Partition of nicotinic acetylcholine receptor in model membranas

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

Villa Carlos Paz, Córdoba, Argentina

Congreso; 44 Reunión Anual de la Sociedad Argentina de Bioquímica y Biología Molecular; 2008

Sociedad Argentina de Bioquímica y Biología Molecular

Nicotinic acetylcholine receptor (AChR) in raft-like model membranes V. Bermudez, S. S. Antollini, G. A. Fernández Nievas, M. I. Aveldaño and F. J. Barrantes INIBIBB, Bahía Blanca. Argentina. E-mail: bermudez@criba.edu.ar Nicotinic acetylcholine receptor (AChR) affinity purified from T. californica membranes and a synthetic peptide corresponding to the M4 transmembrane region (gM4) of the receptor were reconstituted into synthetic liposomes with lipid compositions resembling that of raft domains (PC:SM:Chol, 1:1:1). The preferential localization of AChR or gM4 in such synthetic membranes was analyzed using detergent-resistant (DRM) or detergent-soluble domains (DSM) obtained by treatment with 1% Triton X-100 at 4°C followed by SDS-PAGE and Western blotting. The influence of the ganglioside GM1 on AChR was also analyzed with these techniques. The efficiency of the Forster resonance energy transfer (E) between the protein intrinsic fluorescence and dehydroergosterol (fluorescence cholesterol mimetic) served as a measurement of the protein location in the membrane. Purified AChR displayed no preferential partition between raft and non-raft domains whereas the gM4 peptide preferentially partitioned into ordered domains. The presence of GM1 increased the proportion of AChR in the DRM fractions, and antibody-mediated crosslinking of the ganglioside induced a significant redistribution of the AChR towards the DSM fraction. Thus, the preferential localization of the AChR protein in the membrane may not only be governed by its lipid-protein interface.