Interactions Between SNAP-25 and Synaptotagmin-1 Are Involved in Vesicle Priming, Clamping Spontaneous and Stimulating Evoked Neurotransmission

Abstract

Whether interactions between synaptotagmin-1 (syt-1) and the soluble NSF attachment protein receptors (SNAREs) are required during neurotransmission is debated. We examined five SNAP-25 mutations designed to interfere with syt-1 interactions. One mutation, D51/ E52/E55A, targeted negative charges within region II of the primary interface (Zhou et al., 2015); two mutations targeted region I (D166A and D166/E170A) and one mutation targeted both (D51/E52/E55/D166A). The final mutation (D186/D193A) targeted C-terminal residues not expected to interact with syt-1. An in vitro assay showed that the region I, region II, and region IϩII (D51/E52/E55/D166A) mutants markedly reduced the attachment between syt-1 and t-SNARE-carrying vesicles in the absence of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P 2 ]. In the presence of PI(4,5)P 2 , vesicle attachment was unaffected by mutation. When expressed in Snap-25-null mouse autaptic neurons, region I mutations reduced the size of the readily releasable pool of vesicles, whereas the region II mutation reduced vesicular release probability. Combining both in the D51/E52/E55/D166A mutation abrogated evoked release. These data point to a division of labor between region I (vesicle priming) and region II (evoked release). Spontaneous release was disinhibited by region I mutations and found to correlate with defective complexin (Cpx) clamping in an in vitro fusion assay, pointing to an interdependent role of synaptotagmin and Cpx in release clamping. Mutation in region II (D51/E52/E55A) also unclamped release, but this effect could be overcome by synaptotagmin overexpression, arguing against an obligatory role in clamping. We conclude that three synaptic release functions of syt-1, vesicle priming, spontaneous release clamping, and evoked release triggering, depend on direct SNARE complex interaction.

An Electrostatic Energy Barrier for SNARE-Dependent Spontaneous and Evoked Synaptic Transmission

“…Thus, the electrostatics of the WT SNARE-complex results in an intermediate fusion barrier, which allows further modification by synaptotagmin and complexins. Our model and findings agree with the increased spontaneous release rates and mildly increased vesicular release probability upon neutralization of D186 and D193 (Schupp et al, 2016). The correlated effect on spontaneous and evoked release when inserting negative or positive charges into the complexin accessory helix also agrees with our model (Trimbuch et al, 2014).…”

“…We measured spontaneous and evoked release rates in the presence and absence of syt-1. We found an increase in normalized spontaneous release rates and a reduction in RRP size after either syt-1 KO or knockdown (KD), consistent with previous data using the syt-1 KD (Schupp et al, 2016). However, the data from the syt-1 KO appear at odds with earlier reports of un-changed spontaneous release rates in autaptic neurons (Geppert et al, 1994;Liu et al, 2009;Wierda and Sørensen, 2014).…”

“…The surface charge of the fusion machine might affect the fusion barrier when placed between two negatively charged membranes. To investigate this, we placed mutations in the C-terminal half of the second SNARE domain in SNAP-25 (denoted SN2), which does not interact directly with syt-1 (Brewer et al, 2015;Schupp et al, 2016;Zhou et al, 2015Zhou et al, , 2017 or complexin (Chen et al, 2002). To avoid destabilizing the SNARE-complex, we mutated amino acid residues with side chains facing outward (Sutton et al, 1998) ( Figure 1A).…”

Mechanism of neurotransmitter release coming into focus

“…196 Subsequently, two similar crystal structures of Syt1 C 2 AB bound to the SNARE complex revealed three binding modes 197 that were drastically different from each other and from that defined by NMR spectroscopy, consistent FRET and NMR data showing that there are multiple types of Syt1-SNARE complex interactions. 196,219 One of the binding modes observed in the crystal structures, referred to as the primary interface, was validated by mutagenesis and electrophysiological data, while the other two binding modes most likely arise from crystal contacts (see also 220,221 ). The primary interface involves the two SNARE motifs of SNAP-25, partially overlapping with the C 2 B domain-binding site defined by NMR spectroscopy, but the interacting region of the C 2 B domain is located on the convex side of the b-sandwich, opposite to the concave side containing the polybasic region [ Fig.…”

SNARE Complex Alters the Interactions of the Ca2+sensor Synaptotagmin 1 with Lipid Bilayers

“…The SNARE proteins form a coil-coiled four-helical bundle, which consists of the SV-associated protein synaptobrevin (Syb) and the PM-associated proteins syntaxin (Syx) and SNAP25 also known as t-SNARE. Syt1 can interact with t-SNARE proteins, and multiple studies suggest an important role for Syt1-SNARE interactions during fusion [18][19][20][21][22][23]. However, other studies have argued against this possibility [24,25], and it is still debated how the Syt1-SNARE complex is formed in vivo and what is the role of the Syt1-SNARE interactions in the fusion process [26][27][28][29][30][31][32].…”