Fluidity of the lipids next to the acetylcholine receptor protein of Torpedo membrane fragments. Use of amphiphilic reversible spin-labels

Bienvenüe, Alain; Rousselet, Annie; Kato, G.; Devaux, Philippe F.

doi:10.1021/bi00624a005

Cited by 36 publications

(16 citation statements)

References 13 publications

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“…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]).…”

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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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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“…Our approach to the problem has been to artificially link the spin-labeled fatty acids to intrinsic proteins in native membranes (Devaux et al. 1975;Bienvenue et al. 1977).…”

Section: Mobility Of the Hydrocarbon Chains In Direct Contact With Rhmentioning

confidence: 99%

“…However, in this latter case the apparent correlation time describing the motion is increased by more than two orders of magnitude, showing that lipid-depleted membranes cannot be used to characterize the viscosity of the boundary layer of native membranes. N M R and rod outer segment membranes, Brown et al (1977) concluded that, although the segmental motion of hydrocarbon chains was affected by the proximity of the proteins, all phospholipids can diffuse rapidly in the plane of the membrane and that boundary and free lipids exchange rapidly.Concurrently, using spin-labeled fatty acid derivatives containing specific polar head groups, we were able to explore the hydrophobic environment of different membrane-bound proteins in native membranes (Devaux et al, 1975;Lauquin et al, 1977;Bienvenue et al, 1977). Our general conclusion was that, although evidence of hindrance in the motion of spin-labeled chains accompanies the binding to the proteins, fluidity can be found in the direct environment of such proteins.…”

mentioning

confidence: 99%

Spin-label studies of lipid-protein interactions in retinal rod outer segment membranes. Fluidity of the boundary layer

Favre¹,

Baroin²,

Bienvenüe³

et al. 1979

Biochemistry

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In order to fix spin-labeled acids at the boundary layer of membrane-bound proteins, spin-labeled long-chain derivatives (m,n)MSL (general formula, CH,(CH2),R-(CH2),COO(CH2)2-M, where R is an oxazolidine ring containing a nitroxide and M is a maleimide residue) were synthesized. The spin-labeled molecules bind covalently to at least two different classes of sulfhydryl groups on rhodopsin in disc membrane fragments from bovine retina. One class of sites is hydrophilic and corresponds to the two SH groups labeled readily by N-ethylmaleimide; the second class of sites is only reached by hydrophobic probes. (10,3)MSL binds equally well to the two classes of sites on rhodopsin, whereas (1 , I 4)MSL, more hydrophobic, binds preferentially to the hydrophobic sites. Apparently a third class of SH groups can be labeled if a very large excess of (m,n)MSL is employed, but proteins may be denatured in this latter case. Labels not covalently bound are removed from the membranes by incubation with fatty acid free bovine serum albumin. However, it is found that the probes do not bind only to rhodopsin in the disc membranes. (m,n)MSL also binds covalently to phosphatidylethanolamine in the rod outer segments or in liposomes. This covalent binding to phospholipids is demonstrated by lipid extraction and thin-layer chromatographic analysis. In order to obtain the pure EPR spectra of the spin-labeled fatty acids bound to the protein, the spectra corresponding to phospholipid-bound spin labels have been I t is often admitted that intrinsic membrane proteins are surrounded by a boundary layer or "annulus" of rigidly bound lipid. The immobilization of this shell of lipid has been deduced essentially from EPR experiments involving spin-labeled fatty acids incorporated into reconstituted systems containing variable lipid to protein ratios. Jost et al. (1973) were the first to propose from spin-label experiments the model of a boundary layer of lipid surrounding an intrinsic membrane protein, namely cytochrome oxidase. Later, Hesketh et al. (1976) reported similar experiments with Ca2+-ATPase, while Chapman et al. (1977) showed that gramcidin A can lead to the same EPR results, if this polypeptide is dissolved in a small amount of lipid.Rhodopsin was also tested with spin-labeled fatty acids. Pontus and Delmelle (1975) found evidence of a rigid boundary layer around this hydrophobic protein. However, previously, Hong & Hubbell (1972) had reached a different conclusion from spin-label experiments with rhodopsin reincorporated into phospholipid; they suggested that the acerage viscosity of the membranes was dependent on the lipid to protein ratio. This is in good agreement with recent results put foward by Cherry et al. (1977) from very different experiments involving bacteriorhodopsin. Finally, using proton manuscript receiced A'ocember 21, 1978. This investigation was supported by research grants from the Centre National de la Recherche Scientifique (ERA 690) and the DelEgation G&n&rale a la Recherche Scientifique et Technique (Comm...

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“…Concurrently we used spin-labeled acyl derivatives of specific ligands or covalently bound spin-labeled fatty acids. We demonstrated the possibility that spin labels that are in direct contact with intrinsic proteins, such as the ADP carrier in mitochondria (12,13), the acetylcholine receptor in torpedo membranes (14), or rhodopsin in disc membrane fragments (15), are moving rapidly. We showed that a spin-labeled fatty acid can be anchored to rhodopsin, provided the fatty acid contains a maleimide residue linked to the carboxylic terminal.…”

mentioning

confidence: 99%

Physical modifications of rhodopsin boundary lipids in lecithin-rhodopsin complexes: a spin-label study.

Davoust¹,

Schoot²,

Devaux³

1979

Proc. Natl. Acad. Sci. U.S.A.

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The microviscosity of rhodopsin boundary lipids was studied with a spin-labeled fatty acid covalently attached to rhodopsin, in rhodopsin-egg lecithin vesicles. When the lipid-to-protein ratio was high (500:1, mole to mole), only narrow peaks were visible in electron paramagnetic resonance spectrum at 370C. This enabled us to show that, under these conditions, not more than 10% of the probes have their motion strongly restricted by the proximity of the protein. When (3), suggest that the lipid population is homogeneous at all temperatures. According to the latter authors, the protein decreases the average order parameter of the lipid. From spin-label experiments involving low lipidto-protein ratios, various authors have come to the conclusion that boundary lipids are strongly immobilized around proteins such as cytochrome oxidase (4-7), Ca2+-ATPase (8), lipophilin (9), and rhodopsin (10). These results, however, were criticized by Chapman et al. (11), who tested the interaction of gramicidin A with lipids, using the spin-label method. Concurrently we used spin-labeled acyl derivatives of specific ligands or covalently bound spin-labeled fatty acids. We demonstrated the possibility that spin labels that are in direct contact with intrinsic proteins, such as the ADP carrier in mitochondria (12, 13), the acetylcholine receptor in torpedo membranes (14), or rhodopsin in disc membrane fragments (15), are moving rapidly. We showed that a spin-labeled fatty acid can be anchored to rhodopsin, provided the fatty acid contains a maleimide residue linked to the carboxylic terminal. The fatty acid moiety allows the maleimide to react specifically with sulfhydryl groups of rhodopsin not accessible to hydrophilic reagents. 2755The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

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Fluidity of the lipids next to the acetylcholine receptor protein of Torpedo membrane fragments. Use of amphiphilic reversible spin-labels

Cited by 36 publications

References 13 publications

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

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

Spin-label studies of lipid-protein interactions in retinal rod outer segment membranes. Fluidity of the boundary layer

Physical modifications of rhodopsin boundary lipids in lecithin-rhodopsin complexes: a spin-label study.

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