The sodium dependent catecholamine transporters have a major influence on the net synaptic output of central dopaminergic and noradrenergic systems and are the primary site of action for stimulant drugs of abuse such as cocaine and amphetamine. Our laboratory has isolated a human cDNA encoding a transport activity with the substrate-specificity and pharmacologic properties of a norepinephrine transporter, including sensitivity to both antidepressants and psychomotor stimulants. The investigations proposed in this application use molecular biologic techniques to characterize the structure, function and regulation of catecholamine transport proteins. Additional members of this gene family will be isolated from cDNA libraries using sequence homology and PCR (polymerase chain reaction) strategies based on the sequence of the cloned sodium-dependent norepinephrine transporter. Two candidates have already been identified: a potential glial form of the norepinephrine transporter mRNA and an mRNA encoding a related transporter with a distribution in the CNS consistent with a dopamine carrier. Initial studies in this proposal aim at characterizing the substrate specificity and pharmacologic sensitivity of each transporter cDNA expressed by transfection into a variety of cell types. A panel of specific antisera will be raised against determinants of the predicted proteins to evaluate the anatomic distribution of each gene product, to confirm structural models predicted from sequence analysis and to address key aspects of the cell biology of these proteins. These antisera will be used in conjunction with other molecular techniques to study the regulation of both transport activity and transporter gene expression. An additional goal of this proposal is the construction and characterization of chimeric and mutant transporters to identify functional domains including binding sites for substrates and pharmacologic agents such as cocaine. The availability of cDNA clones encoding catecholamine carriers will allow precise structure-function studies to be undertaken in transfected cells devoid of vesicular storage Compartments and free from the confounding influences of other transport pathways. The detailed characterization of catecholamine transporter structure, cellular physiology and regulation obtained by the above studies should define the initial site of action of stimulant drugs and provide a strong foundation for future studies of the mechanisms of addiction.
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