The amino‐terminal sequence of the 40 000 molecular weight subunit of the acetylcholine receptor protein from <i>Torpedo marmorata</i>

Devillers‐Thiéry, Anne; Changeux, Jean‐Pierre; Paroutaud, Pierre; Strosberg, A. Donny

doi:10.1016/0014-5793(79)81092-3

Cited by 59 publications

(32 citation statements)

References 27 publications

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“…However, with the recently developed new technology of high resolution microsequencing, amino acid sequences could be determined from small quantities of protein. The sequence of 20 amino acids comprising the N-terminal domain of the ␣-subunit of the T. marmorata receptor was established in my laboratory (34). A chemical identity card of the receptor was made available, the first ever established for a neurotransmitter receptor.…”

Section: The Pentameric Organization Of the Nicotinic Receptor And Thmentioning

confidence: 99%

The Nicotinic Acetylcholine Receptor: The Founding Father of the Pentameric Ligand-gated Ion Channel Superfamily

Changeux

2012

Journal of Biological Chemistry

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211

149

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A critical event in the history of biological chemistry was the chemical identification of the first neurotransmitter receptor, the nicotinic acetylcholine receptor. Disciplines as diverse as electrophysiology, pharmacology, and biochemistry joined together in a unified and rational manner with the common goal of successfully identifying the molecular device that converts a chemical signal into an electrical one in the nervous system. The nicotinic receptor has become the founding father of a broad family of pentameric membrane receptors, paving the way for their identification, including that of the GABA A receptors.It has been 42 years since the isolation of the nicotinic acetylcholine receptor (nAChR) 2 from fish electric organ, the first ligand-gated ion channel and the first ion channel ever identified; 25 years since the first GABA A and glycine receptor subunits were cloned and sequenced and concomitantly their homology with the nAChRs recognized; and 5 years since the discovery that closely homologous ligand-gated ion channels are present in prokaryotes (1). In this minireview, I briefly retrace the main steps in the discovery of the nAChR, the titular head of this receptor superfamily. The Concept of Receptor and the Chemical Identification of the Acetylcholine ReceptorThe English physiologist John Newport Langley, working with neuromuscular preparations, proposed in 1905 that muscle tissue possesses "a substance that combines with nicotine and curare . . . receives the stimulus and transmits it." He called the muscle entity the "receptive substance." In the subsequent 50 years, the concept of pharmacological receptors inspired three main lines of research: first, the pharmacological approach aimed at characterizing the specificity of the receptor site by using novel chemical ligands (e.g. the distinction between nicotinic and muscarinic AChRs by Sir Henry Dale); second, the electrophysiological approach exemplified by Bernard Katz and John Eccles aimed at understanding the ionic responses to endogenous neurotransmitter signals; and third, the chemical tradition aimed at the chemical identification of the receptor molecule(s).In the late 1960s, lipids, polysaccharides, proteins, and even nucleic acids were considered as potential receptors. The early independent efforts of Carlos Chagas, Eduardo de Robertis, and David Nachmansohn to identify the receptor for acetylcholine (ACh) in the electric organ of the fish Electrophorus electricus with radioactive ligands were abandoned because their tissue extracts lacked specificity (2). However, in the course of these studies, Nachmansohn recognized the extraordinarily rich content of nicotinic synapses in the electric organ (2). With Ernest Schoffeniels, he devised a method for preparing individual cells, or electroplaques, from the electric organ. This offered the opportunity to investigate, simultaneously, the electrophysiological, pharmacological, and biochemical characteristics of the response to ACh within the same biological system (2). At this time, th...

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Section: The Pentameric Organization Of the Nicotinic Receptor And Thmentioning

confidence: 99%

The Nicotinic Acetylcholine Receptor: The Founding Father of the Pentameric Ligand-gated Ion Channel Superfamily

Changeux

2012

Journal of Biological Chemistry

Self Cite

211

149

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“…On the other hand, the 40000-M, polypeptide, which in the best preparations of reconstituted receptor represents up to 70 "/, of the proteins measured by scanning the gel after electrophoresis, is the only chain labelled by most covalent affinity reagents of the acetylcholine receptor site [2,25 -27,751. This discrepancy may be explained by: (a) difficulties in estimating correct amounts of protein ; (b) half-of-the-sites reactivity [76], although such as phenomenon has never been demonstrated to occur without ambiguity for a-toxin binding; (c) the presence of chemically different 40000-M, chains ; only one of them would carry the acetylcholine binding site but sequencing of the 40000-M, chain did not reveal any heterogeneity up to the 25th amino acid [29,30]; (d) an underestimation of the number of a-toxin sites caused by the possible presence of an endogenous inhibitor, the loss of a specific cofactor, or a partial denaturation of the 40000-M, chain; (e) the loss during denaturing gel electrophoresis of small peptides created by nicking. These last possibilities have to be considered seriously since it is a general observation (see for instance [3,49,75]) that no strict correlation seems to exist between the specific activity (in a-toxin sites) of a given preparation and its state of purity revealed by NaDodSO4 gel electrophoresis.…”

