Biophysical and Ion Channel Functional Characterization of the Torpedo californica Nicotinic Acetylcholine Receptor in Varying Detergent–Lipid Environments
- 1 May 2008
- journal article
- research article
- Published by Springer Science and Business Media LLC in The Journal of Membrane Biology
- Vol. 223 (1), 13-26
- https://doi.org/10.1007/s00232-008-9107-7
Abstract
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 (3α,7α,12α-trihydroxy-5β-cholan-24-oic acid) maintaining stability and supporting ion channel function, and non-lipid-analogue 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) 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.Keywords
This publication has 27 references indexed in Scilit:
- Towards automated screening of two-dimensional crystalsJournal of Structural Biology, 2007
- Automated molecular microscopy: The new Leginon systemJournal of Structural Biology, 2005
- Refined Structure of the Nicotinic Acetylcholine Receptor at 4Å ResolutionJournal of Molecular Biology, 2004
- Emerging structure of the Nicotinic Acetylcholine receptorsNature Reviews Neuroscience, 2002
- Rapid preparation of the nicotinic acetylcholine receptor for crystallization in detergent solutionFEBS Letters, 1988
- A minimum number of lipids are required to support the functional properties of the nicotinic acetylcholine receptorBiochemistry, 1988
- Reconstitution of acetylcholine receptor function in lipid vesicles of defined compositionBiochimica et Biophysica Acta (BBA) - Biomembranes, 1983
- Activation and inactivation kinetics of Torpedo californica acetylcholine receptor in reconstituted membranesBiochemistry, 1982
- Functional Equivalence of Monomeric and Dimeric Forms of Purified Acetylcholine Receptors from Torpedo californica in Reconstituted Lipid VesiclesEuropean Journal of Biochemistry, 1980
- A RAPID METHOD OF TOTAL LIPID EXTRACTION AND PURIFICATIONCanadian Journal of Biochemistry and Physiology, 1959