Biology subjects

Vardar, G.

Publications and source records attributed to Vardar, G..

3 recordsLinked to original sources

bioRxiv2023

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

The SNARE proteins are central in membrane fusion and, at the synapse, neurotransmitter release. However, their involvement in the dual regulation of the synchronous release while maintaining a pool of readily releasable vesicles remains unclear. Using a chimeric approach, we performed a systematic analysis of the SNARE domain of STX1A by exchanging the whole SNARE domain or its N- or C-terminus subdomains with those of STX2. We expressed these chimeric constructs in STX1-null hippocampal mouse neurons. Exchanging the C-terminal half of STX1s SNARE domain with that of STX2 resulted in a reduced RRP accompanied by an increased release rate, while inserting the C-terminal half of STX1s SNARE domain into STX2 lead to an enhanced priming and decreased release rate. Additionally, we found that the mechanisms for clamping spontaneous, but not for Ca2+-evoked release, are particularly susceptible to changes in specific residues on the outer surface of the C-terminus of the SNARE domain of STX1A. Particularly, mutations of D231 and R232 affected the fusogenicity of the vesicles. We propose that the C-terminal half of the SNARE domain of STX1A plays a crucial role in the stabilization of the RRP as well as in the clamping of spontaneous synaptic vesicle fusion through the regulation of the energetic landscape for fusion, while it also plays a covert role in the speed and efficacy of Ca2+-evoked release.

neuroscience↗

bioRxiv2022

Syntaxin-1A modulates vesicle fusion in mammalian neurons via juxtamembrane domain dependent palmitoylation of its transmembrane domain

SNAREs are undoubtedly one of the core elements of synaptic transmission. On the contrary to the well characterized function of their SNARE domains bringing the plasma and vesicular membranes together, the level of contribution of their juxtamembrane domain (JMD) and the transmembrane domain (TMD) to the vesicle fusion is still under debate. To elucidate this issue, we analyzed three groups of STX1A mutations: 1) elongation of STX1As JMD by three amino acid insertions in the junction of SNARE-JMD or JMD-TMD; 2) charge reversal mutations in STX1As JMD; and 3) palmitoylation deficiency mutations in STX1As TMD. We found that both JMD elongations and charge reversal mutations have position dependent differential effects on Ca2+-evoked and spontaneous neurotransmitter release in cultured murine hippocampal neurons. Importantly, we show that STX1As JMD regulates the palmitoylation of STX1As TMD and loss of STX1A palmitoylation either through charge reversal mutation K260E or by loss of TMD cysteines particularly inhibits spontaneous vesicle fusion. Interestingly, the retinal ribbon specific STX3B has a glutamate in the position corresponding to the K260E mutation in STX1A and mutating it with E259K acts as a molecular on-switch. Furthermore, palmitoylation of post-synaptic STX3A can be induced by the exchange of its JMD with STX1As JMD together with the incorporation of two cysteines into its TMD. Forced palmitoylation of STX3A dramatically enhances spontaneous vesicle fusion suggesting that STX1A regulates spontaneous release through two distinct mechanisms: one through the C-terminal half of its SNARE domain and the other through the palmitoylation of its TMD.

neuroscience↗

bioRxiv2021

Reexamination of N-terminal domains of Syntaxin-1 in vesicle fusion from central murine synapses

Syntaxin-1 (STX1) and Munc18-1 are two requisite components of synaptic vesicular release machinery, so much so synaptic transmission cannot proceed in their absence. They form a tight binary complex through two major binding modes: one through STX1s N-peptide, the other through STX1s closed conformation driven by its Habc-domain. However, physiological roles of these two reportedly different binding modes in synapses are still controversial. Here we characterized the roles of STX1s N-peptide, Habc-domain, and open conformation with and without N-peptide deletion using our STX1-null mouse model system and exogenous reintroduction of STX1A mutants. We show, on the contrary to the general view, that the Habc-domain is absolutely required and N-peptide is dispensable for synaptic transmission. However, STX1s N-peptide plays a regulatory role, particularly in the Ca2+-sensitivity and the short-term plasticity of vesicular release, whereas STX1s open conformation governs the vesicle fusogenicity. Strikingly, we also show that neurotransmitter release still proceeds when both the interaction modes between STX1 and Munc18-1 are presumably intervened together, necessitating a refinement of the conceptualization of STX1-Munc18-1 interaction.

neuroscience↗