Sphingomyelin-dependent partition profile of nicotinic acetylcholine receptor in model lipid domains

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

Capital Federal

Conferencia; 5th Special Conference of the International Society for Neurochemistry ?Synapses and dendritic spines in health and disease?; 2012

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 a part 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 external signals 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 whether the different affinities among the different types of SM and Chol determine the preference of the AChR for Lo domains, we first checked whether the 16:0-SM species induces Lo domains different 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 presence of the AChR in detergent-resistant and detergent-soluble fractions (DRMs and DSM, respectively) was determined by SDS-PAGE. AChR in liposomes with brain-SM showed preference (60:40) for the DSM fraction, whereas in 16:0-SM liposomes exhibited almost the opposite behavior (near 66% preference for the DRM fraction). Thus, partition of the AChR in liquid-disorder (Ld) or liquid-ordered (Lo) domains is markedly influenced by the type of sphingomyelin present in the host membrane.