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

BORRONI M.V; KUMARI S; A. CHAUDHRY; B. CHANDA; R. MASSOL; S. MAYOR; BARRANTES, FRANCISCO

Buzios

Congreso; Ier CONGRESO IBRO/LARC DE NEUROCIENCIAS DA AMERICA LATINA, CARIBE E PENÍNSULA IBÉRICA; 2008

IBRO/LARC

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 (BTX) or monoclonal antibodies against the á subunit. Cells were imaged using a combination of wide-field, confocal, and TIRF microscopies. Results. Binding of BTX 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-BTX complexes in narrow, tubular, surface-connected compartments, as shown by differential surface-accessibility of fluorescently-tagged BTX-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. BTX-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.