Dopamine D2 Receptor Regulation of the Dopamine Transporter Expressed in Xenopus laevis Oocytes Is Voltage-Independent
- Department of Pharmacology and Neuroscience Program, University of Colorado Health Sciences Center, Denver, Colorado
Abstract
Presynaptic dopamine D2 receptors (D2Rs) regulate dopamine transporter (DAT) activity in the brain. A potential mechanism was suggested by the observations that somatodendritic D2R activation produces hyperpolarization and the velocity of DAT expressed in Xenopus laevis oocytes varies with changes in membrane potential. To investigate whether D2R regulation of DAT function is voltage-dependent, we coexpressed the long isoform of the human (h) D2R and the hDAT in oocytes. Most DAT substrates fully activate D2Rs at concentrations used to measure uptake. Thus, DAT function was compared under conditions of maximal D2R activation (0.1–10 μM DA) or maximal D2R blockade (DA + 1 μM (−)-sulpiride). D2R activation significantly increased [3H]DA uptake into unclamped oocytes expressing relatively lower velocities. Uptake measured with a saturating concentration of DA suggested a D2R-induced increase in Vmax. The D2R-mediated enhancement of DA uptake was not associated with changes in resting membrane potential and was abolished by pertussis toxin pretreatment. Furthermore, in voltage-clamped oocytes, D2R activation enhanced both DA uptake and DAT-mediated steady-state currents by as much as 70%. Activation of D2Rs resulted in a 59% increase in cell surface binding of the cocaine analog [3H]WIN 35,428; this effect was also abolished by pertussis toxin pretreatment. Saturation experiments confirmed that D2R activation was associated with an increased Bmax and unchangedKi for [3H]WIN 35,428. These results suggest that D2R-induced up-regulation of DAT activity occurs via a voltage-independent mechanism that depends on Gi/o activation and a rapid increase in expression of functional DAT molecules at the cell surface.
Footnotes
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Send reprint requests to: Dr. R. Dayne Mayfield, Institute for Cellular and Molecular Biology, College of Natural Sciences, University of Texas, 2500 Speedway, MBB 1.124, Austin, TX 78712. E-mail: dayne.mayfield{at}mail.utexas.edu
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This work was supported by National Institutes of Health Grants DA04216 and DA00174.
- Abbreviations:
- DAT
- dopamine transporter
- D2R
- dopamine D2 receptor
- DA
- dopamine (3,4,7-dihydroxyphenylethylamine)
- FRB
- frog Ringers' buffer
- GIRK
- G protein-coupled inwardly rectifying potassium channel
- h
- human
- PKC
- protein kinase C
- PTX
- pertussis toxin
- RMP
- resting membrane potential
- WIN 35,428
- 2β-carbomethoxy-3β-(4-fluorophenyl)tropane
- GABA
- γ-aminobutyric acid
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- Received April 21, 2000.
- Accepted September 21, 2000.
- The American Society for Pharmacology and Experimental Therapeutics
