Effects of Lipid-Analog Detergent Solubilization on the Functionality and Lipidic Cubic Phase Mobility of the Torpedo californica Nicotinic Acetylcholine Receptor
Over the past three decades, the Torpedo californica nicotinic acetylcholine receptor (nAChR) has been one of the most extensively studied membrane protein systems. However, the effects of detergent solubilization on nAChR stability and function are poorly understood. The use of lipid-analog detergents for nAChR solubilization has been shown to preserve receptor stability and functionality. The present study used lipid-analog detergents from phospholipid-analog and cholesterol-analog detergent families for solubilization and affinity purification of the receptor and probed nAChR ion channel function using planar lipid bilayers (PLBs) and stability using analytical size exclusion chromatography (A-SEC) in the detergent-solubilized state. We also examined receptor mobility on the lipidic cubic phase (LCP) by measuring the nAChR mobile fraction and diffusion coefficient through fluorescence recovery after photobleaching (FRAP) experiments using lipid-analog and non-lipid-analog detergents. Our results show that it is possible to isolate stable and functional nAChRs using lipid-analog detergents, with characteristic ion channel currents in PLBs and minimal aggregation as observed in A-SEC. Furthermore, fractional mobility and diffusion coefficient values observed in FRAP experiments were similar to the values observed for these parameters in the recently LCP-crystallized β2-adrenergic receptor. The overall results show that phospholipid-analog detergents with 16 carbon acyl-chains support nAChR stability, functionality and LCP mobility.
KeywordsDetergent nAChR Lipidic cubic phase FRAP Fluorescence recovery Planar lipid bilayer
This work was supported by National Institutes of Health (NIH) grants 2RO1GM56371-12, 5T34GM07821-31, Minority Access to Research Careers (MARC) and 2R25GM061151; Research Initiative for Scientific Enhancement (RISE); University of Puerto Rico Río, Piedras Campus Institutional Funds for Research; and the Specialized Neurosciences Research Program (SNRP) U54NS0433011. We acknowledge the contribution to this study by grants 1S10RR 13705-01 and DBI-0923132 to establish and upgrade the Confocal Microscopy Facility at the University of Puerto Rico (CIF-UPR) and the University of Puerto Rico Institutional Funds. We thank Dr. Raymond Stevens for access to the A-SEC instrumentation, Dr. Vadim Cherezov for expert advice in FRAP, Dr. Hernán Martínez for his commentaries about FRAP data analysis and Manuel Delgado-Vélez for critical reading.
- Asmar-Rovira GA, Asseo-García AM, Quesada O, Hanson MA, Cheng A, Nogueras C, Lasalde-Dominicci JA, Stevens RC (2008) Biophysical and ion channel functional characterization of the Torpedo californica nicotinic acetylcholine receptor in varying detergent–lipid environments. J Membr Biol 223:13–26PubMedCrossRefGoogle Scholar
- Guzman GR, Santiago J, Ricardo A, Marti-Arbona R, Rojas LV, Lasalde-Dominicci JA (2003) Tryptophan scanning mutagenesis in the alphaM3 transmembrane domain of the Torpedo californica acetylcholine receptor: functional and structural implications. Biochemistry 42:12243–12250PubMedCrossRefGoogle Scholar
- McArdle PF, Rutherford S, Mitchell BD, Damcott CM, Wang Y, Ramachandran V, Ott S, Chang YP, Levy D, Steinle N (2008) Nicotinic acetylcholine receptor subunit variants are associated with blood pressure; findings in the Old Order Amish and replication in the Framingham Heart Study. BMC Med Genet 9:67PubMedCrossRefGoogle Scholar
- Paas Y, Cartaud J, Recouvreur M, Grailhe R, Dufresne V, Pebay-Peyroula E, Landau EM, Changeux JP (2003) Electron microscopic evidence for nucleation and growth of 3D acetylcholine receptor microcrystals in structured lipid detergent matrices. Proc Natl Acad Sci USA 100:11309–11314PubMedCrossRefGoogle Scholar