Dopamine Transporter Amino and Carboxyl Termini Synergistically Contribute to Substrate and Inhibitor Affinities
Brudnick Neuropsychiatric Research Institute, Department of Psychiatry
Biochemistry | Mental and Social Health | Neuroscience and Neurobiology | Psychiatry | Psychiatry and Psychology
Extracellular dopamine and serotonin concentrations are determined by the presynaptic dopamine (DAT) and serotonin (SERT) transporters, respectively. Numerous studies have investigated the DAT and SERT structural elements contributing to inhibitor and substrate binding. To date, crystallographic studies have focused on conserved transmembrane domains, where multiple substrate binding and translocation features are conserved. However, it is unknown what, if any, role the highly divergent intracellular N and C termini contribute to these processes. Here, we used chimeric proteins to test whether DAT and SERT N and C termini contribute to transporter substrate and inhibitor affinities. Replacing the DAT N terminus with that of SERT had no effect on DA transport Vmax but significantly decreased DAT substrate affinities for DA and amphetamine. Similar losses in uptake inhibition were observed for small DAT inhibitors, whereas substituting the DAT C terminus with that of SERT affected neither substrate nor inhibitor affinities. In contrast, the N-terminal substitution was completely tolerated by the larger DAT inhibitors, which exhibited no loss in apparent affinity. Remarkably, all affinity losses were rescued in DAT chimeras encoding both SERT N and C termini. The sensitivity to amino-terminal substitution was specific for DAT, because replacing the SERT N and/or C termini affected neither substrate nor inhibitor affinities. Taken together, these findings provide compelling experimental evidence that DAT N and C termini synergistically contribute to substrate and inhibitor affinities.
amphetamine, cocaine, dopamine transporter, kinetics, monoamine transporter, psychostimulants, reuptake, selective serotonin reuptake inhibitor (SSRI), serotonin transporter
DOI of Published Version
J Biol Chem. 2017 Jan 27;292(4):1302-1309. doi: 10.1074/jbc.M116.762872. Epub 2016 Dec 16. Link to article on publisher's site.
The Journal of biological chemistry
Sweeney, Carolyn G.; Tremblay, Bradford P.; Stockner, Thomas; Sitte, Harald H.; and Melikian, Haley E., "Dopamine Transporter Amino and Carboxyl Termini Synergistically Contribute to Substrate and Inhibitor Affinities" (2017). Psychiatry Publications and Presentations. 762.