The rapid reuptake of neurotransmitters across presynaptic neural and glial membranes constitutes the principle mechanism for the termination of chemical signals at CNS and -sympathetic synapses. Specific, Na+-driven, high-affinity transporters have been identified for L-glutamate, GABA, glycine, norepinephrine, dopamine, serotonin, adenosine, and the acetylcholine metabolite choline, among others. Defects in carrier structure or regulation are likely to exert profound effects on nervous system development and synaptic function and have been implicated in depression, hypertension, and drug addiction. The ability of transmitter-like neurotoxins, such as MPP+ and 6-hydroxydopamine, to be concentrated within nerve endings by these transporters suggests an involvement in neuropathology. Although the ability of antidepressants and cocaine to block monoamine reuptake focused attention on neurotransmitter transport over two decades ago, the majority of these proteins have yet to be purified and their genes have remained uncharacterized. Through the application of novel molecular biologic strategies, cDNA cloning of the human L-norepinephrine transporter (NET) has been achieved. Ibis breakthrough heralds new opportunities for the understanding of molecular principles involved in monoamine transporter assembly, function, and regulation, of critical importance if we are to understand the functional consequences of alterations or mutations occurring in brain disease. To achieve these goals, the present proposal first seeks to express and characterize cloned NET in transfected mammalian cells to determine whether a single mRNA encodes all the readily observable activities present in the native transporter. Secondly, mutated NET will be to produced and these constructs expressed in mammalian cells to elucidate the organization of functional domains and critical residues. These studies will be greatly advanced by the procurement of transporter and domain-specific antibodies, requiring the overexpression of NET in bacterial and eukaryotic cells and immunization with purified protein or synthetic peptides. These antibodies will permit the confirmation of gross structural integrity following transporter mutations, permit immunoprecipitation of labeled transporters for regulatory studies, and allow for tests of structural models built on primary sequence data. Finally, the present proposal evaluates the presence of transporter diversity, both within the NET family and among other monoamine carriers, utilizing sequence and expression-based cloning strategies, complemented by a determination of cellular organization by situ-hybridization. Insights gleaned should illuminate conserved features of related neurotransmitter transporters and provide a basic understanding of the consequences of altered transporter structure and function in disease.
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