Studies of Protein Interactions Implicated in Neurotransmitter Secretion
Neurons communicate by releasing chemical messengers, called neurotransmitters. Neurotransmitter release occurs when transmitter-containing vesicles, called synaptic vesicles, fuse with the neuronal plasma membrane. Vesicle fusion is tightly regulated by calcium concentrations in the neuron. My research focuses on the biochemical events that underlie neurotransmitter release, specifically calcium-sensitive protein interactions that occur between proteins of the synaptic vesicle and plasma membrane. One of these interactions is between the synaptic vesicle proteins synaptotagmin and SV2. In this project I am trying to determine whether binding to SV2 regulates the interaction of synaptotagmin with other protein implicated in transmitter release. To do this I am performing in vitro protein binding studies with recombinant peptides corresponding to the synaptotagmin binding region of SV2. In my initial experiments I am measuring the effects of SV2 peptide on the ability of recombinant synaptotagmin to interact with neuronal proteins presented in an extract of nerve endings. These experiments will be complemented by immunoprecipitation studies designed to asses the effect of SV2 peptide on the interaction of native synaptotagmin with other proteins. Techniques I am learning include protein binding assays, gel electrophoresis, immunoprecipitation and immunoblot analysis.
