Deficiency of PKA-mediated SNAP-25b phosphorylation destabilizes exocytotic fusion pores and reduces the interactions of t-SNAREs.

Abstract

Ca²⁺-dependent exocytosis initiates with the formation of fusion pores comprising the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex. Although cellular signalling typically occurs in transient oscillations on the order of tens of seconds, it remains unclear how such rapid SNARE phosphorylation influences fusion pore kinetics, analogous to transient regulation observed in ion channels. Here we demonstrate that protein kinase A (PKA)-mediated phosphorylation of SN25b (the neuronal isoform of synaptosome-associated protein of 25 kD) modulates secretion rate and fusion pore kinetics in PC12 cells (rat pheochromocytoma derivatives). Upon acute application of KCl and forskolin, cells overexpressing SN25b exhibited a reduced secretion rate compared to the control. This reduction was occluded by overexpressing a PKA-phosphodeficient mutant, SN25b-T138A, rather than a PKA-phosphomimetic mutant, SN25b-T138E. Notably, SN25b, SN25b-T138A or SN25b-T138E did not alter the fraction of incomplete fusion events or quantal size compared to the control. Further kinetic analysis indicated that SN25b-T138A destabilized initial fusion pores by promoting the closure and dilatation of fusion pores. Mechanistically, in situ proximity ligation assays showed that SN25b-T138A reduced its interaction with the other t-SNARE syntaxin-1 compared to the control and SN25b, correlating with destabilized fusion pores. Moreover, compared to SN25b-T138E, SN25b-T138A decreased whole-cell Ca²⁺ currents and weakened its interaction with synaptobrevin-2 and L-type Ca²⁺ channel subunits. These changes in interaction were associated with increased secretion and full-fusion rate, implying efficient disassembly after dilatation. Together, PKA-mediated phosphorylation of SN25b rapidly modulates fusion pore kinetics in response to transient signalling oscillations, thereby fine-tuning exocytotic efficiency in real time. KEY POINTS: Protein kinase A (PKA)-mediated SNAP-25 phosphorylation rapidly reduces the rate of secretion. PKA-phosphodeficiency of SNAP-25 destabilizes the kinetics of initial fusion pores, correlating with its decreased interaction with syntaxin-1. PKA-phosphodeficiency of SNAP-25 decreases the interaction with synaptobrevin-2 and the L-type calcium channel subunit, leading to efficient priming. PKA-mediated SNAP-25 phosphorylation rapidly regulates fusion pore kinetics and shapes exocytotic kinetics on the order of tens of seconds.