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 a nd inhibitors were investigated by using rotating disk electrode voltammetr y. The Na+ dependence of DA uptake varied with Na+ substitutes, hyperbolic with Li+, almost linear at 1 mu M DA but hyperbolic at 8 mu M DA with choli ne, and sigmoidal with K+. With Na+ substituted by Li+, K-[DA] decreased an d V-app remained constant with increasing [Na+], whereas K[Na+] decreased a nd V-app increased with increasing [DA], suggesting an ordered sequence wit h Na+ binding before DA. Similar trends for the Na+-DA interactions were ob served in the presence of cocaine. Cocaine inhibited DA uptake solely by in creasing K-[DA], with its K-i not significantly different at 55 and 155 mM [Na+], whereas it inhibited Nat stimulation by reducing V-app more than K[N a+] at 1 mu M DA, and V-app only and less potently at 8 mu M DA. Thus, coca ine may compete with DA, not with Na+, for the transporter, and might not f ollow 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 ma inly by reducing V-app but affected cocaine inhibition by elevating K-i. De spite their different patterns for inhibiting DA uptake, nontransportable i nhibitors cocaine, methylphenidate, mazindol, and 1-[2-[bis(4-fluorophenyl) methoxy]ethyl]-4-(3-phenyl-2-propyl)piperazine (GBR12909) showed similarly modest Na+ dependence in their K-i values. In contrast, substrates DA, m-ty ramine, and amphetamine displayed a similarly stronger Na+ requirement for their apparent affinities.