations causing no blockade produced, when given after cyproheptadine (6 mug/ml), a high degree of blockade. At concentrations of 9 to 20 mug/ml, cyproheptadine induced neuromuscular blockade which was slow in onset, more apparent at higher rate of stimulation and was not reversed by neostigmine, choline or tetraethylammonium. In the cat tibialis anterior muscle, it potentiated blockade by tubocurarine, decamethonium and succinylcholine, and blocked acetylcholine twitches. In the chick biventer cervicis muscle, the durg was more effective in blocking indirect twitches than responses to carbachol.     

Acetylcholine receptor-mediated membrane current in oocytes injected with Electrophorus electricus mRNA: analyses of nicotine, succinylcholine, and decamethonium responses on the basis of the minimal model

Nicotinic acetylcholine receptor was synthesized in Xenopus oocytes after injection of the mRNA purified from Electrophorus electricus electroplax. Nicotine, succinylcholine, and decamethonium (agonist)-elicited membrane currents in the injected oocytes were measured electrophysiologically by the voltage-clamping method. The following four different measurements were made to establish the relationship between the agonist concentration and the membrane current: 1) the agonist-induced membrane current before desensitization, 2) the agonist-induced membrane current after desensitization equilibrium, 3) the fraction of the active form of the receptors after desensitization equilibrium, 4) the rate of recovery of desensitized receptors upon removal of the agonist. These results were analyzed on the basis of the minimal model proposed from receptor-mediated ion translocation measurements. The equilibrium and rate constants of the model were evaluated for nicotine, succinylcholine, and decamethonium, and could explain the observed electrical responses in the injected oocyte, i.e. the characteristics of the receptor response caused by these agonists.     

Antibodies against an alpha-bungarotoxin-binding peptide of the alpha-subunit of the acetylcholine receptor

Polyclonal and monoclonal antibodies were raised against a peptide comprising residues 173-204 of the alpha-subunit of the acetylcholine receptor. The polyclonal and pooled monoclonal antibodies inhibited up to 50% of 125I-alpha-bungarotoxin binding to peptide 173-204. Some of the antibodies recognized native receptor but did not significantly affect alpha-bungarotoxin binding. Epitope mapping revealed that the antibodies are directed against residues 183-194 indicating this region is a major determinant of toxin binding. This region is most likely conformationally constrained in the native receptor.     

Profile of the alpha-bungarotoxin-binding regions on the extracellular part of the alpha-chain of Torpedo californica acetylcholine receptor.

The continuous alpha-neurotoxin-binding regions on the extracellular part (residues 1-210) of the alpha-chain of Torpedo californica acetylcholine receptor were localized by reaction of 125I-labelled alpha-bungarotoxin with synthetic overlapping peptides spanning this entire part of the chain. The specificity of the binding was confirmed by inhibition with unlabelled toxin and, for appropriate peptides, with unlabelled anti-(acetylcholine receptor) antibodies. Five toxin-binding regions were localized within residues 1-10, 32-41, 100-115, 122-150 and 182-198. The third, fourth and fifth (and to a lesser extent the first and second) toxin-binding regions overlapped with regions recognized by anti-(acetylcholine receptor) antibodies. The five toxin-binding regions may be distinct sites or, alternatively, different 'faces' in one (or more) sites.     

Monoclonal antibodies directed against a synthetic peptide corresponding to the alpha-bungarotoxin binding region of the acetylcholine receptor

Murine monoclonal antibodies have been produced against a 32 amino acid synthetic peptide corresponding to residues 173-204 on the alpha-subunit of the nicotinic acetylcholine receptor from Torpedo californica. All of the monoclonal antibodies were of the IgM subtype and most cross-reacted with the purified native receptor. None of the antibodies were effective in blocking alpha-bungarotoxin binding to the receptor nor, conversely, did alpha-bungarotoxin interfere with antibody binding. However, two monoclonal antibodies, previously shown to bind near the ligand binding site on the native receptor, did compete partially (50%) with the binding of one of the IgM monoclonal antibodies.