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

Biophysical and Ion Channel Functional Characterization of the Torpedo californica Nicotinic... 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. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The Journal of Membrane Biology Springer Journals

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

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Publisher
Springer-Verlag
Copyright
Copyright © 2008 by Springer Science+Business Media, LLC
Subject
Life Sciences; Human Physiology ; Biochemistry, general
ISSN
0022-2631
eISSN
1432-1424
D.O.I.
10.1007/s00232-008-9107-7
Publisher site
See Article on Publisher Site

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.

Journal

The Journal of Membrane BiologySpringer Journals

Published: Jun 25, 2008

References

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