Contact-dependent aggregation of functional Ca2+ channels, synaptic vesicles and postsynaptic receptors in active zones of a neuromuscular junction

To examine whether Ca2+ channels aggregate in a contact-dependent manner, w e characterized the distribution of synaptic vesicles and postsynaptic rece ptors, and compared it to the location of Ca2+ entry sites, in a Xenopus la evis nerve-muscle coculture preparation using a localized Ca2+ detection me thod. The majority (75%) of Ca2+ entry sites at spontaneously formed nerve- muscle contacts were associated with enhanced immunofluorescence to the syn aptic vesicle protein, SV2. In contrast, only 11% of recorded sites without Ca2+ transients exhibited significant SV2 immunofluorescence. When compari ng the spatial distribution of synaptic markers with that of Ca2+ entry sit es, we found that the majority of Ca2+ entry sites (61%) were associated wi th both enhanced SV2 immunofluorescence and R-BTX fluorescence, thereby ide ntifying putative neurotransmitter release sites where Ca2+ channels, synap tic vesicles and postsynaptic receptors are colocalized. Using polystyrene beads coated with a heparin binding protein known to mediate in vitro posts ynaptic receptor clustering, we show that the location of Ca2+ domains was associated with enhanced SV2 immunofluorescence at neurite-to-bead contacts . We conclude that the localization of functional Ca2+ channels to putative active zones follows a contact-dependent signalling mechanism similar to t hat known to mediate vesicle aggregation and AChR clustering.