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

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.

Importance of cholesterol in dopamine transporter function

“…In fact, two earlier studies (Wu et al . ; Chen et al . ) found, in the presence of Na + , an impact of Zn 2+ on K d rather than B max , whereas Liang et al .…”

Binding of uptake blockers to the neuronal dopamine transporter: further investigation about cationic and anionic requirements

“…This more complete model is consistent with the interactions of Na + and K + with the binding of 2β-carbomethoxy-3β-(4-fluorophenyl)[ 3 H]tropane ([ 3 H]WIN 35,428) to the hDAT (human DAT) expressed in HEK-293 cells (Li and Reith 1999). However, the interactions between Na + , K + and the native DAT could be even more complex, since the inability of Na + to antagonize the K + -induced inhibition of the [ 3 H]WIN 35,428 binding to rat striatal membranes and the shallow curves describing the binding inhibition resulting from increasing K + concentrations were not consonant with a competition between K + and Na + for common binding sites (Reith and Coffey 1993 (Wu et al 1997;Li and Reith 1999).…”

Modeling of the Interaction of Na+ and K+ with theBinding of Dopamine and [3H]WIN 35,428 to the Human Dopamine Transporter

“…The source of the DAT in the present experiments was the HEK‐293‐hDAT cell line developed by the group of Janowsky (Eshleman et al, 1997). The general conditions for growing the cells, working up the membrane preparations, and conducting the binding assays were as described by us previously (Wu et al, 1997). In brief, after cell Iysis, membranes were prepared and homogenized with a Brinkmann Polytron (setting of 6 for 15 s) in an ice‐cold 10 m M sodium phosphate buffer (NaPhos) resulting from mixing primary and half‐strength secondary NaPhos to pH 7.4 at room temperature.…”