Calcium Regulation of Exocytosis in PC12 Cells

The calcium (Ca²⁺) regulation of neurotransmitter release is poorly understood. 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 vesicles were docked at the plasma membrane. Second, we further investigated the mechanism of rescue of botulinum neurotoxin E inhibition of release by recombinant SNAP-25 COOH-terminal coil, which is known to be required in the triggering stage. We confirmed here that Ca²⁺ was required simultaneously with 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 triggering, suggesting that SNARE (soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor) complex assembly was involved in the final Ca²⁺-triggered event. Using this rescue system, we also identified a series of acidic surface SNAP-25 residues that rescued better than wild-type when mutated, due to broadened Ca ²⁺ sensitivity, suggesting that this charged patch may interact electrostatically with a negative regulator of membrane 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 release.