The stability of the primed pool of synaptic vesicles and the clamping of spontaneous neurotransmitter release relies on the integrity of the C-terminal half of the SNARE domain of Syntaxin-1A

Reviewer #1 (Public Review):

In this systematic and elegant structure-function analysis study, the authors delve into the intricate involvement of syntaxin 1 in various pivotal stages of synaptic vesicle priming and fusion. The authors use an original and fruitful approach based on the side-by-side comparison of the specific contributions of the two isoforms syntaxin 1 and syntaxin 2, and their respective SNARE domains, in priming, spontaneous, and Ca2+-dependent glutamate release. The experimental approach, mastered by the authors, offers an ideal means of unraveling the molecular roles played by syntaxins. Although it is not easy to come up with a model explaining all the observed phenotypes, the authors carefully restrict their conclusions to the role of the C-terminal half of the syntaxin1 C-terminal SNARE domain in the maintenance of the RRP and the clamping of neurotransmitter release. The study is carefully carried out, the conclusions are supported by high-quality data, and the manuscript is clearly written. In addition, the study clearly sets new questions that open new paths for future experimental work.

Reviewer #2 (Public Review):

Summary:
The manuscript by Salazar-Lázaro et al. systematically dissects the different functional properties of the SNARE-domains of syntaxin-1 and syntaxin-2. By systematically substituting the SNARE-domain (or its C- or N-terminal half) into the non-cognate counterpart, the authors find that the C-terminal half of the SNARE-complex is especially important for maintaining RRP size and clamping spontaneous release. They also mutate single residues, to further nail down the effect. Overall, this is an interesting manuscript, which sheds light on the functionality of different co-expressed SNARES.

Strengths:
The strength of the manuscript is the systematic dissection, using substitution of either SNARE-domain into the other syntaxin, together with the state-of-the art methods. The authors follow up with a substitution of single and paired residues. This is a large undertaking, which has been very well carried out.

Weaknesses:
No major weaknesses. The large number of experiments paint a somewhat complicated picture. The writing could be improved in places to increase clarity.

Reviewer #3 (Public Review):

Summary:
In this manuscript, Salazar-Lázaro et al. presented interesting data that C-terminal half of the Syx1 SNARE domain is responsible for clamping of spontaneous release, stabilizing RRP, and also Ca2+-evoked release. The authors routinely utilized the chimeric approach to replace the SNARE domain of Syx1 with its paralogue Syx2 and analyzed the neuronal activity through electrophysiology. The data are straightforward and fruitful. The conclusions are partly reasonable.

Strengths:
The electrophysiology data that illustrate the important functions of Syx1 in clamping of spontaneous release, stabilizing RRP, and Ca2+-evoked release were clear and convincing.

Weaknesses:
One obvious weakness is that the authors did not explore the underlying mechanism. I think it is easy for the authors to carry out some simple assays to verify their hypothesis for the mechanism, instead of just talking about it in the discussion section.