Abstract
ComplexinII (CpxII) inhibits non-synchronized vesicle fusion, but the underlying mechanisms have remained unclear. Here, we provide evidence that the far C-terminal domain (CTD) of CpxII interferes with SNARE assembly, thereby arresting tonic exocytosis. Acute infusion of a CTD-derived peptide into mouse chromaffin cells enhances synchronous release by diminishing premature vesicle fusion like full-length CpxII, indicating a direct, inhibitory function of the CTD that sets the magnitude of the primed vesicle pool. We describe a high degree of structural similarity between the CpxII CTD and the SNAP25-SN1 domain (C-terminal half) and show that the CTD peptide lowers the rate of SDS-resistant SNARE complex formation in vitro. Moreover, corresponding CpxII:SNAP25 chimeras do restore complexin's function and even 'superclamp' tonic secretion. Collectively, these results support a so far unrecognized clamping mechanism wherein the CpxII C-terminus hinders spontaneous SNARE complex assembly, enabling the build-up of a release-ready pool of vesicles for synchronized Ca2+-triggered exocytosis.
Highlights
The accumulation of vesicles in a release-ready state is essential for fast transmitter release from secretory cells
Using viral expression of a truncated Cpx-variant or acute infusion of an isolated C-terminal peptide in wt chromaffin cells, we show that the C-terminal domain (CTD) of CpxII is essential and rate-limiting for hindering premature fusion and for augmenting a pool of primed vesicles
We show that the CTD of CpxII maintains tight control over premature vesicles exocytosis to support a pool of release-ready vesicles
Summary
The accumulation of vesicles in a release-ready state is essential for fast transmitter release from secretory cells. The N-terminus of Cpx accelerates evoked release in murine neurons and neuroendocrine cells (Dhara et al, 2014; Maximov et al, 2009; Xue et al, 2007) by increasing the Ca2+-affinity of synchronous secretion, but has no effect at the neuromuscular junction (NMJ) of C. elegans (Hobson et al, 2011; Martin et al, 2011). The accessory a-helix, instead, has been shown to play an inhibitory action in in vivo studies (Cho et al, 2014; Martin et al, 2011; Maximov et al, 2009; Trimbuch et al, 2014; Xue et al, 2007; Yang et al, 2010). A variety of different models for the inhibition by the accessory a-helix have been proposed including direct binding to SNAREs or other proteins (Bykhovskaia et al, 2013; Cho et al, 2014; Giraudo et al, 2009; Krishnakumar et al, 2011; Kummel et al, 2011; Lu et al, 2010; Yang et al, 2010), electrostatic membrane interactions
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