Cholesterol modulation of nicotinic acetylcholine receptor surface mobility.
Nicotinic acetylcholine receptor (AChR) function and distribution are quite sensitive to cholesterol (Chol) levels in the plasma membrane (reviewed by Barrantes in J Neurochem 103 (suppl 1):72-80, 2007). Here we combined confocal fluorescence recovery after photobleaching (FRAP) and confocal fluorescence correlation spectroscopy (FCS) to examine the mobility of the AChR and its dependence on Chol content at the cell surface of a mammalian cell line. Plasma membrane AChR exhibited limited mobility and only ~55% of the fluorescence was recovered within 10 min after photobleaching. Depletion of membrane Chol by methyl-beta-cyclodextrin strongly affected the mobility of the AChR at the plasma membrane; the fraction of mobile AChR fell from 55 to 20% in Chol-depleted cells, whereas Chol enrichment by methyl-beta-cyclodextrin-Chol treatment did not reduce receptor mobility at the cell surface. Actin depolymerization caused by latrunculin A partially restored receptor mobility in Chol-depleted cells. In agreement with the FRAP data, scanning FCS experiments showed that the diffusion coefficient of the AChR was about 30% lower upon Chol depletion. Taken together, these results suggest that membrane Chol modulates AChR mobility at the plasma membrane through a Chol-dependent mechanism sensitive to cortical actin.
Baier
Carlos J
CJ
UNESCO Chair of Biophysics and Molecular Neurobiology, Instituto de Investigaciones Bioquímicas de Bahía Blanca, C.C. 857, B8000FWB, Bahía Blanca, Argentina.
Gallegos
Cristina E
CE
Levi
Valeria
V
Barrantes
Francisco J
FJ
D000199:Actins / Q000378:metabolism*; D000818:Animals; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000494:pharmacology; D016466:CHO Cells; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism*; D002784:Cholesterol / Q000378:metabolism*; D006224:Cricetinae; D003412:Cricetulus; D004058:Diffusion; D036681:Fluorescence Recovery After Photobleaching; D018613:Microscopy, Confocal; D009038:Motion; D055503:Protein Multimerization / Q000187:drug effects; D011978:Receptors, Nicotinic / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D053778:Thiazolidines / Q000494:pharmacology; D047392:beta-Cyclodextrins / Q000494:pharmacology
D000199:Actins; D019086:Bridged Bicyclo Compounds, Heterocyclic; D011978:Receptors, Nicotinic; D053778:Thiazolidines; D047392:beta-Cyclodextrins; C108732:methyl-beta-cyclodextrin; D002784:Cholesterol; C037067:latrunculin A