The overall goal of this program project is to elucidate the precise molecular mechanism and regulation of the fusion machine that drives exocytosis for the controlled release of neurotransmitter at nerve terminals. The assembly of SNARE molecules residing in the synaptic vesicle and presynaptic plasma membrane takes center stage and provides the driving energy for this process. Even though we know the structure of the fully assembled cis-SNARE complex after fusion in atomic detail and have detailed conformational models for several of the SNAREs before fusion, we do not precisely know how (i) they are conditioned with regulatory proteins such as Munc18 and Munc13 to form an active acceptor complex on the plasma membrane, (ii) how this acceptor SNARE complex engages with the synaptic vesicle SNARE upon encounter, and (iii) how this high-energy trans-SNARE complex is ultimately triggered by the synaptic vesicle protein synaptotagmin and calcium to proceed to full assembly and fusion. Three projects led by three expert leaders in the biochemistry, structural biology, and biophysics of neuronal exocytotic membrane fusion are designed to jointly unravel the precise molecular interactions that drive the neuronal fusion machine through the vesicle docking, priming, and fusion steps with the highest possible structural and time resolution. The team will seek to define the structures and configurations of the active presynaptic acceptor SNARE complex and the fusion-restricted trans-SNARE complex between two membranes, and the team will strive to uncover the molecular mechanism, by which calcium-synaptotagmin engages with the membranes and/or complex to release their fusion-restriction. To achieve this goal the team will use a unique combination of approaches ranging from highly innovative biochemical procedures to reconstitute the relevant proteins, EPR, DEER, and NMR spectroscopy to characterize the pertinent structures in membrane environments, and FLIC and single vesicle TIRF microscopy to measure membrane topology and read out fusion on the millisecond timescale.

Public Health Relevance

Rapid regulated neurotransmitter release at the synapse is of central importance for building neuronal circuits in the brain and peripheral nervous tissues. Multiple neurological and neurodegenerative diseases such as epilepsy, depression, and Parkinson's can be traced to defective neurotransmitter release pathways. The release of neurotransmitter is required for signaling between neurons and is accomplished by calcium- regulated rapid exocytosis and fusion of synaptic vesicles at the presynaptic cell membrane. This program project will seek to unravel at unprecedented spatial and temporal resolution the inner workings of the molecular machine that leads to fusion of synaptic vesicles at the neuronal cell membrane and that results in the rapid release of neurotransmitters for neuronal cell signaling. Knowledge gained from this basic research project may ultimately help to combat diseases that are affected by impaired neurotransmitter release in the brain or peripheral tissues.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Program Projects (P01)
Project #
2P01GM072694-11A1
Application #
9209665
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Ainsztein, Alexandra M
Project Start
2005-04-01
Project End
2022-06-30
Budget Start
2017-07-01
Budget End
2018-06-30
Support Year
11
Fiscal Year
2017
Total Cost
Indirect Cost
Name
University of Virginia
Department
Physiology
Type
Schools of Medicine
DUNS #
065391526
City
Charlottesville
State
VA
Country
United States
Zip Code
22904
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Milovanovic, Dragomir; Honigmann, Alf; Koike, Seiichi et al. (2015) Hydrophobic mismatch sorts SNARE proteins into distinct membrane domains. Nat Commun 6:5984

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