Partition profile of the nicotinic acetylcholine receptor in lipid domains upon reconstitution

Vicente Bermúdez; Silvia S. Antollini; Gaspar A. Fernández Nievas; Marta I. Aveldaño; Francisco J. Barrantes

doi:10.1194/jlr.M005132

Partition profile of the nicotinic acetylcholine receptor in lipid domains upon reconstitution[S]

Instituto de Investigaciones Bioquímicas de Bahía Blanca, Consejo Nacional de Investigaciones Científicas y Técnicas, and UNESCO Chair of Biophysics and Molecular Neurobiology, Universidad Nacional del Sur, Buenos Aires, Argentina

1↵To whom correspondence should be addressed. e-mail: silviant{at}criba.edu.ar

Abstract

The nicotinic acetylcholine receptor (AChR) is in intimate contact with the lipids in its native membrane. Here we analyze the possibility that it is the intrinsic properties of the AChR that determine its partition into a given lipid domain. Torpedo AChR or a synthetic peptide corresponding to the AChR γM4 segment (the one in closer contact with lipids) was reconstituted into “raft”-containing model membranes. The distribution of the AChR was assessed by Triton X-100 extraction in combination with fluorescence studies, and lipid analyses were performed on each sample. The influence of rapsyn, a peripheral protein involved in AChR aggregation, was studied. Raft-like domain aggregation was also studied using membranes containing the ganglioside GM1 followed by GM1 crosslinking. The γM4 peptide displays a marked preference for raft-like domains. In contrast, AChR alone or in the presence of rapsyn or ganglioside aggregation exhibits no such preference for raft-like domains, but it does cause a significant reduction in the total amount of these domains. The results indicate that the distribution of the AChR in lipid domains cannot be due exclusively to the intrinsic physicochemical properties of the protein and that there must be an external signal in native cell membranes that directs the AChR to a specific membrane domain.

Footnotes

↵[S] The online version of this article (available at http://www.jlr.org) contains supplementary data in the form of one figure.
This work was supported by grants from the Agencia Nacional de Promoción Científica y Técnológica (FONCYT) (S.S.A. and F.J.B.), the Universidad Nacional del Sur (F.J.B.), and the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET) (F.J.B.).
Abbreviations:

AChR

nicotinic acetylcholine receptor

CDx

methyl-β-cyclodextrin

Chol

cholesterol

CTx

cholera toxin

CTxB

cholera toxin B-subunit

DHE

dehydroergosterol

DOPC

dioleoylphosphatidylcholine

DRM

detergent-resistant membrane

DSM

detergent-soluble membrane

E

energy transfer efficiency

FRET

Förster resonance energy transfer

GM1

ganglioside GM1

GP

generalized polarization

NMJ

neuromuscular junction

PC

phosphatydilcholine

POPA

palmitoyloleylphosphatidic acid

POPC

palmitoyloleylphosphatydilcholine

SM

sphingomyelin

TM

transmembrane