Cholesterol dependence of acetylcholine receptor stability at the cell surface

BORRONI M.V; BARRANTES, FRANCISCO

Foz do Iguaçu ? Brazil

Simposio; XIII International Symposium on Cholinergic Mechanisms; 2008

Introduction. Stability of the nicotinic acetylcholine receptor (AChR) at the cell membrane is important in determining cholinergic response and can be altered under pathological conditions. Cholesterol (Chol) is necessary for ion translocation and formation and maintenance of AChR clusters (reviewed in Barrantes, 2007). AChR internalization proceeds via a novel endocytic pathway in a cell line heterologously expressing adult muscle-type receptor, CHO-K1/A5 (Kumari et al, 2008). Chol depletion (Chol) accelerates AChR endocytosis in these cells (Borroni et al., 2007). Here we explore the possible mechanism leading to enhanced receptor endocytosis. Methods. CHO-K1/A5 cells were grown as in Roccamo et al. (1999), transiently transfected using Lipofectamine, and used for experiments 12 h after transfection. Cell-surface AChR labeling was carried out by incubation of the cells with fluorescent-BTX for 1 h on ice. Cells were examined with a Nikon Eclipse E-600 microscope coupled with a CCD camera. Results. Transient overexpression of dominant negative mutants of dynamin (dyn-K44A) or Rac1 (Rac1N17) under Chol deprivation showed that receptor internalization in Chol- cells is dynamin-independent and Rac1-dependent, as in normal cells. Cells were analyzed for the possible involvement of the actin cytoskeleton. The Chol-dependent accelerated AChR endocytosis was not affected upon disruption of the actin fibers. Transient transfection with a dominant negative mutant of Arf6 delayed the otherwise accelerated internalization of cell-surface receptors. Discussion. These results highlight the importance of Chol in modulating AChR endocytosis and thus contributing to the maintenance of functional receptor levels at the cell surface. The novel endocytic pathway recently described in CHO-K1/A5 cells (Kumari, Borroni et al., 2008) Chol depletion accelerates AChR internalization (Borroni et al., 2007) via. Taken together, this series of experiments showed that AChR endocytosis becomes sensitive to Arf6 activity under conditions of low Chol levels.