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

Abstract

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.

A Juxtamembrane Mutation in the N Terminus of the Dopamine Transporter Induces Preference for an Inward-Facing Conformation

“…Because both V max and K m values for T62D-hDAT were reduced compared with hDAT, the overall catalytic effectiveness of the uptake process in T62D-hDAT was only 30% less than in hDAT. The second-order rate constant (V max /k cat ), however, is much lower than that reported in another study using hDAT-HEK cells (1.2 ϫ 10 7 M Ϫ1 ⅐ s Ϫ1 ) (Earles and Schenk, 1999). Lower temperature (25 versus 37°C) could partially account for this, but other factors could be our construct or the cells.…”

Labeling of Dopamine Transporter Transmembrane Domain 1 with the Tropane Ligand N-[4-(4-Azido-3-[125I]iodophenyl)butyl]-2β-carbomethoxy-3β-(4-chlorophenyl)tropane Implicates Proximity of Cocaine and Substrate Active Sites

“…Together, these findings strongly suggest that this region of DAT is part of the DA active site. Because all of these residues are within or immediately adjacent to the [ 125 I]MFZ 2-24 labeled sequence, our results suggest that cocaine binds to DAT at or near the TM1 DA binding site, where it may compete with DA for access, consistent with the known competitive mechanism of transport inhibition (Reith et al, 1992;Earles and Schenk, 1999).…”

Binding and Transport in Norepinephrine Transporters

“…Although, cocaine is reported to inhibit transporters through a uncompetitive mechanism (56,57). From the literature, Na ϩ removal has no effect on 5HT and DA displacement of high affinity SERT and DAT antagonists (28,31,32).…”