Multiple ionic conductances of the human dopamine transporter: The actions of dopamine and psychostimulants

Electrophysiological and pharmacological studies of a cloned human dopamine transporter (hDAT) were undertaken to investigate the mechanisms of transporter function and the actions of drugs at this target. Using two- electrode voltage-clamp techniques with hDAT-expressing Xenopus laevis oocytes, we show that hDAT can be considered electrogenic by two criteria. (1) Uptake of hDAT substrates gives rise to a pharmacologically appropriate 'transport-associated' current (2) The velocity of DA uptake measured in oocytes clamped at various membrane potentials was voltage-dependent, increasing with hyperpolarization. Concurrent measurement of transport- associated current and substrate flux in individual oocytes revealed that charge movement during substrate translocation was greater than would be expected for a transport mechanism with fixed stoichiometry of 2 Na⁺ and 1 Cl^- per DA⁺ molecule. In addition to the transport-associated current, hDAT also mediates a constitutive leak current, the voltage and ionic dependencies of which differ markedly from those of the transport-associated current. Ion substitution experiments suggest that alkali cations and protons are carried by the hDAT leak conductance. In contrast to the transport-associated functions, the leak does not require Na⁺ or Cl^-, and DAT ligands readily interact with the transporter even in the absence of these ions. The currents that hDAT mediates provide a functional assay that readily distinguishes the modes of action of amphetamine-like 'DA-releasing' drugs from cocaine-like translocation blockers. In addition, the voltage dependence of DA uptake suggests a mechanism through which presynaptic DA autoreceptor activation may accelerate the termination of dopaminergic neurotransmission in vivo.