Inositol pyrophosphates inhibit synaptotagmin-dependent exocytosis

Inositol pyrophosphates such as 5-diphosphoinositol pentakisphosphate (5-IP 7 ) are highly energetic inositol metabolites containing phosphoanhydride bonds. Although 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. We report here that elevated 5-IP 7 levels, caused by overexpression of inositol hexakisphosphate (IP 6 ) kinase 1 (IP6K1), suppressed depolarization-induced neurotransmitter release from PC12 cells. Conversely, IP6K1 depletion decreased intracellular 5-IP 7 concentrations, leading to increased neurotransmitter release. Consistently, knockdown of IP6K1 in cultured hippocampal neurons augmented action potential-driven synaptic vesicle exocytosis at synapses. Using a FRET-based in vitro vesicle fusion assay, we found that 5-IP 7 , but not 1-IP 7 , exhibited significantly higher inhibitory activity toward synaptic vesicle exocytosis than IP 6 . Synaptotagmin 1 (Syt1), a Ca 2+ sensor essential for synaptic membrane fusion, was identified as a molecular 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 understanding of the signaling mechanisms of inositol pyrophosphates.