Partition of nicotinic acetylcholine receptor in model membranes

Abstract

Nicotinic acetylcholine receptor (AChR) affinity-purified from T. californica membranes and a synthetic peptide corresponding to the M4 transmembrane region (ãM4) 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 ãM4 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 this techniques. The efficiency of the Förster resonance energy transfer (E) between the protein intrinsic fluorescence and dehydroergosterol (fluorescent cholesterol mimetic) served as a measure of the protein location in the membrane. Purified AChR displayed no preferential partition between raft and non-raft domains whereas the ãM4 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