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)
Institute
National Institute of General Medical Sciences (NIGMS)
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
1994-08-01
Project End
2016-04-30
Budget Start
2014-05-01
Budget End
2015-04-30
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
Lou, Xiaochu; Kim, Jaewook; Hawk, Brenden J et al. (2017) ?-Synuclein may cross-bridge v-SNARE and acidic phospholipids to facilitate SNARE-dependent vesicle docking. Biochem J 474:2039-2049
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
Na, Jung-Hyun; Lee, Won-Kyu; Kim, Yuyoung et al. (2016) Biophysical characterization of the structural change of Nopp140, an intrinsically disordered protein, in the interaction with CK2?. Biochem Biophys Res Commun 477:181-7
Yin, Linxiang; Kim, Jaewook; Shin, Yeon-Kyun (2016) Complexin splits the membrane-proximal region of a single SNAREpin. Biochem J 473:2219-24
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

Showing the most recent 10 out of 40 publications