Section: Acetylcholine Receptor Site and Local Anesthetic Binding Sitmentioning

confidence: 99%

“…N o significant difference was noticed between the two modes of staining. In addition, after scanning the gels, the surface of the peaks was found to vary linearly with the quantity of protein (up to 5 pg) and the same staining intensity per mass of protein was determined with each of the individual 40000-MI, 50000-M, and 66000-M, chains purified as in [29]. With pure membrane preparations, the height of each peak varied linearly with its surface and was taken as a sufficient parameter to compare the relative amounts of the different polypeptide chains.…”

Section: Qucrntitative Measurements Of the Composition In Po(ypcy7fidmentioning

confidence: 99%

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Reconstitution of a Functional Acetylcholine Receptor

Sobel

Heidmann

Cartaud

et al. 1980

European Journal of Biochemistry

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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 43000-M, protein and dispersed into solution by sodium cholate under conditions where more than 8 5 % 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 40000-M, chain, which is labelled by the affinity reagent 4-(N-maleimido)phenyI [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 cc-'251-bungarotoxin sites. This ratio, the three classes of binding sites, and their regulation by carbamylcholine are conserved through the reconstitution cycle.Investigations into the molecular mechanism of the physiological action of acetylcholine may, taking a reductionist viewpoint, be organised along two lines. In the first, the acetylcholine-sensitive membrane is resolved into its various components, which are subsequently characterized chemically. These components evidently suffice for the occurrence of the physiological response. The demonstration that they are necessary may be accomplished, following the second approach, by the reconstitution of an acetylcholine-sensitive membrane in which the individual molecular species are reintegrated into an artificial environment.Abbreviations. NaDodS04, sodium dodecyl sulfate; Dns-CsCho, [l-(5-dimethyIaminonaphthalene)-sulfonamido]-n-hexanoic acid-8-N-trimethylammonium bromide ethyl ester.To carry out this demonstration, membrane fragments were purified, on the basis of their high content in acetylcholine receptor sites, from electric organ of(see [4]). The method was developed on a macroscopic scale [2] required for biochemical and biophysical studies and the same membranes were shown to respond in vitro to cholinergic agoni...

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“…The AChR has also been solubilized with detergents and purified by affinity chromatography to homogeneity (Raftery et al 1975;Karlin et al 1976;Lindstrom & Patrick, 1974). Amino acid sequence analysis of the purified subunits has started (Raftery et al 1980;Devillers-Thiery et al 1979) and high resolution structural studies are currently under way (Zingsheim et al 1980;Klymkowsky, Heuser & Stroud, 1980). The AChR from Torpedo californica is an integral membrane glycoprotein of which the monomeric form is composed of four glycopeptide subunits termed a, /?, 7 and 8 with apparent molecular weights approximating 40000, 50000, 60000 and 65000 daltons (Reynolds & Karlin, 1978;Lindstrom et al 1979;Raftery et al 1980).…”

Section: (Iii) Monolayers and Planar Bilayersmentioning

confidence: 99%

Functional reassembly of membrane proteins in planar lipid bilayers

Montal

Darszon

Schindler

1981

Quart. Rev. Biophys.

116

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Recent progress in membrane biology has brought us to a stage where it is possible to associate complex biological processes to identifiable membrane proteins. Technical advances in the biochemical characterization and purification of membrane proteins have contributed a wealth of structural information. The reconstitution approach has proved to be valuable in our efforts to understand the molecular mechanisms of membrane transport and energy transduction.

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The amino‐terminal sequence of the 40 000 molecular weight subunit of the acetylcholine receptor protein from Torpedo marmorata

Cited by 59 publications

References 27 publications

The Nicotinic Acetylcholine Receptor: The Founding Father of the Pentameric Ligand-gated Ion Channel Superfamily

The Nicotinic Acetylcholine Receptor: The Founding Father of the Pentameric Ligand-gated Ion Channel Superfamily

Reconstitution of a Functional Acetylcholine Receptor

Functional reassembly of membrane proteins in planar lipid bilayers

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