Nanoscale cluster organization of the acetylcholine receptor: cholesterol comes of age

KELLNER, ROBERT R; BAIER, CARLOS J; BORRONI, VIRGINIA; WILLIG, KATRIN; HELL, STEPHAN W; BARRANTES F.J

Baltimore

Congreso; 51st Annual Meeting of the Biophysical-Society; 2007

Biophysical Society

The effect of cholesterol (Chol) on acetylcholine receptor (AChR) supramolecular organization was studied by a combination of light microscopy approaches. Wide-field and confocal microscopy reveal AChR submicron-sized (240-280 nm) domains that remain stable over a period of hours at the cell-surface membrane of CHO-K1/A5, a mammalian clonal cell line stably expressing adult murine AChR. Acute (30 min, 37°C) exposure to methyl-β-cyclodextrin (CDx), commonly used as a diagnostic tool of endocytic mechanisms, accelerated AChR internalization kinetics. Stimulated emission depletion (STED) fluorescence microscopy, providing resolution beyond the diffraction barrier, resolved the domains into AChR nanoclusters with a peak size distribution of ~55 nm. Chol depletion by CDx altered the short- and long-range organization of the AChR nanoclusters. In the short range, AChRs formed larger nanoclusters, possibly related to the alteration of Chol-dependent protein-protein associations. Ripley?s K-test on STED images disclosed changes in nanocluster distribution on larger scales (0.5-3.5 um), presumably due to abolition of cytoskeletal physical barriers normally preventing the lateral diffusion of AChR nanoclusters. Homeostatic control of Chol content at the plasmalemma may thus modulate cell-surface organization and stability of receptor domains, and fine-tune receptor channel function to temporarily compensate for acute AChR loss from the cell surface.