Preferential partition of nicotinic acetylcholine receptor in model lipid domains

PERILLO, V.L.; PEÑALVA, D.A.; AVELDAÑO, M.I.; BARRANTES, F.J.; ANTOLLINI, S.S.

Buenos Aires

Congreso; 5th ISN Special Conference; 2012

Preferential partition of nicotinic acetylcholine receptor in model lipid domains. Perillo V.L.a; Peñalva, D.A. a; Aveldaño, M.I. a; Barrantes F.J.b; Antollini S.S. a a Instituto de Investigaciones Bioquímicas de Bahía Blanca, Consejo de Investigaciones Científicas y Técnicas (CONICET) / Universidad Nacional del Sur, 8000 Bahía Blanca, Argentina. b Laboratory of Molecular Neurobiology, Inst. Biomedical Res., UCA,and CONICET, 1107 Buenos Aires, Argentina. The preferential location of the nicotinic acetylcholine receptor (AChR) at liquid-ordered domains (Lo), the membrane heterogeneities commonly termed rafts, has been postulated to be an initial step of its clustering mechanism. The typical and predominant raft lipids are sphingomyelin (SM) and cholesterol (Chol). Previous work from our laboratory with AChR protein reconstituted in model systems composed of brain SM, Chol and POPC (1:1:1) did not show any preferential affinity of the AChR for these domains. We concluded that the distribution of the AChR may not depend exclusively on the intrinsic physicochemical properties of the receptor but also on some external signal from the cell membrane (Bermúdez et al., 2010). A recent study showed that Chol exhibits highest affinity for 16:0-SM over any SM with other acyl chain lengths (Jaikishan and Slotte, 2011). To test if the different affinities between the different types of SM and Chol determine the preference of the AChR for Lo domains, we first checked whether this SM species induces Lo domains with differences from those obtained with brain SM and other SM species. Giant unilamellar vesicles (GUVs) were prepared using ternary mixtures of POPC:Chol:SM (1:1:1) with different SM types. Domains were visible only in the case of GUVs prepared using 16:0-SM obtained from rat testis, but not in GUVs with SM extracted from egg yolk, with commercial brain SM, nor with the mixture of 16:0-18:0 SM from rat testis. Affinity-purified AChR from T. californica was reconstituted into liposomes having different SM types: PC:Chol:brain-SM (1:1:1) and PC:Chol:16:0-SM (1:1:1) and treated with 1% Triton X-100 at 4°C. The location of the AChR in detergent resistant and soluble fractions (DRMs and DSM, respectively) was determined by SDS-PAGE. AChR in liposomes with brain-SM showed 60% preference for the DSM fraction, whereas in 16:0-SM liposomes showed almost the opposite (near 66% preference for the DRM). Thus, the AChR was found to occur in liquid-disorder (Ld) or liquid-ordered (Lo) domains depending on the type of sphingomyelin used in the host membrane.