Purified Acetylcholine Receptor Research Articles

Selective photoaffinity labeling of acetylcholine receptor using a cholinergic analogue.

A bisazido derivative was synthesized from bis(3-aminopyridinium)-1,10-decane diiodide and it was shown that it was bound (KD congruent to 2.2 muM) specifically to purified acetylcholine receptor and fulfilled the requirements for a photoaffinity label. Like the parent compound the derivative could transform membrane-bound receptor from a low ligand affinity conformation(s) to a high ligand affinity form (s), a transition which is thought to resemble desensitization processes observed in vivo. Photolysis of 3H-labeled bisazido reagent was carried out in the presence of the receptor. After dodecyl sulfate-polyacrylamide gel electrophoresis of labeled purified receptor two of the four subunits (mol wt 40 000 and 60 000) contained 90% of the bound radioactivity while for membrane-bound receptor the subunits of mol wt 40 000 and 50 000 were labeled. The results favor the assumption that the specific ligand binding sites are located on mol wt 40 000 subunits and labeling of the other subunits reflects (a) their proximity to the ligand-binding site and (b) alterations in subunit topography between membrane-bound and solubilized states.

Studies of nicotinic acetylcholine receptor protein from rat brain: II. Partial purification

The pharmacological specificity of the binding of 125I-labeled α-bungarotoxin to a 1% Emulphogene BC-720 extract of a rat brain particulate fraction has been investigated. The extract contains a component which possesses the binding characteristics of a nicotinic acetylcholine receptor protein. The crude soluble acetylcholine receptor protein was purified by affinity chromatography utilizing the α-neurotoxin of Naja naja siamensis as ligand and 1.0 M carbamylcholine chloride as eluant. A single, batch-wise, affinity chromatography procedure yields an average purification of 510-fold. When this purified material is treated a second time by affinity chromatography, purification as high as 12 600-fold has been obtained. Binding of 125I-labeled α-bungarotoxin to this purified acetylcholine receptor protein is saturable with a K d of 1·10 −8 M. Nicotine and acetylcholine iodide at concentrations of 10 −5 M inhibit 125I-labeled toxin-acetylcholine receptor protein complex formation by 41 and 61% respectively. At 10 −4 M, carbamylcholine chloride and (+)-tubocurarine chloride give respectively 52 and 82% inhibition. Eserine sulfate and atropine sulfate have no effect on complex formation at a concentration of 10 −4 M. These data support the isolation of partially purified nicotinic acetylcholine receptor protein.

Immunological comparison of acetylcholine receptors and their subunits from species of electric ray

Rabbit antibodies were produced against purified acetylcholine receptor and each of the four acetylcholine receptor subunits from Torpedo californica. Using the technique of double diffusion in agar, cross reactivities were observed between these antibodies and purified acetylcholine receptor and receptor subunits from Torpedo marmorata, Torpedo nobiliana, and Narcine brasiliensis, as well as from Torpedo californica. The specificity of each of the four anti-subunit antibodies and the conservation of subunit antigenic determinants in the four electric rays studied are demonstrated.

Phosphorylation of acetylcholine receptor by endogenous membrane protein kinase in receptor-enriched membranes of Torpedo californica.

THE molecular basis of synaptic transmission is a fundamental problem in neurobiology. Although neurotransmitters produce striking changes in post-synaptic ion permeability, little is known about the molecular events following binding of neurotransmitter to its receptor. It has been postulated that the effects of neurotransmitters on post-synaptic membranes are mediated by changes in protein phosphorylation1,2. There has been no direct evidence for neurotransmitter-dependent changes in protein phosphorylation in membranes prepared from innervated tissues, however. We investigated this problem using an acetylcholine receptor-enriched membrane fraction prepared from the electric organ of Torpedo californica and found that several membrane polypeptides are phosphorylated when this fraction is incubated with γ-32P-ATP and 100 mM K+ (ref. 3). In particular, we demonstrated that a polypeptide of 65,000 molecular weight (MW) is phosphorylated and that phosphorylation of this and other polypeptides is inhibited by cholinergic ligands. A 65,000 MW polypeptide in the same membrane preparation also reacted with antibody prepared against the purified acetylcholine receptor. This suggested that a component of the acetylcholine receptor itself could be phosphorylated by an endogenous membrane protein kinase. Here we present proof that the 65,000 MW component of the acetylcholine receptor is a substrate for an endogenous membrane protein kinase.

