Protein tyrosine kinase inhibitors alter human dopamine transporter activity in Xenopus oocytes

The dopamine (DA) transporter (DAT) regulates dopaminergic synaptic transmi ssion by controlling extracellular levels of DA. Thus, understanding signal ing mechanisms that alter DAT function is critical for understanding dopami nergic 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 t ransporter. Using a relatively high concentration of DA (10 muM), we found that several PTK inhibitors, namely, genistein, lavendustin A, and tyrphost in 25 (10 muM), 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 o f the DAT, with no change in K-m. The effects of PTK inhibition on hDAT-ass ociated currents were also measured. All three PTK inhibitors attenuated su bstrate transport-associated currents to similar extents as DA uptake. In c ontrast, the potent Src inhibitor 4-amino-5-(4-chlorophenyl)- 7-(t-butyl) p yrazolo[3,4-d] pyrimidine (PP2) did not significantly inhibit either DA upt ake or transport-associated currents. PTK inhibitors decreased hDAT-associa ted leak currents, however in a more variable manner than for uptake and tr ansport-associated currents. Genistein also decreased cell surface binding of [H-3] WIN 35,428 to hDAT by 48% of control. Together, these data provide several lines of evidence suggesting that PTK inhibition rapidly reduces h DAT activity via redistribution of the transporter away from the cell surfa ce. Thus, PTKs likely represent another component of cellular signaling cas cades that acutely regulate neurotransmitter transporters.