The overall goal of this research project is to gain an understanding of the role of the second messenger inositol-1,4,5-trisphosphate (IP3) in olfactory transduction. Work in our laboratory has concentrated on the olfactory system of the channel catfish as a model. In this model system odorants transiently increase intracellular levels of IP3. Preliminary studies using this model have led us to propose a hypothesis whereby intracellular IP3 binds to a specific receptor site located in the plasma membrane of the olfactory cilia. Furthermore this receptor site is thought to be an ion channel. To test this hypothesis we will use a combination of biochemical and electrophysiological methods. We will characterize the affinity, specificity and subcellular location of the IP3 binding site using radiolabeled IP3 in combination with other inositol phosphate analogs. The IP3 receptor is isolated olfactory cilia will be solubilized and purified. Electrophysiological studies will determine the properties of the IP3-gated channel in isolated cilia patches and compare these properties to solubilized and purified receptors as a mechanism for monitoring successful purification. The binding specificities of the purified and chemically modified receptor will be studied. Ion flux and channel activity of native and chemically modified receptors will be studied to assess the effects of modification on receptor function. Antibodies will be prepared against the IP3 receptor to study the location, biochemical and functional properties of the receptor protein, and for use as tools in future molecular biological studies.
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