Da. Digregorio et al., Contact-dependent aggregation of functional Ca2+ channels, synaptic vesicles and postsynaptic receptors in active zones of a neuromuscular junction, EUR J NEURO, 14(3), 2001, pp. 533-546
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.