Neurons transmit information by releasing neurotransmitters into the synaptic cleft. Release is triggered by increases in intracellular Ca2+ concentration and is mediated by the fusion of transmitter-filled synaptic vesicles with the presynaptic plasma membrane. While these aspects of synaptic transmission are well established, the molecular mechanism that couples Ca2+ to exocytosis is not known. The synaptic vesicle protein synaptotagmin binds Ca2+ and is essential for rapid and efficient Ca2+-triggered exocytosis. However, little is known concerning the molecular mechanism(s) by which synaptotagmin operates in the release process. The long term goal of our research is to elucidate the biochemical function of synaptotagmin in excitation- secretion coupling. To address this question, three Specific Aims are proposed: first, we will continue to delineate the synaptotagmin signaling pathway by identifying and characterizing synaptotagmin.target protein complexes. These studies include extensions of previously identified synaptotagmin.effector interactions, the characterization of preliminary effectors, and the identification of novel effectors. Second, we will determine whether individual synaptotagmin.effector interactions participate in Ca2+-triggered exocytosis. These studies will involve a detailed analysis of the structural determinants that mediate synaptotagmin.effector assembly. Binding domain data will be used to design peptides that potently and specifically inhibit discrete interactions in vitro. The function of individual interactions will then be determined by assessing the effects of the peptides on Ca2+- triggered exocytosis from semi-intact secretory cells. Third, we will determine the association kinetics of individual Ca2+- dependent synaptotagmin.effector interactions. From this analysis we can determine the temporal order of these interactions and discern which interactions are rapid enough to trigger release. This real time analysis will make us of synaptotagmin inserted into artificial liposomes and represents an initial step in the reconstitution of the molecular machinery that underlies Ca2+-triggered membrane fusion. A better understanding of the mechanisms of neuronal exocytosis will provide a framework for studying how this process is modulated and thus contributes to synaptic plasticity in both normal and pathophysiological states. Finally, defining this mechanism should ultimately provide targets for treatment of diseases in which synaptic transmission is impaired.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM056827-03
Application #
6151195
Study Section
Physiology Study Section (PHY)
Program Officer
Shapiro, Bert I
Project Start
1998-02-01
Project End
2003-01-31
Budget Start
2000-02-01
Budget End
2001-01-31
Support Year
3
Fiscal Year
2000
Total Cost
$208,286
Indirect Cost
Name
University of Wisconsin Madison
Department
Physiology
Type
Schools of Medicine
DUNS #
161202122
City
Madison
State
WI
Country
United States
Zip Code
53715
Zhang, Zhenjie; Bhalla, Akhil; Dean, Camin et al. (2009) Synaptotagmin IV: a multifunctional regulator of peptidergic nerve terminals. Nat Neurosci 12:163-71
Dean, Camin; Liu, Huisheng; Dunning, F Mark et al. (2009) Synaptotagmin-IV modulates synaptic function and long-term potentiation by regulating BDNF release. Nat Neurosci 12:767-76
Liu, Huisheng; Dean, Camin; Arthur, Christopher P et al. (2009) Autapses and networks of hippocampal neurons exhibit distinct synaptic transmission phenotypes in the absence of synaptotagmin I. J Neurosci 29:7395-403
Abdulreda, Midhat H; Bhalla, Akhil; Rico, Felix et al. (2009) Pulling force generated by interacting SNAREs facilitates membrane hemifusion. Integr Biol (Camb) 1:301-10
Chicka, Michael C; Hui, Enfu; Liu, Huisheng et al. (2008) Synaptotagmin arrests the SNARE complex before triggering fast, efficient membrane fusion in response to Ca2+. Nat Struct Mol Biol 15:827-35
Shahin, Victor; Datta, Debajyoti; Hui, Enfu et al. (2008) Synaptotagmin perturbs the structure of phospholipid bilayers. Biochemistry 47:2143-52
Jackson, Meyer B; Chapman, Edwin R (2008) The fusion pores of Ca(2+)-triggered exocytosis. Nat Struct Mol Biol 15:684-689
Liu, Tingting; Wang, Tingting; Chapman, Edwin R et al. (2008) Productive hemifusion intermediates in fast vesicle fusion driven by neuronal SNAREs. Biophys J 94:1303-14
Abdulreda, Midhat H; Bhalla, Akhil; Chapman, Edwin R et al. (2008) Atomic force microscope spectroscopy reveals a hemifusion intermediate during soluble N-ethylmaleimide-sensitive factor-attachment protein receptors-mediated membrane fusion. Biophys J 94:648-55
Gaffaney, Jon D; Dunning, F Mark; Wang, Zhao et al. (2008) Synaptotagmin C2B domain regulates Ca2+-triggered fusion in vitro: critical residues revealed by scanning alanine mutagenesis. J Biol Chem 283:31763-75

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