Synaptotagmin III在PC12中 藉由鈣離子結合至C2AB區塊調控胞吐作用的動態變化

Synaptotagmin (Syt) protein family consists of at least seventeen isoforms. Most of them have been reported as vesicular Ca2+ sensors by Ca2+ binding to their C2AB domains. For example, Syt I plays a role in regulating the kinetics of vesicular exocytosis. However, the functions of other isoforms remain to be identified. Previously, we found that Syt III is upregulated in retinal output neurons (retinal ganglion cells, RGCs) and optic nerves during a critical period (postnatal day P6 in rats) of visual circuit development. However, how Syt III functions in regulated exocytosis remains unclear. Here, we investigated if Syt III involves in Ca2+-regulated exocytosis by Ca2+ binding to its C2AB domains. We used PC12 cells as the model to examine Syt III’s function in regulated exocytosis, by combining molecular perturbation, immunofluorescence staining, and amperometry (a single-vesicle technique to measure real-time exocytosis). By mutating aspartate to asparagine at the site 386 and 520 of Syt III, we blocked the Ca2+ binding ability in the C2AB domains (D386, 520N, designated Syt III-C2AB*). After overexpressing control vector, wild-type Syt III, or Syt III-C2AB* in PC12 cells, we analyzed single exocytotic events by amperometry. We found that Syt III was poorly colocalized with dense-core vesicles (DCVs) but localized to plasma membrane. Moreover, overexpressing Syt III increased secretion rate after long-time stimulus compared to control or Syt III-C2AB*, suggesting that Syt III may involve in vesicle recruitment by Ca2+ binding to its C2AB domains. From co-immunoprecipitation, we found that binding to Ca2+ can enhance the interaction between Syt III and SN25, accounting for a decrease in the prespike foot (PSF) duration. These results suggested that in contrast to the conventional role of Syt family, Syt III functions as a “plasma membrane” Ca2+ sensor in modulating the kinetics of regulated exocytosis, via Ca2+ binding to its C2AB domains.