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-a (CSPa) is a chaperone to ensure protein folding. Loss of CSPa function associates with many neurological diseases. However, its function in modulating regulated exocytosis remains elusive. Although cspaknockouts exhibit impaired synaptic transmission, overexpression of CSPa in neuroendocrine cells inhibits secretion. These seemingly conflicting results lead to a hypothesis that CSPa may undergo a modification that switches its function in regulating neurotransmitter and hormone secretion. Previous studies implied that CSPa 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 CSPa (CSPa-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 CSPa (WT), the CSPa phosphodeficient mutant (S10A), or the CSPa 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-andrun 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-and-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: CSPa may modulate fusion pore dynamics in a phosphorylation-dependent manner. Therefore, through changing its phosphorylated state influenced by diverse cellular signalings, CSPa may have a great capacity to modulate the rate of regulated exocytosis.