Experimental autoimmune myasthenia gravis in rats. Modification by thymectomy and prednisolone.

In rats immunized with purified acetylcholine receptor protein (AChRP) from Torpedo electroplax, a defect of neuromuscular transmission physiologically identical to that seen in myasthenia gravis developed. The most sensitive index of the neuromuscular blockage was miniature end-plate potential (MEPP) amplitude. As early as 24 hours after inoculation with AChRP, the thymus showed reactive changes that are probably nonspecific. Removal of the thymus before or within three days after immunization delayed, but did not prevent, development of reduced MEPP amplitude. Prednisolone given within 35 days after immunization produced reversal of MEPP reduction within 24 hours, but had no such immediate effect when given 15 days later. It is probable that prednisolone acted by reducing the immunologic responsiveness of the animals during the developing phase of the defect of neuromuscular transmission.

Cholinergic sites in skeletal muscle: interaction with concanavalin A.

Abstract– The interaction of normal and denervated skeletal muscle cholinergic sites with the lectin concanavalin A and concanavalin A‐Sepharose are detailed. Concanavalin A blocks the binding of 125I‐α‐bungarotoxin to both the high and low affinity sets of cholinergic sites described previously. The characteristics of the block of 125I‐α‐bungarotoxin binding to the high affinity set (acetylcholine receptor) is not competitive. The data suggest that the concanavalin binds multivalently to the macro‐molecular complex containing the ACh receptor site and sterically prevents the α‐bungarotoxin binding. The interaction of both sets of cholinergic sites with concanavalin A‐Sepharose was also studied. The macromolecule(s) containing both the high and low affinity sets of sites bind to the concanavalin A‐Sepharose. The data indicate a multivalent association with the affinity resin. Following the affinity procedure, a partial purification in both sets of sites is effected. The equilibrium binding of 125I‐diiodo‐α‐bungarotoxin to the preparations from the affinity procedure (both normal and denervated muscle) was examined. The KD of the α‐bungarotoxin binding to the high affinity sets of sites (acetylcholine receptor) in both normal and denervated preparations changes from ∼10−9mol/l to ∼ 10−10 mol/l following purification. No change in the KD of the α‐bungarotoxin binding to the low affinity set of sites was observed following purification. The 125l‐α‐bungarotoxin binding to the partially purified acetylcholine receptor was blocked by unlabelled α‐bungarotoxin, concanavalin A, d‐tubocurarine and carbamylcholine.

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.

Cellular immunity in myasthenia gravis. Response to purified acetylcholine receptor and autologous thymocytes.

To determine the potential importance of an immune response directed against the acetylcholine receptor in myasthenia gravis, we studied cell-mediated immunity to receptor as measured by lymphocyte stimulation in 21 myasthenic patients and 21 controls, including five with amyotrophic lateral sclerosis. The mean (+/- S.E.M.) stimulation index was 5.3 +/- 1.3 for patients and 1.2 +/- 0.3 for controls (P less than 0.005). Fourteen patients had indexes greater than 2.0 (nine of 11 males, five of 10 females, 10 of 11 elderly patients, and five of six with thymoma). Stimulation index correlated with disease activity (rs = 0.71, P less than 0.01). Peripheral blood lymphocytes from one of three young female myasthenic patients responded to autologous thymocytes but not to receptor; peripheral blood lymphocytes from the other two responded to receptor but not to autologous thymocytes. Our findings are further evidence that autoimmunity to the acetylcholine receptor plays a central part in myasthenia gravis.

Reconstitution of a purified acetylcholine receptor.

