Protein tyrosine kinase inhibitors alter human dopamine transporter activity in Xenopus oocytes.
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 microM), we found that several PTK inhibitors, namely, genistein, lavendustin A, and tyrphostin 25 (10 microM), decreased DA uptake velocity by 58, 41, and 30% of control, respectively. Furthermore, genistein potently inhibited DA uptake with a K(i) = 68 nM. Kinetic analysis confirmed that genistein decreased the V(max) of the DAT, with no change in K(m). 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 [(3)H]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.
Doolen
S
S
Department of Pharmacology and Neuroscience Program, University of Colorado Health Sciences Center, Denver, Colorado, USA. suzanne.doolen@uchsc.edu
Zahniser
N R
NR
D000818:Animals; D001665:Binding Sites; D002352:Carrier Proteins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D003042:Cocaine / Q000031:analogs & derivatives* / Q000494:pharmacology; D004298:Dopamine / Q000378:metabolism; D050483:Dopamine Plasma Membrane Transport Proteins; D018765:Dopamine Uptake Inhibitors / Q000494:pharmacology; D004594:Electrophysiology; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D019833:Genistein / Q000494:pharmacology; D006801:Humans; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; D009865:Oocytes / Q000187:drug effects / Q000378:metabolism; D010636:Phenols / Q000494:pharmacology; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors*; D014162:Transfection; D014316:Tritium; D020032:Tyrphostins / Q000494:pharmacology; D014982:Xenopus laevis
D002352:Carrier Proteins; D050483:Dopamine Plasma Membrane Transport Proteins; D018765:Dopamine Uptake Inhibitors; D004791:Enzyme Inhibitors; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; D010636:Phenols; C493214:SLC6A3 protein, human; D020032:Tyrphostins; D014316:Tritium; C079836:tyrphostin 25; C062898:lavendustin A; C021279:(1R-(exo,exo))-3-(4-fluorophenyl)-8-methyl-8- azabicyclo(3.2.1)octane-2-carboxylic acid, methyl ester; D019833:Genistein; D011505:Protein-Tyrosine Kinases; D003042:Cocaine; D004298:Dopamine