The goal of this research proposal is to develop a better understanding of the mechanisms underlying synaptic transmission through the characterization and mutational analysis of Hrs, a protein recently implicated in neurotransmitter release. Docking and fusion of synaptic vesicles with the presynaptic membrane is thought to require a core complex formed by three proteins: synaptobrevin, SNAP-25, and syntaxin. In an attempt to identify proteins that interact with this core complex in mouse, Bean et al. (1997) performed a yeast two hybrid screen with SNAP-25 as bait. This led to the identification of Hrs, a cytoplasmic protein that is highly expressed in the brain, specifically binds SNAP-25, and contains an FYVE zinc finger domain commonly found in proteins involved in vesicle transport in yeast and vertebrates. Furthermore, addition of recombinant Hrs to permeabilized PCl2 cells inhibited neurotransmitter release, suggesting that Hrs plays a role in this process. We have recently cloned and sequenced the Drosophila homolog of hrs, and have mapped the gene to a well-characterized region in the Drosophila genome. Here, we propose to examine the in vivo role of Hrs in neurotransmitter release in Drosophila. First, we will determine the tissue and subcellular localization of Hrs and perform a biochemical analysis of the interactions of Hrs with other proteins involved in synaptic transmission. We will then isolate mutations in Hrs and characterize them molecularly and genetically. Finally, and most importantly, electrophysiological studies will be carried out to determine the effects of these mutations on neurotransmitter release in vivo. The dissection of the function of Hrs through electrophysiological and biochemical analysis should yield important insights into the mechanisms of synaptic vesicle release. An understanding of this mechanism may contribute to medically relevant issues such as drug action at the synapse, disease states associated with changes in neurotransmitter release, and the processes underlying learning and memory.
