Vol. 290, Issue 3, 940-949, September 1999

Cationic Modulation of Human Dopamine Transporter: Dopamine Uptake and Inhibition of Uptake¹

Nianhang Chen , Clarence G. Trowbridge and Joseph B. Justice, Jr.

Department of Pharmacology, Nanjing Medical University, Nanjing, Peoples Republic of China (N.C.); and Department of Chemistry, Emory University, Atlanta, Georgia (N.C., C.G.T., J.B.J.)

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 µM DA but hyperbolic at 8 µM 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 µM DA, and V_app only and less potently at 8 µM 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)piperazine (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.