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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, we characterized the distribution of synaptic vesicles and postsynaptic receptors, and compared it to the location of Ca2+ entry sites, in a Xenopus laevis nerve‐muscle coculture preparation using a localized Ca2+ detection method. The majority (75%) of Ca2+ entry sites at spontaneously formed nerve–muscle contacts were associated with enhanced immunofluorescence to the synaptic vesicle protein, SV2. In contrast, only 11% of recorded sites without Ca2+ transients exhibited significant SV2 immunofluorescence. When comparing the spatial distribution of synaptic markers with that of Ca2+ entry sites, we found that the majority of Ca2+ entry sites (61%) were associated with both enhanced SV2 immunofluorescence and R‐BTX fluorescence, thereby identifying putative neurotransmitter release sites where Ca2+ channels, synaptic vesicles and postsynaptic receptors are colocalized. Using polystyrene beads coated with a heparin binding protein known to mediate in vitro postsynaptic 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 that known to mediate vesicle aggregation and AChR clustering.