A Scientist Development Award is requested to support research on the transmembrane topology of the biogenic amine re-uptake transporters. This family of transporters represents the molecular targets for cocaine, amphetamines, and antidepressants in the human brain. The amino acid sequences predicted from cloned transporter cDNAs suggest that each forms twelve hydrophobic transmembrane domains, connected inside and outside the cell by hydrophilic loops of varying sizes. The importance of these loops in substrate and drug binding will be investigated, using site-directed mutagenesis to delete them or to form chimeric proteins between members of the family with different substrate specificities. Antigenic epitopes and protease sites will also be added to allow investigation of topology by standard biochemical methods. The three-dimensional model resulting from this topological information should contribute to the elucidation of the substrate binding site, as well as the binding sites for cocaine and the antidepressants, which directly compete with substrate binding. Eventually this knowledge may allow the design of more specific and potent antidepressants. Additional studies will focus on the potential phosphorylation of the transporters by protein kinases, using kinase activators and inhibitors, as well as in vivo labeling with inorganic 32P. Later, mutagenesis studies will be used to determine the functional significance of phosphorylation if it is detected. These studies will include the use of human, neuronally derived cell lines expressing the human transporter isoforms. Knowledge of the interactions of these proteins with second messenger systems may shed light on such complex processes as drug addiction or the etiology of depression. The studies proposed here will fulfill three moor career objectives for the candidate: 1) to use an extensive background in membrane transport and molecular biology to move to the drug abuse and mental health field, 2) to gain expertise in techniques necessary to express and investigate transport proteins in cultured cells, and 3) to gain further experience before embarking on a fully independent research program.
