The major objective of this proposal is to define the cellular and subcellular mechanism involved in the neuronal storage and release of serotonin (5-HT). Central and peripheral serotonergic neurons will be studied and in addition, a neurectodermally-derived paraneuron (the parafollicular cell of the thyroid) will also be examined. These three cell types all contain an intravesicular protein serotonin binding protein (SBP), that binds 5-HT with a high affinity under intracellular conditions. This protein is not found in 5-HT storing cells that are not of neurectodermal origin. Two forms of SBP, differing in molecular weight (45kDa and 56kDa) have been purified and characterized. Both are present in all 3 cell types although evidence suggests that the 56kDa form may be more prevalent in cell bodies and preterminal axons, while the 45kDa form appears to be more concentrated in nerve terminals. We propose that the 56kDa form of SBP which can be phosphorylated, is the precursor of the 45kDa material which cannot. In addition to testing this hypothesis, we will investigate the functional significance of the inhibition of 5-HT binding by SBP phosphorylation and the regulation of phosphorylation by Ca+2. In order to do this, the intracellular Ca+2 concentration will be manipulated pharmacologically and monitored with Quin-2. A photoaffinity probe, ANPA-5-HT, will be used to covalently label SBP either inside axon terminals or on their surface. This will be done to examine the possibility that SBP is retained by neurons when synaptic vesicles release their transmitter by exocytosis and is recycled. Maps of tryptic digests of the 45kDa and 56kDa forms of SBP will be constructed in order to examine the structural homologies between them. Monoclonal and polyclonal antibodies to SBP (as well as anti-idiotypic antibodies to 5-HT, which may react with SBP and 5-HT receptors) will be obtained and used both to study properties of the molecules and to explore the cellular and subcellular localization of SBP. Co-localization of 5-HT and SBP is predicted. Finally, the ontogency of 5-HT and SBP will be examined in relation to the appearance of synaptic vesicles. These studies of the brain and peripheral model systems should provide insights into critical elements of the cellular biology of serotonergic neurons that in turn should help in understanding those clinical conditions in which serotonergic function may be abnormal. In addition these studies should provide information necessary for designing pharmacological agents to manipulate serotonergic mechanisms.
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