Detergent-partition profile of nicotinic acetylcholine receptor in reconstituted ?raft? model membranes.

BERMÚDEZ, V.; ANTOLLINI, S. S.; FERNÁNDEZ NIEVAS, G. A.; AVELDAÑO, M. I.; BARRANTES, F. J.

Montevideo , Uruguay

Congreso; ICBP-2007 6th International Conference of Biological Physics and 5th Southern Cone Biophysics Congress; 2007

Biological membranes are thought to contain lipid lateral heterogeneities or microdomains termed ?rafts?, composed of unsaturated phosphatidylcholines (PC), sphingomyelins (SM), and cholesterol (Chol).  In order to investigate whether the nicotinic acetylcholine receptor (AChR) associates with this type of lipid domain, affinity purified AChR from T. californica was  reconstituted into lipid mixtures having the composition of a ?raft?-forming (PC:SM:Chol, 1:1:1) mixture, and the partition of the receptor protein between detergent (1% Triton X-100, 4°C)-resistant (DRM) or detergent-soluble domains (DSM) assessed by SDS-PAGE and Western blotting. To characterize the role that AChR clustering has on the distribution of the protein in these biochemical fractions, AChR was reconstituted in the presence of purified rapsyn, a soluble receptor-associated protein purported to contribute to AChR aggregation, treated with Triton X-100 and analyzed as described above. The role of ?raft? lipid aggregation on AChR distribution was analyzed using model lipid mixtures containing the ganglioside GM1 (a bona fide ?raft? lipid), and inducing GM1 aggregation by cholera toxin B + anti-cholera toxin antibody-mediated cross-linking. The results indicate that 1) purified AChR displays no preferential partition into either fraction; 2) incorporation of rapsyn favors the partition of the AChR in DRM fractions; 3) the presence of GM1 also increases the proportion of AChR in the DRM fractions, and, more importantly, 4) antibody-mediated cross-linking of this ganglioside induces a redistribution of the AChR, increasing the partition of protein in DSM fractions. Thus ganglioside aggregation and AChR clustering appear to have opposite effects on AChR detergent partition profile.