ATPase N-ethylmaleimide-sensitive factor mediated calcium dependent fusion pore closure and endocytosis

For a long time, it remained an unresolved question how the fusion pore is regulated to close after fusion occurs during kiss-and-run. Our study focuses on the physiological function of NSF in modulating single vesicle fusion events in live cells, using whole-cell capacitance recording coupled with live-cell confocal imaging. Our results demonstrates that in both male Sprague-Dawley rat adrenal chromaffin endocrine cells and dorsal root ganglion neurons, inhibiting or diminishing the ATPase activity of NSF significantly impedes fusion pore closure, inhibits various forms of endocytosis, and disrupts the replenishment of readily releasable pool. Interestingly, NSF is exclusively required for calcium-dependent exo-endocytosis but not for the calcium-independent process. In response to calcium influx, NSF disassembles the trans-SNARE complex intermediate via its ATPase activity, which regulates fusion pore closure and subsequently mediates slow, fast, and overshoot endocytosis. Our findings revealed a key mechanism for fusion pore regulation in calcium-evoked exo-endocytosis coupling. N-ethylmaleimide-sensitive factor (NSF) mediated disassembly of trans-SNARE complexes is identified as the mechanism regulating fusion pore closure. This activity is critical for Ca²⁺dependent exo-endocytosis coupling and vesicle pool maintenance.