PHARMACOLOGICAL HETEROGENEITY OF THE CLONED AND NATIVE HUMAN DOPAMINETRANSPORTER - DISASSOCIATION OF [H-3] WIN 35,428 AND [H-3] GBR 12,935BINDING

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 p harmacological profile of numerous dopamine transport substrates and i nhibitors on [H-3]DA uptake with the binding of [H-3]WIN 35,428 and [H -3]GBR 12,935 to COS-7 cells transiently expressing the cloned human D A transporter. [3H]DA uptake and [3H]WIN 35,428 binding was specific, saturable, and to a single class of binding sites with an estimated K- m/V-max of similar to 2 mu M and 6 pmol/min/10(5) cells for DA uptake and K-d/B-max values of similar to 10 nM and 113 fmol/10(5) cells for [H-3]WIN 35,428. [H-3]DA uptake was inhibited in a concentration-depen dent and uniphasic manner by dopaminergic agents with an appropriate r ank order of potency for the DA transporter. Although most uptake bloc kers inhibited [H-3]WIN 35,428 binding in a uniphasic manner, WIN 35,4 28, Lu 19,005, D-amphetamine, and DA clearly displayed the presence of both high and low affinity components. Comparison of the K-i values f or the inhibition of [H-3]DA uptake with [H-3]WIN 35,428 binding revea ls that, for uptake blockers and D-amphetamine, it is the high affinit y component that shares pharmacological identity with effects on DA up take (r = 0.9985), whereas for DA it is the low affinity site. In stri king contrast, however, [H-3]GBR 12,935 binding to COS-7 cells could n ot be made to exhibit a pharmacological profile indicative of the DA t ransporter and suggests that the site regulating functional [H-3]DA up take may not be identical with sites labeled by [H-3]GBR 12,935 in the se cells. Moreover, these sites appear unrelated to those previously d escribed in native membranes as ''piperazine acceptor'' or P450 protei ns. Comparison of K-i values and rank order of potency for the inhibit ion of [H-3]WIN 35,428 or [H-3]GBR 12,935 binding to human caudate mem branes reveals pharmacological homology, but not identity, with that o f the cloned DA uptake process. Taken together, these data suggest tha t 1) [H-3]WIN 35,428 recognizes two sites of the DA transporter, of wh ich only one appears to represent the functional state of the protein, and 2) [H-3]WIN 35,428 and [H-3]GBR 12,935 do not appear to bind the same functional form/ state of the DA transporter. Whether the noniden tity of binding sites is a manifestation of some post-translational re gulatory event (e.g., phosphorylation/accessory binding protein) or ca used by the existence of multiple molecular forms of the DA transporte r is currently unknown.