Phosphomimetic Mutation of Cysteine String Protein-α Increases the Rate of Regulated Exocytosis by Modulating Fusion Pore Dynamics in PC12 Cells

Background: Cysteine string protein-alpha (CSP alpha) is a chaperone to ensure protein folding. Loss of CSP alpha function associates with many neurological diseases. However, its function in modulating regulated exocytosis remains elusive. Although csp alpha-knockouts exhibit impaired synaptic transmission, overexpression of CSP alpha in neuroendocrine cells inhibits secretion. These seemingly conflicting results lead to a hypothesis that CSP alpha may undergo a modification that switches its function in regulating neurotransmitter and hormone secretion. Previous studies implied that CSP alpha undergoes phosphorylation at Ser(10) that may influence exocytosis by altering fusion pore dynamics. However, direct evidence is missing up to date. Methodology/Principal Findings: Using amperometry, we investigated how phosphorylation at Ser(10) of CSP alpha (CSP alpha-Ser(10)) modulates regulated exocytosis and if this modulation involves regulating a specific kinetic step of fusion pore dynamics. The real-time exocytosis of single vesicles was detected in PC12 cells overexpressing control vector, wild-type CSP alpha (WT), the CSP alpha phosphodeficient mutant (S10A), or the CSP alpha phosphomimetic mutants (S10D and S10E). The shapes of amperometric signals were used to distinguish the full-fusion events (i.e., prespike feet followed by spikes) and the kiss-and-run events (i.e., square-shaped flickers). We found that the secretion rate was significantly increased in cells overexpressing S10D or S10E compared to WT or S10A. Further analysis showed that overexpression of S10D or S10E prolonged fusion pore lifetime compared to WT or S10A. The fraction of kiss-alpha nd-run events was significantly lower but the frequency of full-fusion events was higher in cells overexpressing S10D or S10E compared to WT or S10A. Advanced kinetic analysis suggests that overexpression of S10D or S10E may stabilize open fusion pores mainly by inhibiting them from closing. Conclusions/Significance: CSP alpha may modulate fusion pore dynamics in a phosphorylation-dependent manner. Therefore, through changing its phosphorylated state influenced by diverse cellular signalings, CSP alpha may have a great capacity to modulate the rate of regulated exocytosis.