The neuron has a remarkable ability to release quanta of neurotransmitters in less than a millisecond in response to the pulse of Ca2+. This fast synchronized release constitutes the fundamental basis of major brain activities. The release requires vesicle fusion, which is orchestrated by the fusion machine SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins, a priming agent Complexin (Cpx), and the Ca2+ switch Synatotagmin 1 (Syt1). This research project uses the innovative single-vesicle fusion assay to investigate the mechanism by which SNARE-dependent vesicle fusion is regulated by Cpx, Syt1, and Ca2+. The single-vesicle fusion assay makes it possible to dissect the sequential intermediate stages 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 with millisecond time resolution. With this powerful approach, we intend to reconstitute synchronized vesicle fusion in a test tube. Specifically, we test a mechanistic model proposing that Cpx arrests an intermediate stage called hemifusion and that Syt1 delivers the final blow to the fusion machinery at the Ca2+ spike. In parallel, this research program uses site-directed spin labeling (SDSL) and electron paramagnetic resonance (EPR), a powerful technique for the investigation of the protein structure at the membrane interface. With EPR we intend to comprehend the structural basis for the regulation of SNARE-dependent fusion necessary for synchronization. The combined approach of the single fusion assay and SDSL EPR will provide important insights into the mechanism by which the synchronized fast release is choreographed 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)
Type
Research Project (R01)
Project #
5R01GM051290-20
Application #
8654337
Study Section
Biochemistry and Biophysics of Membranes Study Section (BBM)
Program Officer
Ainsztein, Alexandra M
Project Start
Project End
Budget Start
Budget End
Support Year
20
Fiscal Year
2014
Total Cost
Indirect Cost
Name
Iowa State University
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
City
Ames
State
IA
Country
United States
Zip Code
50011
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
Yang, Yoosoo; Kim, Jaewook; Kim, Hye Yun et al. (2015) Amyloid-β Oligomers May Impair SNARE-Mediated Exocytosis by Direct Binding to Syntaxin 1a. Cell Rep 12:1244-51
Lai, Ying; Zhao, Lin; Bu, Bing et al. (2015) Lipid molecules influence early stages of yeast SNARE-mediated membrane fusion. Phys Biol 12:025003
Lai, Ying; Lou, Xiaochu; Diao, Jiajie et al. (2015) Molecular origins of synaptotagmin 1 activities on vesicle docking and fusion pore opening. Sci Rep 5:9267
Choi, Bong-Kyu; Kim, Jae-Yeol; Cha, Moon-Yong et al. (2015) β-Amyloid and α-synuclein cooperate to block SNARE-dependent vesicle fusion. Biochemistry 54:1831-40
Lou, Xiaochu; Shin, Jaeil; Yang, Yoosoo et al. (2015) Synaptotagmin-1 is an antagonist for Munc18-1 in SNARE zippering. J Biol Chem 290:10535-43
Shin, Jaeil; Lou, Xiaochu; Kweon, Dae-Hyuk et al. (2014) Multiple conformations of a single SNAREpin between two nanodisc membranes reveal diverse pre-fusion states. Biochem J 459:95-102
Kim, Jaewook; Yang, Yoosoo; Song, Seung Soo et al. (2014) Beta-amyloid oligomers activate apoptotic BAK pore for cytochrome c release. Biophys J 107:1601-8
Yang, Yoosoo; Kim, Se-Hyun; Heo, Paul et al. (2014) SNARE zippering is hindered by polyphenols in the neuron. Biochem Biophys Res Commun 450:831-6
Lai, Ying; Kim, Sunae; Varkey, Jobin et al. (2014) Nonaggregated α-synuclein influences SNARE-dependent vesicle docking via membrane binding. Biochemistry 53:3889-96

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