Cholesterol effects on muscletype nicotinic acetylcholine receptor distribution and dynamics studied with single-molecule STORM microscopy and Bayesian analysis

SANCHEZ FRANCISCO; PABLO CAMINO; ALEJO MOSQUIERA; BARRANTES FRANCISCO JOSE

Buenos Aires

Congreso; 2nd Congress of the Federation of Latin-American and Caribbean Societies for Neuroscience (FALAN); 2016

Federation of Latin-American and Caribbean Societies for Neuroscience (FALAN)

The lipid microenvironment and the neutral lipid cholesterolin particular, have been shown to exert modulatory effectson the supramolecular organization and dynamics ofthe neurotransmitter receptor for acetylcholine (nAChR)(Barrantes, Regulation of the nicotinic acetylcholinereceptor by cholesterol as a boundary lipid, In: CholesterolRegulation of Ion Channels and Receptors, John Wiley& Sons. pp. 183-204 (2012)). Adult muscle-type nAChRsheterologously expressed in the CHO-K1/A5 cell line werestudied using single-molecule superresolution microscopyunder various experimental conditions involvingacute cyclodextrin-mediated cholesterol depletion orenrichment. Cells were labeled either with a monovalentligand, fluorescent Alexa Fluor555 alpha-bungarotoxin(BTX), or the monoclonal antibody mAb35 against thenAChR alpha1 subunit (mAb) followed by Texas Red-labeledsecondary antibodies. In both cases nanometer-sizedsingle molecules and aggregates thereof could be imagedwith nanometer precision and the number of localizations,their density and other structural parameters as well as thesingle-particle trajectories, mean square displacements(MSD) and apparent diffusion coefficients of the nAChRat the cell surface measured within minutes of changes incholesterol levels. We conclude that cholesterol levels affectthe distribution and dynamics of the nAChR at the cellsurface.