The principal objective of this proposal is to understand the molecular mechanism of serotonin uptake by the mammalian serotonin transporter. The serotonin transporter utilizes the Na+ electrochemical potential to transpor6t neurotransmitters into the cell against their concentration gradient, so that a low extracellular neurotransmitter concentration can be maintained. The molecular mechanism that underlies this process is not known. The proposed set of experiments will help to establish an integrated picture of transporter function and identify structural domains that participate in specific steps of the transport cycle. According to this study's working hypothesis, the transporter protein contains a channel-like lumen flanked by extracellular and intracellular gates. The opening and closing of these gates is governed by the binding of organic substrate and ions in the lumen. The energetic coupling between neurotransmitter transport and the electrochemical potential of ions is a result of the sequential opening and closing of the gates as well as the dependance of neurotransmitter binding on the binding of co-transporting ions. The proposed research will utilize electrophysiology and other functional measurements in order to characterize mutated transporters, and to identify and study functional domains that contribute to specific aspects of the transport process. We will investigate domains in the protein that (1) participate in transporter gating, (2) form the transporter lumen, and (3) form and are actively involved with ion and serotonin binding sites. Studies have shown that an alterations in the activity of the human serotonin transporter is associated with several mental disorders and possibly with alcohol and cocaine abuse. The transporter is also a major target for therapeutic drugs such as fluoxetine, methylphenidate, and amphetamine. Understanding the roles of the serotonin transporter in normal brain function, mental disorders, and drug abuse requires more insight into the biophysical and molecular mechanisms of transporter function.