Abstract
The dopamine (DA) transporter (DAT) regulates dopaminergic synaptic transmission by controlling extracellular levels of DA. Thus, understanding signaling mechanisms that alter DAT function is critical for understanding dopaminergic neurotransmission. We have expressed the human DAT (hDAT) in Xenopus laevis oocytes to test the hypothesis that protein tyrosine kinases (PTKs) acutely regulate DAT function by altering cell surface expression of the transporter. Using a relatively high concentration of DA (10 μM), we found that several PTK inhibitors, namely, genistein, lavendustin A, and tyrphostin 25 (10 μM), decreased DA uptake velocity by 58, 41, and 30% of control, respectively. Furthermore, genistein potently inhibited DA uptake with a Ki = 68 nM. Kinetic analysis confirmed that genistein decreased the Vmaxof the DAT, with no change in Km. The effects of PTK inhibition on hDAT-associated currents were also measured. All three PTK inhibitors attenuated substrate transport-associated currents to similar extents as DA uptake. In contrast, the potent Src inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) did not significantly inhibit either DA uptake or transport-associated currents. PTK inhibitors decreased hDAT-associated leak currents, however in a more variable manner than for uptake and transport-associated currents. Genistein also decreased cell surface binding of [3H]WIN 35,428 to hDAT by 48% of control. Together, these data provide several lines of evidence suggesting that PTK inhibition rapidly reduces hDAT activity via redistribution of the transporter away from the cell surface. Thus, PTKs likely represent another component of cellular signaling cascades that acutely regulate neurotransmitter transporters.
Footnotes
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Send reprint requests to: Dr. Suzanne Doolen, 4200 E. 9th Ave., Box C-236, Department of Pharmacology, University of Colorado Health Sciences Center, Denver, CO 80262. E-mail:suzanne.doolen{at}uchsc.edu
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This work was supported by National Institute of Health Grant DA04216, a National Research Service Award DA05956 to S.D., and an Instrument Society of America DA00174 to N.R.Z.
- Abbreviations:
- DA
- dopamine
- CNS
- central nervous system
- DAT
- dopamine transporter
- GAT
- γ-aminobutyric acid transporter
- SERT
- serotonin transporter
- NET
- norepinephrine transporter
- PKC
- protein kinase C
- hDAT
- human dopamine transporter
- PTK
- protein tyrosine kinase
- RPTK
- receptor protein tyrosine kinase
- BDNF
- brain-derived growth factor
- FRB
- frog Ringer's buffer
- 3-PPP
- R-(+)-3-(3-hydroxyphenyl)-N-propylpiperidine
- [3H]WIN 35,428
- β-carbomethoxy-3β-(4-fluorophenyl)[3H]tropane
- EGFR
- epidermal growth factor receptor
- GBR 12909
- 1-[2-(bis[4-fluorophenyl]methoxy)ethyl]-4-[3-phenyl-propyl]piperazine
- PP2
- 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine
- Received August 18, 2000.
- Accepted November 28, 2000.
- The American Society for Pharmacology and Experimental Therapeutics
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