This proposal outlines approaches to attain molecular and immunological probes for the high-affinity serotonin transport system, in order to analyze its structural and ultrastructural features. Such studies are needed to enhance current understanding of the mechanistic role(s) of neurotransmitter transport systems in neuronal signal transmission, neuropathology and drug abuse-related neurotoxicity. From these insights may come better diagnoses and treatments for certain neurological disorders, as well as for cocaine- and methamphetamine-addiction. To facilitate these experimental studies, novel cellular models of high- affinity serotonin (5-HT) transport have been developed by gene transfer in our laboratory. These models are two clonal cell lines derived from transfection of human DNA into mouse L-M fibroblasts. Both cell lines exhibit high-affinity 5-HT transport systems with physiological, pharmacological and kinetic characteristics that conform to those of known high-affinity 5-HT transport systems; untransfected L-M cells do not manifest such 5-HT uptake properties. Presumably, the observed transport phenotypes in both transfectant cells are due to expression of the human 5- HT transporter gene. Further, the membrane presence of the 5-HT transport systems in these cells are exogenous to the cell host and likely to elicit an immune response by a mouse host in vivo (while the fibroblastic antigens of the transfectant cells will escape such response). Thus, these transfectant models greatly facilitate experimental strategies to: (1) systematically delineate the human gene(s) encoding the high-affinity serotonin transporter from the transfectant genomes by expression cloning; and (2) develop monoclonal antibodies specific for this transporter by using intact transfectant cells as immunogens for hybridoma development and screening. Analysis of the cloned gene, as well as its corresponding cDNAs, will yield for the first time both the gene organization and the primary sequence of the 5-HT transporter. The protein gene can be applied to mutagenesis studies aimed at identifying structural domains critical to transport function. Identified antibodies will be used to histologically and ultrastructurally examine the location of high-affinity 5-HT transport systems within well-defined serotonergic neurocircuitries of the CNS.
