Broad Objectives: (l) Identify the chemical contents of individual synaptic vesicles (SVs) and relate this to the physiological study, (2) Discriminate between the different models of exocytosis, and (3) Study at the single SV level how the molecular machineries are changed during learning. In a variety of pathologies, excessive activation of glutamate receptors may mediate neuronal injury or death. One of the possible sources of this is abnormal release from vesicles. The specific research aims include: (1) Identifying the neurotransmitters of individual SVs, (2) Studying the transporters in vesicles, and (3) Following the SV membrane proteins and transporters during in vitro exocytosis and relating this to (1) above and to identified SV membrane proteins. The following methods will be used in my research. An optical trap will hold the vesicles for spectroscopy, perform a molecular binding assay, and transfer the vesicles for chemical analysis. Spectroscopy of voltage and pH indicators in the small volumes of single SVs may call for single molecule detection techniques and nonlinear optical microscopy. Fusion will be modeled and studied by the fusion of natural vesicles in a model membrane while spectroscopy is performed. We will measure the binding strength of the protein complex during its development by trapping the vesicle. The SV membrane proteins will be detected by optical chromatographic immunoassay. In this way the functionality of the transporters and other membrane proteins of the vesicle can be related to identified SV membrane proteins. To probe synaptic vesicle content by capillary electrophoresis the vesicle will be lysed and its contents derivatized, separated and detected by laser spectroscopy.
| Peleg, G; Ghanouni, P; Kobilka, B K et al. (2001) Single-molecule spectroscopy of the beta(2) adrenergic receptor: observation of conformational substates in a membrane protein. Proc Natl Acad Sci U S A 98:8469-74 |
