The applicant's long term goals are to study the regulation of neuronal transmission in the central nervous system and to understand the role of pharmacological agents in modifying neuronal transmission. Specifically, the goals are to study the mechanisms of catecholamine storage, release and reuptake within the presynaptic neuron. Catecholaminergic reuptake into presynaptic neurons following synaptic release is the primary mechanism of terminating the response to catecholamines at post-synaptic receptors. Specific reuptake of norepinephrine, dopamine, and serotonin into the presynaptic neuron is achieved by the high affinity, energy-dependent co-transport of the neurotransmitter along with sodium ions through specific membrane-bound proteins. The importance of catecholamine uptake proteins in our society is best exemplified by the impact of various competitive inhibitors of these proteins. These inhibitors include cocaine, amphetamine, and the tri- cyclic antidepressants. The stimulatory action and the abuse potential of these drugs is related directly to their ability to bind and block catecholamine uptake sites. To study the pharmacology of these inhibitory agents, it is necessary to isolate the class of transporter proteins that they inhibit from other proteins involved in catecholamine metabolism. Protein purification strategies have failed to provide structural information regarding these transport proteins. The molecular cloning of the catecholamine transporters is essential for understanding the pharmacology of their action and their inhibition by drugs of abuse. Because the catecholamine uptake transporter can be expressed and can concentrate catecholamines into heterogenous cells, it is ideally suited for some expression cloning techniques. Frog oocyte assays will be used initially to define mRNA's encoding the transporter and to characterize parameters of uptake. cDNA libraries, enriched for the catecholamine transporter clones, will be expressed to high level in eukaryotic cells. Mammalian cloning systems will be used to identify single cells expressing the cDNA for the catecholamine uptake transporter. Recovery of the transporter cDNA from these cells will allow complete sequence determination, and characterization of the transporter protein itself. Expression of the cDNA in heterogenous cells will allow pharmacological investigation of both catecholamine uptake and its inhibition by cocaine.
