The serotonin transporter is responsible for terminating the action of serotonin that has been released into the synaptic cleft. The activity of this transmembrane protein is blocked by antidepressant drugs and cocaine, and modulated by amphetamine derivatives, some of which are neurotoxic. The mechanism by which the transporter accumulates serotonin within the cell is understood in broad terms. The energy for transport comes from Na+, C1 and K= ions flowing downhill across the plasma membrane. Now that the primary structure of the serotonin transporter is known from cDNA cloning, it is important to understand how the various functions of the transporter, including its interactions with drugs of abuse, are related tot he sequence and ultimately to the structure. This application proposes two approaches to this problem. The first approach will yield information on the transmembrane orientation and functional importance of hydrophilic """"""""loops"""""""" believed to connect transmembrane domains. Mutated forms of the transporter containing unique tagged sequence elements will be expressed in mammalian cells and analyzed biochemically and physiologically. The accessibility of various parts of the transporter protein to the cell surface will be determined using a variety of membrane-impermeant reagents. At the same time the transport properties and drug sensitivities of the tagged mutants will be tested. In addition to providing a better picture of the transmembrane topology, this approach will address basic questions of transporter dynamics, and help to localize drug binding sites on the transporter surface. The second approach will examine the effects of stably expressing the serotonin transporter at high levels in different cell types. These studies will use inducible promoters to test the hypothesis that expression of the transporter is inhibitory to cell growth. In particular, the ability of amphetamines to stimulate transporter activity, and consequently dissipate transmembrane ion gradients, will be addressed. These studies are expected to shed light on the ways that the serotonin transporter mediates degeneration of nerve terminals in the presence of amphetamine derivatives.

Agency
National Institute of Health (NIH)
Institute
National Institute on Drug Abuse (NIDA)
Type
Research Project (R01)
Project #
2R01DA007259-04
Application #
2119636
Study Section
Drug Abuse Biomedical Research Review Committee (DABR)
Project Start
1991-12-15
Project End
1999-12-31
Budget Start
1995-02-01
Budget End
1995-12-31
Support Year
4
Fiscal Year
1995
Total Cost
Indirect Cost
Name
Yale University
Department
Pharmacology
Type
Schools of Medicine
DUNS #
082359691
City
New Haven
State
CT
Country
United States
Zip Code
06520
Zhang, Yuan-Wei; Tavoulari, Sotiria; Sinning, Steffen et al. (2018) Structural elements required for coupling ion and substrate transport in the neurotransmitter transporter homolog LeuT. Proc Natl Acad Sci U S A 115:E8854-E8862
Zhang, Yuan-Wei; Turk, Benjamin E; Rudnick, Gary (2016) Control of serotonin transporter phosphorylation by conformational state. Proc Natl Acad Sci U S A 113:E2776-83
Tavoulari, Sotiria; Margheritis, Eleonora; Nagarajan, Anu et al. (2016) Two Na+ Sites Control Conformational Change in a Neurotransmitter Transporter Homolog. J Biol Chem 291:1456-71
Sandtner, Walter; Stockner, Thomas; Hasenhuetl, Peter S et al. (2016) Binding Mode Selection Determines the Action of Ecstasy Homologs at Monoamine Transporters. Mol Pharmacol 89:165-75
Fenollar-Ferrer, Cristina; Stockner, Thomas; Schwarz, Thomas C et al. (2014) Structure and regulatory interactions of the cytoplasmic terminal domains of serotonin transporter. Biochemistry 53:5444-60
Rudnick, Gary; Krämer, Reinhard; Blakely, Randy D et al. (2014) The SLC6 transporters: perspectives on structure, functions, regulation, and models for transporter dysfunction. Pflugers Arch 466:25-42
Porton, B; Greenberg, B D; Askland, K et al. (2013) Isoforms of the neuronal glutamate transporter gene, SLC1A1/EAAC1, negatively modulate glutamate uptake: relevance to obsessive-compulsive disorder. Transl Psychiatry 3:e259
Rudnick, Gary (2013) How do transporters couple solute movements? Mol Membr Biol 30:355-9
Schicker, Klaus; Uzelac, Zeljko; Gesmonde, Joan et al. (2012) Unifying concept of serotonin transporter-associated currents. J Biol Chem 287:438-45
Bulling, Simon; Schicker, Klaus; Zhang, Yuan-Wei et al. (2012) The mechanistic basis for noncompetitive ibogaine inhibition of serotonin and dopamine transporters. J Biol Chem 287:18524-34

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