The nervous system synthesizes and conveys information by electrical activity and release of chemical messengers. The coupling between electrical and chemical signalling is initiated by calcium (Ca) ion influx through voltage-gated Ca channels. Through a sequence of poorly understood or completely unknown steps, Ca triggers exocytosis of transmitter- or hormone-containing vesicles. In the past two years, explosive progress has occurred in the isolation and sequencing of proteins involved in the fusion and retrieval of vesicle membrane from plasma membrane. Almost nothing is known of their function. The proposed work will probe the function of several of the recently identified proteins. Ca-coupled secretion of catecholamines and peptide hormones will be studied in chromaffin cells and isolated nerve endings of the rat neurohypophysis with patch clamp methods and the capacitance detection technique. With this technique, we can monitor small changes in surface membrane area resulting from exo- or endocytosis, while simultaneously controlling and measuring Ca entry. The capacitance detection technique has both superb temporal resolution, reporting events in the msec range, and sensitivity, discriminating single vesicle fusions. The whole-cell recording mode provides the opportunity to manipulate the intracellular milieu, by introducing Ca chelators, second messengers, and proteins into the cell. Ca levels will be manipulated by using protocols which open voltage-gated Ca channels or by photolysis of caged Ca or caged Ca- chelator compounds. We will begin by investigating the properties of two small G proteins Rab3 and Rab5, implicated in exocytosis and endocytosis, respectively. All members of the Rab sub-group of Ha-ras like G proteins are linked to specific steps of membrane traffic. The function and regulation of Rab3 and Rab5 will be tested by eliminating the proteins by antisense oligonucleotides, acutely introducing excess protein in the pipette, and chronically overexpressing the proteins and mutants created by site directed mutagenesis. Synaptic transmission machinery is rapidly being revealed as much more complex than anyone could have predicted even one or two years ago. Along with unraveling the basic processes of secretion, the new information is revealing an incredibly rich potential for short and long term modulation of these processes. The proposed work represents a basic step toward fully understanding synaptic transmission.
