Multisubtrate mechanism for the inward transport of dopamine by the human dopamine transporter expressed in HEK cells and its inhibition by cocaine.

Rotating disk electrode voltammetry was used to measure the time-resolved inward transport of dopamine into human embryonic kidney cells expressing the human transporter for dopamine and a kinetic mechanism of transport is hypothesized. Dopamine transport in this preparation was highly concentrative, with a 10(6)-10(7) inward bias, first order in dopamine and the K(m) and V(max) were found to be 1.6 microM and 18 pmol/sec x 10(6) cells), respectively. The hDAT turnover was estimated to be approximately 18 s(-1) and the second order rate constant of association of dopamine with hDAT was approximately 10(7) M(-1)s(-1). Dopamine transport was found to have a second order dependence on Na(+) (K(Na) approximately 100 mM) and a first order dependence on Cl(-) (K(Cl) approximately 12 mM). Multisubstrate analyses suggested that hDAT operates with an ordered kinetic mechanism in which Na(+) binds first to the transporter protein, dopamine second, and Cl(-) last before translocation of dopamine into or across the membrane. Cocaine competitively inhibited dopamine transport (reaction order of unity and K(i) approximately 0.34 microM) with no discernible effect at the Na(+) and Cl(-) binding sites. These results differ from those of previous studies conducted in preparations of the striatum and nucleus accumbens. Comparisons of the variant results are made and an analysis of the differing apparent kinetic mechanisms is presented.