Assembly and clustering of acetylcholine receptors containing GFP-tagged ε or γ subunits: Selective targeting to the neuromuscular junction in vivo

Acetylcholine receptor (AChR) γ and ε subunits were tagged by green fluorescent protein (GFP) to analyse assembly and targeting in live muscle fibers at the neuromuscular junction. N- or C-terminal fusion polypeptides showed no fluorescence upon transfection of HEK cells. When GFP was inserted into the cytoplasmic loop connecting putative transmembrane regions M3 and M4, the γ/GFP and ε/GFP subunits were fluorescent and formed together with the α, β, and δ subunits GFP-tagged AChR complexes that were integrated into the plasma membrane. As the AChR were also clustered by rapsyn, the results indicate that the cytoplasmatic domains of the γ and ε subunits may not be required for assembly and rapsyn-dependent clustering. The γ/GFP and ε/GFP subunit-containing receptors were expressed in X. laevis oocytes and have affinities for acetylcholine similar to that of the wild-type receptors. Direct gene transfer into single muscle fibers reveals that γ/GFP or ε/GFP polypeptides are expressed at the site of injection and are transported within the endoplasmatic reticulum. When reaching subsynaptic regions, both γ/GFP or ε/GFP subunits compete with endogenous ε subunits to assemble GFP-tagged receptors, which are selectively targeted to the postsynaptic membrane.