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.
DiGregorio
D A
DA
Department of Physiology, UCLA School of Medicine, Los Angeles, CA 90095, USA.
Negrete
O
O
Jeromin
A
A
Peng
H B
HB
Vergara
J L
JL
D000818:Animals; D015220:Calcium Channels / Q000502:physiology*; D002450:Cell Communication / Q000502:physiology; D004396:Coloring Agents; D019085:Fluorescent Antibody Technique, Direct; D005456:Fluorescent Dyes; D007091:Image Processing, Computer-Assisted; D007150:Immunohistochemistry; D008856:Microscopy, Fluorescence; D009469:Neuromuscular Junction / Q000502:physiology*; D009865:Oocytes / Q000378:metabolism; D011950:Receptors, Cholinergic / Q000187:drug effects / Q000378:metabolism; D017981:Receptors, Neurotransmitter / Q000502:physiology*; D013572:Synaptic Vesicles / Q000502:physiology*; D014981:Xenopus
D015220:Calcium Channels; D004396:Coloring Agents; D005456:Fluorescent Dyes; D011950:Receptors, Cholinergic; D017981:Receptors, Neurotransmitter