Transient Cholesterol Effects on Nicotinic Acetylcholine Receptor Cell-Surface Mobility

To what extent do cholesterol-rich lipid platforms modulate the supramolecular organization of the nicotinic acetylcholine receptor (AChR)? To address this question, the dynamics of AChR particles at high density and its cholesterol dependence at the surface of mammalian cells were studied by combining total internal reflection fluorescence microscopy and single-particle tracking. AChR particles tagged with a monovalent ligand, fluorescent alpha-bungarotoxin (alpha BTX), exhibited two mobile pools: i) a highly mobile one undergoing simple Brownian motion (16%) and ii) one with restricted motion (similar to 50%), the rest being relatively immobile (similar to 44%). Depletion of membrane cholesterol by methyl-alpha-cyclodextrin increased the fraction of the first pool to 22% and 33% after 15 and 40 min, respectively; the pool undergoing restricted motion diminished from 50% to 44% and 37%, respectively. Monoclonal antibody binding results in AChR crosslinking-internalization after 2 h; here, antibody binding immobilized within minutes similar to 20% of the totally mobile AChR. This proportion dramatically increased upon cholesterol depletion, especially during the initial 10 min (83.3%). Thus, antibody crosslinking and cholesterol depletion exhibited a mutually synergistic effect, increasing the average lifetime of cell-surface AChRs similar to 10 s to similar to 20 s. The instantaneous (microscopic) diffusion coefficient D2-4 of the AChR obtained from the MSD analysis diminished from similar to 0.001 mu m(2) s(-1) to similar to 0.0001-0.00033 mu m(2) s(-1) upon cholesterol depletion, similar to 30% of all particles falling into the stationary mode. Thus, muscle-type AChR exhibits heterogeneous motional regimes at the cell surface, modulated by the combination of intrinsic (its supramolecular organization) and extrinsic (membrane cholesterol content) factors.