The neuron has a remarkable ability to release quanta of neurotransmitters in less than amillisecond in response to the pulse of Ca2+. This fast synchronized release constitutes thefundamental basis of major brain activities. The release requires vesicle fusion, which isorchestrated by the fusion machine SNARE (soluble N-ethylmaleimide-sensitive factorattachment protein receptor) proteins, a priming agent Complexin (Cpx), and the Ca2+ switchSynatotagmin 1 (Syt1). This research project uses the innovative single-vesicle fusion assay to investigatethe mechanism by which SNARE-dependent vesicle fusion is regulated by Cpx, Syt1, andCa2+. The single-vesicle fusion assay makes it possible to dissect the sequential intermediatestages of vesicle fusion, overcoming major drawbacks of conventional bulk fusion assays.The technique also allows us to track the dynamic transitions in a single fusion event withmillisecond time resolution. With this powerful approach, we intend to reconstitutesynchronized vesicle fusion in a test tube. Specifically, we test a mechanistic modelproposing that Cpx arrests an intermediate stage called hemifusion and that Syt1 delivers thefinal blow to the fusion machinery at the Ca2+ spike. In parallel, this research program usessite-directed spin labeling (SDSL) and electron paramagnetic resonance (EPR), a powerfultechnique for the investigation of the protein structure at the membrane interface. With EPRwe intend to comprehend the structural basis for the regulation of SNARE-dependent fusionnecessary for synchronization. The combined approach of the single fusion assay and SDSL EPR will provideimportant insights into the mechanism by which the synchronized fast release ischoreographed in the neuron.

Public Health Relevance

Neurotransmitter release at synapses underlies major brain activities, such as cognition, memory, and emotion. Dysfunction of the release machinery is linked to many gruesome mental diseases, including schizophrenia, epilepsy, attention deficit syndrome (ADS), and autism. SNAREs are the newly emerged drug targets for neuromuscular diseases. A new line of SNARE inhibitors has been identified as a result of this project. The outcomes of proposed studies will help expand the repertoire on which the development of drugs for mental diseases may be based.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM051290-18
Application #
8224527
Study Section
Biochemistry and Biophysics of Membranes Study Section (BBM)
Program Officer
Ainsztein, Alexandra M
Project Start
1994-08-01
Project End
2016-04-30
Budget Start
2012-05-01
Budget End
2013-04-30
Support Year
18
Fiscal Year
2012
Total Cost
$281,936
Indirect Cost
$83,944
Name
Iowa State University
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
005309844
City
Ames
State
IA
Country
United States
Zip Code
50011
Kweon, Dae-Hyuk; Kong, Byoungjae; Shin, Yeon-Kyun (2018) Search for a minimal machinery for Ca2+-triggered millisecond neuroexocytosis. Neuroscience :
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Su, Chih-Chia; Yin, Linxiang; Kumar, Nitin et al. (2017) Structures and transport dynamics of a Campylobacter jejuni multidrug efflux pump. Nat Commun 8:171
Choi, Bong-Kyu; Kim, Jae-Yeol; Cha, Moon-Yong et al. (2017) Retraction of ""?-Amyloid and ?-Synuclein Cooperate To Block SNARE-Dependent Vesicle Fusion"". Biochemistry 56:1026
Khounlo, Ryan; Kim, Jaewook; Yin, Linxiang et al. (2017) Botulinum Toxins A and E Inflict Dynamic Destabilization on t-SNARE to Impair SNARE Assembly and Membrane Fusion. Structure 25:1679-1686.e5
Xue, Chaoyou; Zhu, Yicheng; Zhang, Xiangmei et al. (2017) Real-Time Observation of Target Search by the CRISPR Surveillance Complex Cascade. Cell Rep 21:3717-3727
Lou, Xiaochu; Shin, Yeon-Kyun (2016) SNARE zippering. Biosci Rep 36:
Kim, Jaewook; Zhu, Yicheng; Shin, Yeon-Kyun (2016) Preincubation of t-SNAREs with Complexin I Increases Content-Mixing Efficiency. Biochemistry 55:3667-73
Heo, Paul; Yang, Yoosoo; Han, Kyu Young et al. (2016) A Chemical Controller of SNARE-Driven Membrane Fusion That Primes Vesicles for Ca(2+)-Triggered Millisecond Exocytosis. J Am Chem Soc 138:4512-21
Lee, Tae-Sun; Lee, Joo-Young; Kyung, Jae Won et al. (2016) Inositol pyrophosphates inhibit synaptotagmin-dependent exocytosis. Proc Natl Acad Sci U S A 113:8314-9

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