MULTIPLE IONIC CONDUCTANCES OF THE HUMAN DOPAMINE TRANSPORTER - THE ACTIONS OF DOPAMINE AND PSYCHOSTIMULANTS

Electrophysiological and pharmacological studies of a cloned human dop amine transporter (hDAT) were undertaken to investigate the mechanisms of transporter function and the actions of drugs at this target. Usin g two-electrode voltage-clamp techniques with hDAT-expressing Xenopus laevis oocytes, we show that hDAT can be considered electrogenic by tw o criteria. (1) Uptake of hDAT substrates gives rise to a pharmacologi cally appropriate ''transport-associated'' current. (2) The velocity o f 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 indi vidual oocytes revealed that charge movement during substrate transloc ation was greater than would be expected for a transport mechanism wit h fixed stoichiometry of 2 Na+ and 1 Cl- per DA(+) molecule. In additi on to the transport-associated current, hDAT also mediates a constitut ive leak current, the voltage and ionic dependencies of which differ m arkedly from those of the transport-associated current. ion substituti on experiments suggest that alkali cations and protons are carried by the hDAT leak conductance. In contrast to the transport-associated fun ctions, the leak does not require Na+ or Cl-, and DAT ligands readily interact with the transporter even in the absence of these ions. The c urrents that hDAT mediates provide a functional assay that readily dis tinguishes the modes of action of amphetamine-like ''DA-releasing'' dr ugs from cocaine-like translocation blockers. In addition, the voltage dependence of DA uptake suggests a mechanism through which presynapti c DA autoreceptor activation may accelerate the termination of dopamin ergic neurotransmission in vivo.