Expansion of calcium microdomains regulates fast exocytosis at a ribbon synapse.
We investigated the Ca2+ signal regulating fast exocytosis at the ribbon synapse of retinal bipolar cells by using total internal reflection fluorescence microscopy to image fluorescent Ca2+ indicators and interference reflection microscopy to monitor exocytosis. Depolarization generated Ca2+ "microdomains" that expanded over the time scale during which the rapidly releasable pool (RRP) of vesicles was released (<40 ms). Replacing mobile Ca2+ buffers in the terminal with 10 mM EGTA prevented expansion of microdomains and decreased the number of rapidly releasable vesicles by a factor of 2. Conversely, decreasing the concentration of EGTA in the terminal to 0.1 mM increased the apparent width of a Ca2+ microdomain from 580 nm to 930 nm and increased the size of the RRP size by a factor of 1.5. The [Ca2+] over the area that the microdomain expanded was estimated to be 2-7 microM. These results indicate that vesicles within the RRP are located hundreds of nanometers from Ca2+ channels, and that fusion of these vesicles can be triggered by low micromolar levels of Ca2+. Variable distances between docked vesicles and Ca2+ channels at the active zone, therefore, provide an explanation for the heterogeneous release probability of vesicles comprising the RRP.
Beaumont
Vahri
V
Medical Research Council Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, United Kingdom.
Llobet
Artur
A
Lagnado
Leon
L
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D004533:Egtazic Acid; D004594:Electrophysiology; D005089:Exocytosis / Q000502:physiology*; D006054:Goldfish; D008856:Microscopy, Fluorescence; D008857:Microscopy, Interference; D051245:Retinal Bipolar Cells / Q000502:physiology*; D015398:Signal Transduction / Q000502:physiology*; D013569:Synapses / Q000502:physiology*; D013572:Synaptic Vesicles / Q000378:metabolism
D004533:Egtazic Acid; D002118:Calcium