Biophysical and Ion Channel Functional Characterization of the Torpedo Californica Nicotinic Acetylcholine Receptor in Varying Detergent-Lipid Environments

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Autor
Asmar-Rovira, Guillermo A.
Asseo-García, Aloysha M.
Quesada, Orestes
Hanson, Michael A.
Nogueras, Carlos
Lasalde-Dominicci, José A.
C. Stevens, Raymond
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ArticleFecha
2009-05-01Metadatos
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The nicotinic acetylcholine receptor (nAChR) of Torpedo electric rays has been extensively characterized over the last three decades. However, the molecular mechanisms by which detergents influence membrane protein stability and function remain poorly understood, and elucidation of the dynamic detergent-lipid-protein interactions of solubilized membrane proteins is a largely unexplored research field. This study examined nine detergents upon nAChR solubilization and purification, to assess receptor lipid composition using GC (Gas Chromatography)-FID (Flame Ionization) and/or GC-MSD (Mass Selective Detectors), stability and aggregation state using A-SEC (Analytical Size-Exclusion Chromatography) and EM (Electron Microscopy), and planar lipid bilayers to measure ion channel function. 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-analog detergents [CHAPS (3-[(3-Cholamidopropyl)-dimethylammonio]-1-propane sulfonate), FC-12 (n-Dodecylphosphocholine) and sodium cholate (3α,7α,12α-Trihydroxy-5β-cholan-24-oic acid)] maintaining stability and supporting ion channel function, while non-lipid analog detergents [Cymal-6 (6-Cyclohexyl-1-hexyl-β-d-maltoside), DDM (n-Dodecyl-β-d-maltopyranoside), LDAO (Lauryldimethylamine-N-oxide) and OG (n-Octyl-β-d-glucopyranoside)] showed decreased stability and significant reduction or loss of 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 when compared to lipid-analog detergents. Overall, these results show that the nAChR can be stable and functional in lipid-analog detergents or in detergents that retain moderate amounts of residual native lipids, while the opposite is true about non-lipid analog detergents. These results highlight the importance of careful biophysical characterization of membrane proteins for future functional or structural studies in the detergent-solubilized state.
Citación
Asmar-Rovira, G. A., Asseo-García, A. M., Quesada, O., Hanson, M. A., Nogueras, C., Lasalde-Dominicci, J. A., & Stevens, R. C. (2008). Biophysical and ion channel functional characterization of the Torpedo californica nicotinic acetylcholine receptor in varying detergent-lipid environments. The Journal of Membrane Biology, 223(1), 13–26. http://doi.org/10.1007/s00232-008-9107-7Colecciones
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