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¹ National Centre for Biological Sciences, Tata Institute of Fundamental Research, Bangalore 560 065, India
² Instituto de Investigaciones Bioquímicas de Bahía Blanca, United Nations Educational, Scientific, and Cultural Organization Chair of Biophysics and Molecular Neurobiology, Bahia Blanca B8000FWB, Argentina
³ National Institute of Immunology, New Delhi 110067, India

Correspondence to Satyajit Mayor: mayor{at}ncbs.res.in

Endocytosis of the nicotinic acetylcholine receptor (AChR) is a proposed major mechanism of neuromodulation at neuromuscular junctions and in the pathology of synapses in the central nervous system. We show that binding of the competitive antagonist -bungarotoxin (BTX) or antibody-mediated cross-linking induces the internalization of cell surface AChR to late endosomes when expressed heterologously in Chinese hamster ovary cells or endogenously in C2C12 myocytes. Internalization occurs via sequestration of AChR–BTX complexes in narrow, tubular, surface-connected compartments, which are indicated by differential surface accessibility of fluorescently tagged BTX–AChR complexes to small and large molecules and real-time total internal reflection fluorescence imaging. Internalization occurs in the absence of clathrin, caveolin, or dynamin but requires actin polymerization. BTX binding triggers c-Src phosphorylation and subsequently activates the Rho guanosine triphosphatase Rac1. Consequently, inhibition of c-Src kinase activity, Rac1 activity, or actin polymerization inhibits internalization via this unusual endocytic mechanism. This pathway may regulate AChR levels at ligand-gated synapses and in pathological conditions such as the autoimmune disease myasthenia gravis.

S. Kumari and V. Borroni contributed equally to this paper.

A. Chaudhry's present address is Dept. of Immunology, University of Washington School of Medicine, Seattle, WA 98195.

B. Chanda's present address is Dept. of Physiology, University of Wisconsin, Madison, WI 53706.

R. Massol's present address is GI Cell Biology Laboratories, Children's Hospital Boston, Harvard Medical School, Boston, MA 02115.

Abbreviations used in this paper: BTX, -bungarotoxin; AChR, acetylcholine receptor; CNS, central nervous system; EEA1, early endosomal antigen 1; GPI, glycosyl-phosphatidylinositol; LAMP, lysosomal-associated membrane protein; NMJ, neuromuscular junction; Pak, p21-associated kinase; PBD, Pak-binding domain; PE, phycoerythrin; SA, streptavidin; Tf, transferrin; TfR, Tf receptor; TIRF, total internal reflection fluorescence.

© 2008 Kumari et al. This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.jcb.org/misc/terms.shtml). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).

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