Calcium regulation of exocytosis in PC12 cells

The calcium (Ca2+) regulation of neurotransmitter release is poorly underst ood. Here we investigated several aspects of this process in PC12 cells. We first showed that osmotic shock by 1 M sucrose stimulated rapid release of neurotransmitters from intact PC12 cells, indicating that most of the vesi cles were docked at the plasma membrane. Second, we further investigated th e mechanism of rescue of botulinum neurotoxin E inhibition of release by re combinant SNAP-25 COOH-terminal coil, which is known to be required in the triggering stage. We confirmed here that Ca2+ was required simultaneously w ith the SNAP-25 peptide, with no significant increase in release if either the peptide or Ca2+ was present during the priming stage as well as the tri ggering, suggesting that SNARE (soluble N-ethylmaleimide-sensitive fusion p rotein attachment protein receptor) complex assembly was involved in the fi nal Ca2+-triggered event. Using this rescue system, we also identified a se ries of acidic surface SNAP-25 residues that rescued better than wild-type when mutated, due to broadened Ca2+ sensitivity, suggesting that this charg ed patch may interact electrostatically with a negative regulator of membra ne fusion, Finally, we showed that the previously demonstrated stimulation of exocytosis in this system by calmodulin required calcium binding, since calmodulin mutants defective in Ca2+-binding were not able to enhance relea se.