This proposal describes studies designed to understand the mechanism of neurotransmitter transporters. These transporters are responsible for terminating the action of neurotransmitters released at synapses. In addition, the serotonin transporter is responsible for accumulation of serotonin in peripheral blood cells (platelets and basophils), placenta and lung. The serotonin transporter is the biological target for clinically useful antidepressant drugs, such as imipramine and fluoxetine. the dopamine transporter is believed to be the physiological target for cocaine. Amphetamines, including MDA and MDMA (ecstasy) act by reversing the normal operation of these transporters and causing efflux of serotonin, norepinephrine, and dopamine. Recently, cDNA clones have been isolated which code for a family of Na+ and C1- dependent transporters including those for GABA, glycine, proline, norepinephrine, serotonin and dopamine. Our long range goal is to understand how these transporters work as they move their neurotransmitter substrate across the plasma membrane. In this application, we propose to examine the relationship between the primary structure of the transporter and its function. We will use two approaches. The first is to examine the functional similarities and differences between transporters in this family, and to correlate sequence conservation between transporters with functional similarities. The second approach is to mutate amino acid residues in the transporter sequence that are correlated with a given function and then examine the consequences for various functional properties of the transporter. by this coupled approach we expect to learn more about the way these transporters work and also to identify specific amino acids required for transporter function.