Interactions between nicotinic acetylcholine receptor and lipid domains.

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

Rosario, Santa Fe, Argentina.

Congreso; XLII Reunión Anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular; 2006

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular

Biochemical and fluorescence spectroscopy methods were used to investigate the interactions between the nicotinic acetylcholine receptor (AChR) and purported “rafts” lipid in model membranes. Affinity purified AChR from T. californica and a synthetic peptide (gM4) corresponding to the M4 transmembrane region, which in the whole receptor interacts with neighboring lipid molecules, were reconstituted into liposomes with a “raft” lipid composition (phospholipid:sphingomyelin:cholesterol 1:1:1). Partition of AChR or gM4 between detergent-resistant membranes (DRM) or soluble domains were assessed by Triton X-100 treatment at 4°C, centrifugation, and SDS-PAGE. The efficiency of the Förster resonance energy transfer (E) between the intrinsic fluorescence of the protein (or the peptide) and dehydroergosterol (fluorescence cholesterol mimetic, used as a reporter of DRM) was used as an assay of the protein distribution in the membrane. The fluorescence probe Laurdan was employed as a control acceptor fluorophore since it shows no preferential partition into any domain in particular. Laurdan generalized polarization (GP) was used to learn about the biophysical properties of the membranes. The results indicate that whereas the gM4 peptide alone partitions preferentially within DRM domains, the whole AChR displays no preferential interactions with these domains.