Cholesterol modulates the biophysical properties of cell membranes and influences the function and trafficking of membrane proteins. A fundamental phenomenon that remains obscure is how the plasma membrane lipid composition regulates the functional pool of membrane receptors. A remarkable model to study this phenomenon is the nicotinic acetylcholine receptor (nAChR), since the nAChR functional pool has been estimated to be but a small fraction of the total number of receptors present in the plasmalemma. Studies on the regulation of the functional pool of the Torpedo californica nAChR expressed in Xenopus laevis oocytes, using a novel lipid-exposed mutation (áC418W) that produces a congenital myasthenic syndrome, demonstrated that cholesterol depletion causes a remarkable gain in macroscopic current of the áC418W mutant with no significant change observed in the wild type (WT) receptors. Electrophysiological techniques, confocal microscopy and biochemical analysis were used to define the mechanism responsible for the increased áC418W macroscopic response seen after cholesterol depletion. The present data suggest that a substantial fraction of the áC418W nAChRs expressed in Xenopus laevis ooctyes is “trapped” in a non-activable state (non functional pool) and that membrane cholesterol depletion results in the relocation of these receptors to the functional pool. Co-localization, co-fractionation and co-immunoprecitation of the áC418W nAChR and the membrane raft marker protein caveolin-1 (cav1) are consistent with the notion that interactions at lipid-exposed domains with lipid membrane microdomains regulate the functional pool of the acetylcholine receptor with potential implications as a novel mechanism to fine-tune cholinergic transmission in the nervous system and in the pathogenesis associated to the áC418W nAChR.

The nicotinic acetylcholine receptor (nAChR) of Torpedo electric rays has been extensively characterized over the last three decades. However, high-resolution structural studies have been hampered by the lack of mechanistic molecular models that describe how detergents influence membrane protein stability and function. Furthermore, elucidation of the dynamic detergent–lipid–protein interactions of solubilized membrane proteins is a largely unexplored research field. This study examines the effects of nine detergents on: (1) nAChR-lipid composition (gas chromatography with flame ionization; GC-FID and/or mass selective detectors; GC-MSD), (2) stability and aggregation state (analytical size exclusion chromatography; A-SEC and electron microscopy; EM) and (3) ion channel function (planar lipid bilayers). Detergent solubilization of nAChR-enriched membranes did not result in significant native lipid depletion or destabilization. Upon purification, native lipid depletion occurred in all detergents, with lipid-analogue detergents CHAPS {(3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate}, FC-12 (n-dodecylphosphocholine) and sodium cholate (3á,7á,12á-trihydroxy-5â-cholan-24-oic acid) maintaining stability and supporting ion channel function, and non-lipid-analogue detergents Cymal-6 (6-cyclohexyl-1-hexyl-â-D-maltoside), DDM (n-dodecyl-â-D-maltopyranoside), LDAO (lauryldimethylamine-N-oxide) and OG (n-octyl-â-d-glucopyranoside) decreasing stability and significantly reducing or completely suppressing ion channel function. Anapoe-C12E9 (polyoxyethylene-[9]-dodecyl ether) and BigCHAP (N,N’-bis-[3-d-gluconamidopropyl] cholamide) retained residual amounts of native lipid, maintaining moderate stability and ion channel function compared to lipid-analogue detergents. Therefore, the nAChR can be stable and functional in lipid-analogue detergents or in detergents that retain moderate amounts of residual native lipids, but not in non-lipid-analogue detergents. Electronic supplementary material The online version of this article (doi:10.1007/s00232-008-9107-7) contains supplementary material, which is available to authorized users.