Inositol pyrophosphates inhibit synaptotagmin-dependent exocytosis

Abstract

SNARE complex is an essential component of intracellular membrane fusion, a fundamental phenomenon in biological processes. It is known that synaptoagmin-1 (syt1) as a $Ca^{2+}$ trigger is a key protein for membrane fusion. Nevertheless, the effect of SNAREs and syt1 reconstituted into vesicles is still veiled. Here, I did research about the function of SNAREs and synaptotagmin-1, respectively, using single and bulk vesicle interaction system. The SNARE complex was not related to $Ca^{2+}$, but syt1 did by penetration to t-vesicles. In addition, transmembrane domain of syt1 is a critical factor for membrane fusion as well as C2AB domain of it. Inositol pyrophosphates such as $5-IP_7$ (5-diphosphoinositol pentakisphosphate) are highly energetic inositol metabolites containing phosphoanhydride bonds. While inositol pyrophosphates are known to regulate various biological events, including growth, survival and metabolism, the molecular sites of $5-IP_7$ action in vesicle trafficking have remained largely elusive. Based on results of PC12 cells and hippocampal neurons, employing a fluorescence resonance energy transfer (FRET)-based in vitro vesicle fusion assay, I found that $5-IP_7$, but not $1-IP_7$, exhibited significantly higher inhibitory activity toward synaptic vesicle exocytosis than did inositol hexakisphosphate ($IP_6$). syt1 essential for synaptic membrane fusion, was identified as a molecu-lar target of $5-IP_7$. Notably, $5-IP_7$ showed a 45-fold higher binding affinity for syt1 compared with $IP_6$. In addition, $5-IP_7$-dependent inhibition of synaptic vesicle fusion was abolished by increasing $Ca^{2+}$ levels. Thus, $5-IP_7$ appears to act through syt1 binding to interfere with the fusogenic activity of $Ca^{2+}$. These findings reveal a role of $5-IP_7$ as a potent inhibitor of syt1 in controlling the synaptic exocytotic pathway and expand our un-derstanding of the signaling mechanisms of inositol pyrophosphates.