Some Structural Properties of the Cholinergic Receptor Protein in Its Membrane Environment Relevant to Its Function as a Pharmacological Receptor

Changeux, J P; Benedetti, Laura; Bourgeois, Jean-Pierre; Brisson, Alain; Cartaud, Jean; Devaux, PF; Grünhagen, Hans-Heinrich; Moreau, Marc; Popot, Jean‐Luc; Sobel, André; Weber, Michel

doi:10.1101/sqb.1976.040.01.023

Cited by 87 publications

(44 citation statements)

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“…Electron microscopy after negative staining of the purified acetylcholine receptor preparation shows an homogeneous population of 8-nm rosette-like particles similar to those observed with highly purified acetylcholine-receptor-rich membranes and with purified acetylcholine receptor protein from Electrophorus electricus [5].…”

Section: Criteria Of Homogeneitymentioning

confidence: 93%

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Large‐Scale Purification of the Acetylcholine‐Receptor Protein in Its Membrane‐Bound and Detergent‐Extracted Forms from Torpedo marmorata Electric Organ

Sobel¹,

Weber²,

Changeux³

1977

European Journal of Biochemistry

370

215

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A method is described for the large-scale purification of membrane fragments very rich in acetylcholine (nicotinic) receptor from the electric organ of Torpedo marmorata. The preparations of purified membrane fragments have a specific activity of more than 4000 nmol a-toxin binding sites/g protein and give only four main polypeptide bands by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Observations by electron microscopy show that the purified preparation of receptor-rich membrane fragments is composed of only one class of membrane fragments covered with 8-nm rosettes identified as acetylcholine receptor molecules.This preparation is used as a starting material for the detergent solubilization and the large-scale purification of the acetylcholine receptor protein, without using affinity chromatography. A sucrose gradient centrifugation of a Triton X-100 extract of receptor-rich membranes done in the presence of 2-mercaptoethanol yields large quantities of receptor protein in a homogeneous form as indicated by polyacrylamide gel electrophoresis, isoelectric focussing and electron microscopy.Polyacrylamide gel electrophoresis of the purified protein in the presence of sodium dodecyl sulfate reveals three bands of apparent molecular weights 40 000

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Section: Criteria Of Homogeneitymentioning

confidence: 93%

“…Moreover, the development of a fractionation method giving membrane fragments particularly rich in rechptor protein rendered it accessible to physicochemical investigations in a membrane-bound and still functional state [5].…”

mentioning

confidence: 99%

Large‐Scale Purification of the Acetylcholine‐Receptor Protein in Its Membrane‐Bound and Detergent‐Extracted Forms from Torpedo marmorata Electric Organ

Sobel¹,

Weber²,

Changeux³

1977

European Journal of Biochemistry

370

215

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“…The receptor-rich membrane fragments purified from T. marmorata electric organ are most probably derived from the cytoplasmic membrane of the electroplaque which underlies the nerve terminal [2,25,26,42,461. Their content in acetylcholine receptor protein is indeed close to that expected from the determination of the density of nicotinic receptor sites estimated by autoradiography in situ on the subsynaptic membrane (see [46,47]).…”

Section: Discussionmentioning

confidence: 99%

“…Indeed, in spite of the possible occurrence of a fluid hydrophobic phase in the vicinity of the acetylcholine receptor site [71], the receptor molecules appear strongly immobilized in the subsynaptic membrane both in vitvo [72] and in vivo [47,, suggesting the existence of protein-protein interactions. Moreover, a binding site for local anesthetics is present in the receptor-rich membrane fragments where it modulates the binding properties of the acetylcholine site (for a review, see [2]). Recent experiments indicate that the binding site for local anesthetics is carried by a polypeptide chain different from the receptor protein , m s u strict0 [17-191.…”

Section: Discussionmentioning

confidence: 99%

Interaction of the Acetylcholine (Nicotinic) Receptor Protein from Torpedo marmorata Electric Organ with Monolayers of Pure Lipids

Popot¹,

Demel²,

Sobel³

et al. 1978

European Journal of Biochemistry

115

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Membrane fragments rich in cholinergic (nicotinic) receptor protein were purified from the electric organ of Torpedo marmoruta. Their lipid composition is essentially characterized by the prominence of cholesterol, phosphatidylethanolamine and phosphatidylcholine, long-chain fatty acyl constituents, and the absence of sphingomyelin. Solubilised receptor was purified from these fragments and the concentration of sodium cholate lowered by dialysis to 0.01 76 (w/v). When this preparation was injected under a lipid monolayer, an increase of surface pressure developed, which was not observed with the detergent alone nor in the absence of lipid film. When covalently radiolabelled receptor preparations were injected at a constant surface pressure the radioactivity recovered with the film was proportional to the increase in area. It is concluded that the pressure or area increases are due to the penetration of the cholinergic receptor protein into the lipid film.Incorporation experiments into films formed from various pure lipids showed that the protein interacts more readily with cholesterol than with ergosterol, phosphatidylcholine, or other phospholipids. Its affinity is also higher for long-chain phosphatidylcholines than for short-chain ones. The degree of unsaturation and fluidity of the 3-sn-phosphatidylcholine (lecithin) films are of secondary importance. Parallel experiments with covalently and non-covalently labelled receptor preparations showed that part of the protein recovered with the film lost its a-toxin binding ability during the penetration.Similar data were obtained with the receptor purified from Electrophorus electricus electric organ.The acetylcholine receptor protein from fish electric organ is a well-characterized [I -41 membrane protein available in sizable quantities in a highly purified form. During the past few years several attempts have been made to incorporate the detergent-solubilized protein into artificial lipid membranes and to regain both a selective transport of cations and its regulation by nicotinic effectors (for reviews see . Because of the limited success of these attempts and their all-or-none character, several parallel approaches have been developed to circum- 250 mM NaC1, 5 mM KC1, 4 mM CaC12; 2 mM MgC12, 1.5 mM sodium phosphate buffer, pH 7.0; Ekctrophorus Ringer's solution : 160 mM NaC1, 5 mM KC1, 2 mM CaC12, 2 mM MgCl2, 2.5 mM sodium phosphate buffer, p H 7.0. Names and abbreviations for lipids follow the recent recommendations of the IUPAC-IUB Commission on Biochemical Nomenclature, see Eur. J . Biochem. 7Y, 11-21 (1977).vent the many difficulties encountered. One was to recover the binding properties [8,9] and conformational transitions [9] of the receptor protein that are strongly modified as a consequence of the detergent solubilization [S, 10-161 and/or purification. A second one is to determine if the solubilized protein carries the structure responsible for specific translocation of cations (ionophore) using specific ligands [17 -191. A third approach is to charac...

