| SNAP-25 and synaptotagmin are key components involved in regulated exocytosis. We performed chemical cross-linking studies and found that synaptotagmins I and IX associate with SNAP-25 during Ca2+-dependent exocytosis in PC12 cells. In addition, we identified C-terminal amino acids in SNAP-25 (Asp179, Asp186, Asp193) that are required for the Ca2+-dependent synaptotagmin binding. To study the functionality of SNAP-25, we developed a replacement assay that applies botulinum neurotoxin (BoNT) type E to inactivate endogenous SNAP-25 in PC12 cells and replaces with a BoNT E-resistant version. Replacement of SNAP-25 in PC12 cells with SNAP-25 containing C-terminal Asp mutations led to a loss-of-function in regulated exocytosis at the Ca2+-dependent fusion step. These results indicate that the Ca2+-dependent interaction of synaptotagmin with SNAP-25 is essential for the Ca2+ -dependent triggering of membrane fusion.; To further investigate the functional role of synaptotagmin, a proposed Ca2+ sensor, we utilized antisense RNA to generate a PC12 cell line lacking synaptotagmin I. This cell line exhibited normal dense-core vesicle biogenesis and distribution, but showed deficiency in norepinephrine uptake through the plasma membrane transporter, indicating that synaptotagmin I may regulate the trafficking of the neurotransmitter transporter. PC12 cells lacking synaptotagmin I did not affect the Ca2+ sensitivity of regulated exocytosis, suggesting that there is an alternative Ca2+ sensor. Moreover, PC12 cells lacking synaptotagmin I demonstrated enhanced Ca 2+-dependent exocytosis and overexpression of synaptotagmin I in this cell line restored regulated exocytosis to the same level as wild-type PC12 cells, indicating a possible negative role of synaptotagmin I in regulated exocytosis. Therefore, PC12 cell line lacking synaptotagmin I may be a useful tool to study regulated exocytosis. |
