A Mechanism for Exocytotic Arrest by the Complexin C-Terminus

Abstract

Hindering premature vesicular fusion is key to build up a molecularly primed ready releasable pool. Complexin-II (CpxII), a 16 KD cytosolic protein, inhibits non-synchronous vesicle fusion at release sites, and by that accumulates a ready releasable pool of primed vesicles that are released in synchrony upon immediate elevation of intracellular calcium. Functionally, CpxII is equipped with four independent yet synergetic domains. A central helix mediates the CpxII-SNARE (N-ethylmaleimide-sensitive factor (NSF) attachment protein receptors) complex interaction, while an accessory helix is important for the stability of the central helix and for reducing premature fusion. The upstream N-terminal domain facilitates exocytosis, whereas the downstream C-terminal domain (CTD) inhibits premature fusion of vesicles. The molecular mechanisms how the CTD blocks asynchronous release are still unclear. With the help of high resolution membrane capacitance measurements coupled with calcium imaging, carbon fiber amperometry, and biochemical assays, we show that infusion of CpxII far CTD -derived peptide into mouse chromaffin cells is able to boost the synchronous release by reducing the premature vesicle fusion. A similar phenotype is also observed by over-expressing the full length WT protein. Using in vitro binding assays, we show that the CTD interacts with SNARE proteins. Moreover, we found that the CTD peptide lowers the rate of SDS resistant SNARE complex formation. We also show that the CTD shares a high degree of similarity to the SNARE protein SNAP25-SN1 domain. Furthermore chimeras of CpxII:SNAP25-SN1 fully restore CpxII function, and even “superclamp” tonic secretion. Collectively, these results provide evidence for a clamping mechanism in which the CTD of CpxII prevents the spontaneous assembly of SNARE complexes, enabling the build-up of a ready releasable pool for synchronised Ca2+-triggered exocytosis.