Nicotinic Acetylcholine Receptor is Internalized via a Rac-dependent, Dynamin-Independent Endocytic Pathway

BORRONI, V.; KUMARI, S.; CHAUDHRY, A.; CHANDA, B.; MASSOL, R.; MAYOR, S.; BARRANTES, F.J.

Búzios, RJ, Brasil

Congreso; I Congresso IBRO/LARC de Neurociências da América Latina, Caribe e Península IbéricaBo; 2008

IBRO/LARC

Nicotinic Acetylcholine Receptor is Internalized via a Rac-dependent, Dynamin-Independent Endocytic Pathway Borroni M.V.1; Kumari S.2; A. Chaudhry3; B. Chanda4; R. Massol5; S. Mayor2; Barrantes, Francisco6 - 1INIBIBB - ; 2NCBS - Cell Biology; 3University of Washington School of Medicine, Seattle, USA - Dept of Immunology; 4University of Wisconsin, Madison, USA - Department of Physiology; 5Hospital Boston, Harvard Medical School - GI Cell Biology Laboratories; 6UNESCO Chair Biophys. & Mol. Neurobiol. - x Objectives. Endocytosis of the nicotinic acetylcholine receptor (AChR) is a proposed mechanism of neuromodulation at neuromuscular junctions and in the central nervous system. Here we characterize muscle-type AChR internalization in mammalian cells. Methods. To label AChR we used fluorescence derivatives of the competitive antagonist á-bungarotoxin (áBTX) or monoclonal antibodies against the á subunit. Cells were imaged using a combination of wide-field, confocal, and TIRF microscopies. Results. Binding of áBTX or antibody-mediated crosslinking induces the internalization of cell-surface AChR to late endosomes when expressed heterologously in CHO cells or endogenously in C2C12 myocytes. Internalization occurs via sequestration of AChR-áBTX complexes in narrow, tubular, surface-connected compartments, as shown by differential surface-accessibility of fluorescently-tagged áBTX-AChR complexes to small and large molecules, and real-time TIRF imaging. Internalization occurs in the absence of clathrin, caveolin or dynamin, but requires actin-polymerization. áBTX-binding triggers c-Src phosphorylation, and subsequently activates the Rho GTPase Rac1. Consequently, inhibition of c-Src kinase activity, Rac1 activity or actin polymerization inhibits internalization via this unusual endocytic mechanism. Conclusions. AChR is internalized via a novel, Rac-dependent, dynamin-independent endocytic pathway. This pathway may regulate AChR levels at ligand-gated synapses. Keywords: AChR endocytosis bungarotoxin Supported by: CONICET ANPCyT