Dialysis of the purified acetylcholine receptor from Torpedo californica electroplax with lipids from the same organ results in a vesicular membrane system in which the receptor is embedded in the bilayer and oriented so that most of the neurotoxin-binding sites appear to be on the outer surface. The reconstituted vesicles are chemically excitable by acetylcholine and carbamylcholine, as measured by 22Na+ efflux. The excitability is specifically blocked by the antagonist alpha-bungarotoxin. These results demonstrate that the purified reconstituted receptor system not only can specifically bind neurotransmitter but can also trigger ion translocation. It therefore has the properties necessary to effect postsynaptic depolarization in vivo.

Interaction between calcium and ligand-binding sites of the purified acetylcholine receptor studied by use of a fluorescent lanthanide

The acetylcholine receptor isolated from Torpedo ocellata binds about 10 moles of a fluorescent lanthanide, terbium, per mole α-bungarotoxin-binding site, a process which is accompanied by a fluorescence enhancement (λexcitation 295 nm, λemission 546 nm) which allows detection of receptor-Tb 3+ complexes at μM concentrations. In presence of calcium two types of terbium-binding site are revealed, both with terbium dissociation constants of 18 ± 0.5 μM. About 60% of the sites bind calcium with an apparent dissociation constant of 1.1 ± 0.1 mM. Sites which interact with calcium also interact with activators of neural transmission, carbamylcholine and decamethonium, but not with the inhibitors, d-tubocurarine and α-bungarotoxin. Whether the displacement of calcium by chemical mediators is directly responsible for activator-induced changes in ion permeability of neural membranes is an important question raised by our experiments. The results show that fluorescent lanthanides can be an important tool in such studies.

Carbamylcholine and acetylcholine-sensitive, cation-selective ionophore as part of the purified acetylcholine receptor.

Black lipid membranes were formed with oxidized cholesterol in the presence of either the acetylcholine receptor, purified from the electric organ of the electric ray Torpedo californica or its tryptic digest. In both cases, conductance of cations increased and was dependent on the concentration of the receptor protein. Conductance of Ca++ was dependent on the concentration, but addition of carbamylcholine gave no reproducible of consistent effects. Only in the case of the tryptic digest of the acetylcholine receptor did carbamylcholine and acetylcholine consistently induce monovalent cation selective conductance (PNa,K: PCl=4.4). The induced monovalent cationic conductance due to carbamylcholine (10 muM) varied from 10- to over 100-fold. Curare (10muM) prevented the action of carbamylcholine. Na-dodecyl sulfate gel electrophoresis of the acetylcholine receptor, before and after tryptic digestion, indicated that this mild enzyme treatment hydrolyzed the receptor molecule subunits. Nevertheless, the receptor molecule retained its full binding of [acetyl(-3)H]acetylcholine; and analytical gel electrophoresis indicated that it remained intact possibly through hydrogen, hydrophobic and disulfice bonding.

Synthesis of acetylcholine receptor during differentiation of cultured embryonic muscle cells.

Acetylcholine receptor, a component of the specialized muscle membrane, appears during the differentiation of embryonic myogenic cells in tissue culture. Demonstration of incorporation of the radioactive precursor L-[35S]methionine into purified receptor polypeptides is presented as evidence for its synthesis de novo. The identity of the purified radioactive species is established by cosedimentation of the [35S]receptor with [3H]alpha-toxin binding activity on sucrose gradients and by crossreaction with antiserum to purified acetylcholine receptor of Electrophorus electricus.

Passive transfer of experimental autoimmune myasthenia by lymph node cells in inbred guinea pigs.

Passive transfer of experimental autoimmune myasthenia (EAM) was performed with lymph node cells from donor guinea pigs immunized with purified acetylcholine receptor (AChR) from Torpedo californica. Recipient animals revealed the same clinical signs and electromyographic patterns as observed in actively challenged animals. These phenomena are parallel to the clinical manifestations of the human disease myasthenia gravis, in which cellular response to AChR was recently demonstrated.

