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.