Journal of Membrane Biology

, Volume 223, Issue 1, pp 13–26 | Cite as

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

  • Guillermo A. Asmar-Rovira
  • Aloysha M. Asseo-García
  • Orestes Quesada
  • Michael A. Hanson
  • Anchi Cheng
  • Carlos Nogueras
  • José A. Lasalde-Dominicci
  • Raymond C. Stevens
Article

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

nAChR Detergent Ion channel function Lipid Planar lipid bilayer Biophysical characterization 

Supplementary material

232_2008_9107_MOESM_ESM.doc (82 kb)
(DOC 82 kb)

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Copyright information

© Springer Science+Business Media, LLC 2008

Authors and Affiliations

  • Guillermo A. Asmar-Rovira
    • 1
  • Aloysha M. Asseo-García
    • 2
  • Orestes Quesada
    • 2
  • Michael A. Hanson
    • 1
  • Anchi Cheng
    • 1
  • Carlos Nogueras
    • 2
  • José A. Lasalde-Dominicci
    • 2
  • Raymond C. Stevens
    • 1
  1. 1.Departments of Cell Biology and Molecular BiologyThe Scripps Research InstituteLa JollaUSA
  2. 2.Departments of Biology, Chemistry and Physical ChemistryUniversity of Puerto RicoRío PiedrasPuerto Rico

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