Experimental autoimmune myasthenia induced in monkeys by purified acetylcholine receptor.

MYASTHENIA GRAVIS (MG) is a neuromuscular junction disease of man characterised by muscular weakness and fatigability.

Some molecular properties of an isolated acetylcholine receptor ion-translocation protein

An improved procedure for isolation and purification of acetylcholine receptor from Torpedo californica electroplax membranes is described. The purified material contains the neurotransmitter recognition site and a second binding subsite which complexes inorganic cations and bis-quaternary cholinergic analogs. In addition to the transmitter recognition site the isolated macromolecule contains the molecular features necessary for ion-translocation during postsynaptic depolarization, since a chemically excitable membrane can be formed from purified acetylcholine receptor and Torpedo phospholipids.

Purified acetylcholine receptor: its reconstitution to a chemically excitable membrane.

Association of purified acetylcholine receptor from Torpedo californica electroplax with lipids from the same organism results in a vesicular membrane system in which the receptor protein is oriented so that all neurotoxin binding sites appear to be on the outer surface. The reconstituted system is chemically excitable by acetylcholine and carbamylcholine, as measured by (22)Na(+) efflux. This excitability is specifically blocked by the antagonist alpha-bungarotoxin. These results demonstrate that the purified receptor macromolecule contains not only the specific neurotransmitter binding site but also the molecular elements necessary for ion translocation in order to effect postsynaptic depolarization.

Affinity-labeling of purified acetylcholine receptor from [formula omitted

The receptor for acetylcholine purified from electric tissue of Torpedo californica has been assayed both by affinity-alkylation and by neurotoxin binding. The specific activity by the latter method is about twice that by the former. Four major components of apparent molecular weights of 39,000, 48,000, 58,000 and 64,000 are separated by dodecyl sulfate-acrylamide gel electrophoresis. Reduction and affinity-alkylation of the receptor with a tritiated quaternary ammonium maleimide derivative results in the exclusive labeling of the 39,000 dalton subunit. This subunit, it is concluded, contains all or part of the acetylcholine binding site.

Affinity of cholinergic ligands for the partially purified acetylcholine receptor from mammalian skeletal muscle

