Abstract
The human dopamine transporter (hDAT) regulates synaptic dopamine (DA) levels and is the site of action of abused and therapeutic drugs. Here we study the effect of a threonine residue (Thr62 in hDAT) that is highly conserved within a canonical phosphorylation site (RETW) in the juxtamembrane N-terminal region of monoamine transporters. In stably transfected human embryonic kidney 293T cells, expression of T62D-hDAT was reduced compared with hDAT or T62A-hDAT. T62D-hDAT displayed dramatically reduced [3H]dopamine up-take but exhibited a higher basal dopamine efflux compared with hDAT or T62A-hDAT, as determined by measurements of [3H]dopamine efflux and amperometry. The high constitutive efflux in T62D-hDAT precluded the measurement of amphetamine-stimulated [3H]dopamine efflux, but when dopamine was added internally into voltage-clamped T62D-hDAT cells, amphetamine-induced efflux comparable with hDAT was detected by amperometry. In accordance with findings that Zn2+ can rescue reduced DA uptake in mutant transporters that are predominantly inward-facing, micromolar concentrations of Zn2+ markedly potentiated [3H]dopamine uptake in T62D-hDAT and permitted the measurement of amphetamine-stimulated dopamine efflux. These results suggest that T62D-hDAT prefers an inward-facing conformation in the transition between inward- and outward-facing conformations. For T62A-hDAT, however, the measured 50% reduction in both [3H]dopamine uptake and [3H]dopamine efflux was consistent with a slowed transition between inward- and outward-facing conformations. The mechanism underlying the important functional role of Thr62 in hDAT activity suggested by these findings is examined in a structural context using dynamic simulations of a three-dimensional molecular model of DAT.
Footnotes
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This work was supported by the National Institutes of Health National Institute on Drug Abuse [Grants DA11697, DA12408, DA14684, DA022413, DA023694]. This work used the DNA Sequencing Core of the Michigan Diabetes Research and Training Center, supported by the National Institutes of Health National Institute of Diabetes and Digestive and Kidney Diseases [Grant 5P60-DK20572].
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ABBREVIATIONS: DAT, dopamine transporter; AMPH, amphetamine; DA, dopamine; HEK, human embryonic kidney; KRH, Krebs-Ringer-HEPES; PKC, protein kinase C; PKA, cAMP-dependent protein kinase; TM, transmembrane domain; ANOVA, analysis of variance; cGPK, cGMP-dependent protein kinase; MD, molecular dynamics; FEP, free energy perturbation; GBR12935, 1-[2-(diphenylmethoxy)ethyl]-4-(3-phenylpropyl)-piperazine; IL, intracellular loop.
- Received May 12, 2008.
- Accepted December 12, 2008.
- The American Society for Pharmacology and Experimental Therapeutics
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