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“…It has been emphasized that a necessary first step for a successful 'reconstitution' was the recovery of the binding properties of the receptor protein [9]. Several binding properties of the membranebound receptor are now known, in particular: (a) preincubation of the membrane-bound receptor with agonists causes a slow increase to affinity [lo], which appears related to the 'desensitization' of the permeability response [25] ; (b) local anaesthetics enhance at equilibrium [19] the affinity of the membrane-bound receptor for agonists and, as we have shown in this paper, accelerate the affinity change caused by agonists (see also [25]); (c) a fluorescent local anaesthetic, quinacrine, may be used to monitor structural changes associated with the binding of agonists (for a full discussion of the subject, see [26]). …”

Section: Discussionmentioning

confidence: 99%

Recovery of Some Functional Properties of the Detergent-Extracted Cholinergic Receptor Protein from Torpedo marmorata after Reintegration into a Membrane Environment

Briley¹,

Changeux

1978

Eur J Biochem

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The change of affinity of the acetylcholine receptor for agonists and the influence of local anaesthetics has been studied in detail in receptor-rich membranes. These properties are changed after solubilisation by ionic detergents.A method for reproducibly reintegrating the receptor protein into a lipid environment is described. Reintegration of the receptor results in partial recovery of the binding and fluorescence properties of the membrane-bound receptor protein. In particular, the slow affinity change caused by agonists can be recovered but not the effect of local anaesthetics on this change. The fluorescence response to cholinergic ligands of the reintegrated receptor protein labelled with quinacrine does not appear identical to that found with the native receptor-rich membranes. It is suggested that the failure to recover the sensitivity to local anaesthetics is at the origin of the difficulties to regain functional reconstitution.Many attempts to reconstitute a functional 'excitable' membrane from preparations of purified nicotinic receptor protein from fish electric organ have been reported in the literature (for review see [l] and [2]). Crude detergent extracts of membrane preparations, rich in acetylcholine receptor protein [3], receptor protein purified by affinity chromatography in the presence of detergent [4,5] and even proteolipid fractions prepared directly from the electric organ [6,7] have been used and in several instances recovery of a permeability response to cholinergic agonists was found [3,4,8] but the reproducibility of these results is often questionable [1,2].As a first step to understanding the several parameters involved, our approach has been to study 'the binding properties of the receptor protein for cholinergic ligands in the membrane-bound and detergentsolubilised states and reintegrated into a membrane environment. Conditions have been previously defined under which a reversible transition between highaffinity and low-affinity states of the receptor protein takes place by varying the concentration of detergent [9]. Subsequently, it was shown that such transitions between affinity states exist in 'native' receptor-rich membrane fragments [lo] and are also regulated by cholinergic agonists. In the course of these studies it was also noticed that, in the same membrane fragments, local anaesthetics stabilize, at equilibrium, the receptor in its high-affinity state.In this paper we describe the reintegration of the receptor protein from Torpedo miirmorata electric organ into a lipid environment and demonstrate that, under these conditions, the agonist-binding characteristics of the membrane-bound receptor and the agonist-dependent affinity change can be recovered. The characteristic effect of local anaesthetics on this transition, however, cannot be demonstrated with the reintegrated receptor protein. The failure to recover the sensitivity to local anaesthetics might be at the origin of the difficulty to regain the full permeability response. MATERIALS AND METHODS Preparation ...

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Some Structural Properties of the Cholinergic Receptor Protein in Its Membrane Environment Relevant to Its Function as a Pharmacological Receptor

Cited by 87 publications

References 1 publication

Large‐Scale Purification of the Acetylcholine‐Receptor Protein in Its Membrane‐Bound and Detergent‐Extracted Forms from Torpedo marmorata Electric Organ

Large‐Scale Purification of the Acetylcholine‐Receptor Protein in Its Membrane‐Bound and Detergent‐Extracted Forms from Torpedo marmorata Electric Organ

Interaction of the Acetylcholine (Nicotinic) Receptor Protein from Torpedo marmorata Electric Organ with Monolayers of Pure Lipids

Recovery of Some Functional Properties of the Detergent-Extracted Cholinergic Receptor Protein from Torpedo marmorata after Reintegration into a Membrane Environment

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