Tae-Sun Leea,b,c,1, Joo-Young Leed,1, Jae Won Kyunge, Yoosoo Yangf, Seung Ju Parkd, Seulgi Leed, Igor Pavlovicg, Byoungjae Kongh, Yong Seok Jhoi, Henning J. Jessenj, Dae-Hyuk Kweonh, Yeon-Kyun Shink, Sung Hyun Kime,l,2, Tae-Young Yoonb,c,2, and Seyun Kimd,m,2
aDepartment of Physics, Korea Advanced Institute of Science and Technology, Daejeon 34141, Korea;
bCenter for Nanomedicine, Institute for Basic Science (IBS), Yonsei University, Seoul 30722, Korea;
cYonsei-IBS Institute, Yonsei University, Seoul 30722, Korea;
dDepartment of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon 34141, Korea;
eDepartment of Biomedical Science, Graduate School, Kyung Hee University, Seoul 02447, Korea;
fBiomedical Research Institute, Korea Institute of Science and Technology, Seoul 02792, Korea;
gDepartment of Chemistry, University of Zurich, 8057 Zurich, Switzerland;
hDepartment of Genetic Engineering and Center for Human Interface Nanotechnology, Sungkyunkwan University, Suwon 16419, Korea;
iAsia-Pacific Center for Theoretical Physics, Pohang, Gyeongbuk 37673, Korea;
jInstitute of Organic Chemistry, Albert-Ludwigs-University Freiburg, Freiburg 79104, Germany;
kDepartment of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, Ames, IA 50011;
lDepartment of Physiology, School of Medicine, Neurodegeneration Control Research Center, Kyung Hee University, Seoul 02447, Korea;
mKorea Advanced Institute of Science and Technology Institute for the BioCentury, Korea Advanced Institute of Science and Technology, Daejeon 34141, Korea
Inositol pyrophosphates such as 5-diphosphoinositol pentakisphosphate (5-IP7) 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-IP7 action in vesicle trafficking have remained largely elusive. We report here that elevated 5-IP7 levels, caused by overexpression of inositol hexakisphosphate (IP6) kinase 1 (IP6K1), suppressed depolarization-induced neurotransmitter release from PC12 cells. Conversely, IP6K1 depletion decreased intracellular 5-IP7 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-IP7, but not 1-IP7, exhibited significantly higher inhibitory activity toward synaptic vesicle exocytosis than IP6. Synaptotagmin 1 (Syt1), a Ca2+ sensor essential for synaptic membrane fusion, was identified as a molecular target of 5-IP7. Notably, 5-IP7 showed a 45-fold higher binding affinity for Syt1 compared with IP6. In addition, 5-IP7-dependent inhibition of synaptic vesicle fusion was abolished by increasing Ca2+ levels. Thus, 5-IP7 appears to act through Syt1 binding to interfere with the fusogenic activity of Ca2+. These findings reveal a role of 5-IP7 as a potent inhibitor of Syt1 in controlling the synaptic exocytotic pathway and expand our understanding of the signaling mechanisms of inositol pyrophosphates.
inositol pyrophosphate, synaptotagmin, synaptic vesicle exocytosis
1T.-S.L. and J.-Y.L. contributed equally to this work.
2To whom correspondence may be addressed.
Author contributions: T.-S.L., J.-Y.L., J.W.K., Y.Y., S.H.K., T.-Y.Y., and S.K. designed research; T.-S.L., J.-Y.L., J.W.K., Y.Y., S.J.P., S.L., and B.K. performed research; I.P. and H.J.J. contributed new reagents/analytic tools; T.-S.L., J.-Y.L., J.W.K., Y.Y., Y.S.J., D.-H.K., Y.-K.S., S.H.K., T.-Y.Y., and S.K. analyzed data; and T.-S.L., J.-Y.L., Y.Y., S.H.K., T.-Y.Y., and S.K. wrote the paper.