FEBS LettersVolume 46, Issue 1-2 p. 145-148 Full-length articleFree Access Affinity of cholinergic ligands for the partially purified acetylcholine receptor from mammalian skeletal muscle J.O. Dolly, J.O. Dolly Department of Biochemistry, State University of New York, Buffalo, New York 14214, USASearch for more papers by this authorE.A. Barnard, E.A. Barnard Department of Biochemistry, State University of New York, Buffalo, New York 14214, USASearch for more papers by this author J.O. Dolly, J.O. Dolly Department of Biochemistry, State University of New York, Buffalo, New York 14214, USASearch for more papers by this authorE.A. Barnard, E.A. Barnard Department of Biochemistry, State University of New York, Buffalo, New York 14214, USASearch for more papers by this author First published: September 15, 1974 https://doi.org/10.1016/0014-5793(74)80355-8Citations: 14AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL References 1 C.W. Porter, T.H. Chiu, J. Wieckowski, E.A. Barnard, Nature, New Biology, 241, (1973), 3– 7. 2 C.W. Porter, E.A. Barnard, T.H. Chiu, J. Membrane Biol., 14, (1973), 383– 402. 3 E.X. Albuquerque, E.A. Barnard, C.W. Porter, J.E. Warnick, Proc. Natl. Acad. Sci. U.S., (1974), in press 4 T.H. Chiu, J.O. Dolly, E.A. Barnard, Biochem. Biophys. Res. Commun., 51, (1973), 205– 213. 5 E.A. Barnard, T.H. Chiu, J.O. Dolly, C.W. Porter, Trans. Amer. Soc. Neurochem., 4, (1973), 49– 6 J.O. Dolly, E.A. Barnard, Federation Proc., 33, (1974), 580– 7 Barnard, E. A., Dolly, J. O., Porter, C. W. and Albuquerque, E. X., Federation Proc., in press. 8 Dolly, J. O. and Barnard, E. A., Biochemistry, submitted for publication. 9 G. Kemp, J.O. Dolly, E.A. Barnard, C.E. Wenner, Biochem. Biophys. Res. Commun., 55, (1973), 1044– 1050. 10 Chiu, T. H. and Barnard, E. A., manuscript in preparation. 11 B. Fulpius, S. Cha, R. Klett, E. Reich, FEBS Letters, 24, (1972), 323– 326. 12 J. Schmidt, M.A. Raftery, Anal. Biochem., 52, (1973), 349– 354. 13 A.S. Mildvan, R.A. Leigh, Biochim. Biophys. Acta., 89, (1964), 393– 397. 14 M. Weber, J.P. Changeux, Mol. Pharmacol., 10, (1973), 15– 34. 15 B.W. Fulpius, R.P. Klett, E. Reich, Robertis E. de J. Schacht Neurochemistry of Cholinergic Receptors (1974), Raven Press New York 19– 29. 16 J.C. Meunier, J.P. Changeux, FEBS Letters, 32, (1973), 143– 148. 17 G.I. Franklin, L.T. Potter, FEBS Letters, 28, (1972), 101– 106. 18 T. Moody, J. Schmidt, M.A. Raftery, Biochem. Biophys. Res. Commun., 53, (1973), 761– 772. 19 M.E. Eldefrawi, A.T. Eldefrawi, S. Seifert, R.D. O'Brien, Arch. Biochem. Biophys., 150, (1972), 210– 218. 20 J. Patrick, S.F. Heinemann, J. Lindstrom, D. Schubert, J.H. Steinbach, Proc. Natl. Acad. Sci. U.S., 69, (1972), 2762– 2766. 21 T.H. Chiu, A.J. Lapa, E.A. Barnard, E.X. Albuquerque, Exp. Neurol., 43, (1974), 399– 413. 22 P. Kruckenberg, H. Bauer, Pfluegers Arch., 326, (1971), 184– 192. Citing Literature Volume46, Issue1-2September 15, 1974Pages 145-148 ReferencesRelatedInformation

Studies on Purified Eel Acetylcholine Receptor and Anti-Acetylcholine Receptor Antibody

Rabbit antiserum against purified Electrophorus electricus acetylcholine receptor is studied using an immunoprecipitin assay to measure either antibody titer or concentration of toxin-binding sites in solubilized receptor preparations. This antiserum, unlike control serum, blocks the electrophysiological response of the electroplax to carbamylcholine. Toxin (alpha-neurotoxin, Naja naja) and several cholinergic ligands produce partial inhibition of the reaction of antiserum with purified acetylcholine receptor. Evidence is presented that some of the toxin-binding sites on receptor, purified by affinity chromatography on toxin-agarose conjugates, are occluded by toxin. In addition, evidence is presented that antireceptor antiserum will cause precipitation of more toxin-binding sites present in an initial extract than in purified receptor preparations.

Studies on the structure of the acetylcholine receptor from Topredo marmorata

The purified acetylcholine receptor of Torpedo marmorata has been characterized by sedimentation velocity measurements on dilute solutions using an ultracentrifuge and scanner. Several preparations were studied and all exhibited sedimentation coefficients in the vicinity of 24S. In a number of experiments the receptor could be resolved into two sedimenting boundaries of 18S and 26S, corresponding to minimum molecular weights of about 5 × 10 5 and 10 6, respectively. Additions of sodium dodecyl sulfate or Triton X-100 resulted in marked decreases in sedimentation coefficient, while treatment with Lubrol-WX had only a slight effect on the S values. Small changes in S 20,w were produced by guanidine hydrochloride alone, although addition of dithiothreitol with 6 M guanidine hydrochloride resulted in an 8.8S component. Electrophoresis in sodium dodecyl sulfate gave one principal band with a molecular weight of 46,000.