Sodium-dependent dopamine transporter
dopamine:sodium symporter activity
Pharmacological heterogeneity of the cloned and native human dopamine transporter: disassociation of [3H]WIN 35,428 and [3H]GBR 12,935 binding.
Controversy exists as to whether the functional state of the dopamine (DA) transporter is identical to sites mediating the specific binding of selective DA transporter radioligands. Therefore, we compared the pharmacological profile of numerous dopamine transport substrates and inhibitors on [3H]DA uptake with the binding of [3H]WIN 35,428 and [3H]GBR 12,935 to COS-7 cells transiently expressing the cloned human DA transporter. [3H]DA uptake and [3H]WIN 35,428 binding was specific, saturable, and to a single class of binding sites with an estimated Km/Vmax of approximately 2 microM and 6 pmol/min/10(5) cells for DA uptake and Kd/Bmax values of approximately 10 nM and 113 fmol/10(5) cells for [3H]WIN 35,428. [3H]DA uptake was inhibited in a concentration-dependent and uniphasic manner by dopaminergic agents with an appropriate rank order of potency for the DA transporter. Although most uptake blockers inhibited [3H]WIN 35,428 binding in a uniphasic manner, WIN 35,428, Lu 19,005, D-amphetamine, and DA clearly displayed the presence of both high and low affinity components. Comparison of the Ki values for the inhibition of [3H]DA uptake with [3H]WIN 35,428 binding reveals that, for uptake blockers and D-amphetamine, it is the high affinity component that shares pharmacological identity with effects on DA uptake (r = 0.9985), whereas for DA it is the low affinity site. In striking contrast, however, [3H]GBR 12,935 binding to COS-7 cells could not be made to exhibit a pharmacological profile indicative of the DA transporter and suggests that the site regulating functional [3H]DA uptake may not be identical with sites labeled by [3H]GBR 12,935 in these cells. Moreover, these sites appear unrelated to those previously described in native membranes as "piperazine acceptor" or P450 proteins. Comparison of Ki values and rank order of potency for the inhibition of [3H]WIN 35,428 or [3H]GBR 12,935 binding to human caudate membranes reveals pharmacological homology, but not identity, with that of the cloned DA uptake process. Taken together, these data suggest that 1) [3H]WIN 35,428 recognizes two sites of the DA transporter, of which only one appears to represent the functional state of the protein, and 2) [3H]WIN 35,428 and [3H]GBR 12,935 do not appear to bind the same functional form/state of the DA transporter. Whether the nonidentity of binding sites is a manifestation of some post-translational regulatory event (e.g., phosphorylation/accessory binding protein) or caused by the existence of multiple molecular forms of the DA transporter is currently unknown.