Cationic modulation of human dopamine transporter: dopamine uptake and inhibition of uptake

Effects of cations on dopamine (DA) uptake into cells expressing the human dopamine transporter and on inhibition of DA uptake by various substrates and inhibitors were investigated by using rotating disk electrode voltammetry. The Na(+) dependence of DA uptake varied with Na(+) substitutes, hyperbolic with Li(+), almost linear at 1 microM DA but hyperbolic at 8 microM DA with choline, and sigmoidal with K(+). With Na(+) substituted by Li(+), K([DA]) decreased and V(app) remained constant with increasing [Na(+)], whereas K([Na+]) decreased and V(app) increased with increasing [DA], suggesting an ordered sequence with Na(+) binding before DA. Similar trends for the Na(+)-DA interactions were observed in the presence of cocaine. Cocaine inhibited DA uptake solely by increasing K([DA]), with its K(i) not significantly different at 55 and 155 mM [Na(+)], whereas it inhibited Na(+) stimulation by reducing V(app) more than K([Na+]) at 1 microM DA, and V(app) only and less potently at 8 microM DA. Thus, cocaine may compete with DA, not with Na(+), for the transporter, and might not follow a strictly ordered reaction with Na(+). With Na(+) substituted by K(+), K([DA]) or K([Na+]) became insensitive to Na(+) or DA. K(+) impaired the DA uptake mainly by reducing V(app,) but affected cocaine inhibition by elevating K(i). Despite their different patterns for inhibiting DA uptake, nontransportable inhibitors cocaine, methylphenidate, mazindol, and 1-[2-[bis(4-fluorophenyl)methoxy]ethyl]-4-(3-phenyl-2-propyl)piperazi ne (GBR12909) showed similarly modest Na(+) dependence in their K(i) values. In contrast, substrates DA, m-tyramine, and amphetamine displayed a similarly stronger Na(+) requirement for their apparent affinities.