The long-term objective of the proposed research is characterization of the transport systems (porter) directly involved in the accumulation of catecholamines and serotonin in synaptic vesicles and storage granules. We propose to prepare mouse monoclonal antibodies to two forms of the porter recently clone from brain and PC12 cells using synthetic peptides as immunogens. Monoclonal antibodies to the porter have not been described previously, and will be useful for a wide range of investigations involving the detection, purification, and characterization of porter isoforms. Antibodies will be used to identify and characterize isoforms of the porter, first in the adrenal and then in other catecholamine and serotonin containing tissues. Differences not attributable to primary sequence will be determined using lectin and immunoaffinity chromatography, specific deglycosylation methods, and isoelectric focusing, along with immunochemical detection methods. Functional heterogeneity of porter isoforms expressed in a heterologous system will be assessed by examination of inhibitor binding parameters, substrate specificities and kinetic parameters for the major isoforms. The binding of tritiated methyl reserpate, a new radiolabeled analog of reserpine, will be characterized. Preliminary studies suggest it binds reversibly and specifically to the initial substrate binding site of the porter, and a ligand with these desirable properties has not been described previously. Using the tritiated forms of methyl reserpate and the noncompetitive transport inhibitor dihydrotetrabenazine, the relationships between binding sites for substrates and various inhibitors will be examined in competition studies, and the stoichiometry of various sites will be determined. The topological orientation of these sites will be assessed by use of impermeant hydrophilic analogs of tetrabenazine and reserpine. The effects of energization in the presence and absence of substrate or inhibitors on the quaternary structure, conformation and orientation of the porter will be evaluated using reversible crosslinking agents, hydrophobic and membrane surface probes, and immunochemical methods. Electrochemical measurements of substrate transport by the porter reconstituted in a planar lipid bilayer will be attempted. This would allow testing of various kinetic models for transport, and could serve as a general method for the study of other catecholamine transport systems as well. Dr. R. Mark Wightman, an expert in the field of analytical electrochemistry, will act as a consultant in these studies. The proposed investigations could lead to more selective treatments for central nervous system disorders involving catecholamines or serotonin. Furthermore, the role of the vesicular porter in such disorders is unknown. A thorough characterization of the molecular forms of the porter present in various tissue, as well as elucidation of any related functional differences, are crucial steps in resolving this questions.
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