Cholesterol and glycosphingolipid modulation of cellsurface

BARRANTES FRANCISCO,; BAIER JAVIER,; BONINI IDA,; GALLEGOS CRISTINA,; PEDICONI MARIA,; THORSTEN LANG,; GARBUS INGRID,; DE LOS SANTOS, BEATRIZ; PICARDI VICTORIA

no se

Congreso; Annual Meeting of the International Society for Neurochemistry; 2003

International Society for Neurochemistry

Lipid domains can affect membrane functions by concentrating signaling molecules and receptors in particular regions of the cell surface. We investigated the effects of acute and chronic cholesterol( Chol)and glycosphingolipid modification on the distribution of the AChR and lipid domains in model non-polarized cells. Changes in AChR distribution at the cell surface were followed by fluorescence microscopy. Cyclodextrin-mediated Chol depletion reduced a-bungarotoxin fluorescence at the surface of intact cells and in plasma membrane sheets. Inhibition of glycosphingolipid biosynthesis by selective agents (FB-1, DL-PDMP or ISP-1) in a control cell line, CHO-K1/A5, also modified the AChR-associated fluorescence. Lipid (domain) fluorescence did not colocalize with AChR-associated fluorescence in membrane sheets. The latter was concentrated in small, well-resolved submicron-sized fluorescent spots that we interpret as AChR microdomains constituted by small numbers of receptor molecules, analogous to those found in preinnervation stages along neuromuscular junction development. We were also able to resolve the functional consequences of Chol modification in living mammalian cells at the single-channel level using patch-clamp recording. Chol depletion caused a gain-offunction, whereas Chol enrichment decreased channel mean open time, indicating that Chol and glycosphingolipid content are regulatory elements that finely tune receptor targetting and function.