Cations Affect [3H]Mazindol and [3H]WIN 35,428 Binding to the Human Dopamine Transporter in a Similar Fashion


Address correspondence and reprint requests to Dr. M. E. A. Reith at Department of Biomedical and Therapeutic Sciences, College of Medicine, University of Illinois, Box 1649, Peoria, IL 61656, U.S.A.


Abstract: The present study addresses the possibility that there are different cocaine-related and mazindol-related binding domains on the dopamine transporter (DAT) that show differential sensitivity to cations. The effects of Zn2+, Mg2+, Hg2+, Li+, K+, and Na+ were assessed on the binding of [3H]mazindol and [3H]WIN 35,428 to the human (h) DAT expressed in C6 glioma cells under identical conditions for intact cell and membrane assays. The latter were performed at both 0 and 21°C. Zn2+ (30–100 µM) stimulated binding of both radioligands to membranes, with a relatively smaller effect for [3H]mazindol; Mg2+ (0.1–100 µM) had no effect; Hg2+ at ∼3 µM stimulated binding to membranes, with a relatively smaller effect for [3H]mazindol than [3H]WIN 35,428 at 0°C, and at 30–100 µM inhibited both intact cell and membrane binding; Li+ and K+ substitution (30–100 mM) inhibited binding to membranes more severely than to intact cells; and Na+ substitution was strongly stimulatory. With only a few exceptions, the patterns of ion effects were remarkably similar for both radioligands at both 0 and 21°C, suggesting the involvement of common binding domains on the hDAT impacted similarly by cations. Therefore, if there are different binding domains for WIN 35,428 and mazindol, these are not affected differentially by the cations studied in the present experiments, except for the stimulatory effect of Zn2+ at 0 and 21°C and Hg2+ at 0°C.

Abbreviations used: DA, dopamine; DAT, DA transporter; hDAT, human DAT; C6-hDAT, C6 glioma cells expressing hDAT; GBR 12783, 1-[2-(diphenylmethoxy)ethyl]-4-(3-phenyl-2-propenyl)piperazine; GBR 12935, 1-[2-(diphenylmethoxy)ethyl]-4-(3-phenylpropyl)piperazine; WIN 35,428, 2β-carbomethoxy-3β-(4-fluorophenyl)tropane.