Ca++-triggered synchronized release of the neurotransmitter at the synapse, which underlies neuronal communication and synaptic plasticity, requires membrane fusion. This remarkable process is controlled by an exquisitely orchestrated array of protein-protein interactions. SNARE (soluble N- ethylmaleimide-sensitive factor attachment protein receptor) assembly is the central event that may drive membrane fusion. A Ca++-sensor synaptotagmin I (SytI) and complexins impinge upon SNARE assembly to control the timing and the size of the release. The present project uses the newly developed single fusion assay based on total internal reflection (TIRF) microscopy to investigate the mechanism by which SNARE-dependent fusion is regulated by SytI, complexins, and Ca++. The single fusion assay makes it possible to dissect and characterize individual fusion intermediates in unprecedented detail. Further, this technique allows us to track the dynamic transitions in a single fusion event with msec time resolution. With this powerful new fusion assay, we will dissect how the fusion regulators modulate the individual fusion steps. Further, the present project uses site-directed spin labeling (SDSL) and electron paramagnetic resonance (EPR), an established technique for the investigation of structures and topologies of membrane proteins. On the basis of various EPR results, a structural model of the protein in the native- like phospholipid bilayer is generated at the backbone resolution. In this project, conformational changes of SNARE complexes induced by SytI, complexins, and Ca++ are determined using SDSL EPR to gain insights into the structural basis of the synchronized release. The combined approach employing the single fusion assay and SDSL EPR will provide insights into mechanism whereby the synchronized release is manufactured in the neuron. Dysfunction of synchronous neurotransmitter release is linked to hideous mental illnesses such as schizophrenia, autism, and epilepsy, as well as to less serious illnesses including behavioral disorders. The outcomes of the proposed research will lead to the understanding of the mechanism whereby the synchronous release is controlled, which will eventually lead to the mechanism-based design of drugs for metal illnesses.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM051290-17
Application #
7994152
Study Section
Special Emphasis Panel (ZRG1-BCMB-B (02))
Program Officer
Ainsztein, Alexandra M
Project Start
1994-08-01
Project End
2012-04-30
Budget Start
2010-12-01
Budget End
2012-04-30
Support Year
17
Fiscal Year
2011
Total Cost
$257,864
Indirect Cost
Name
Iowa State University
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
005309844
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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