CATIONS AFFECT [H-3] MAZINDOL AND [H-3] WIN-35,428 BINDING TO THE HUMAN DOPAMINE TRANSPORTER IN A SIMILAR FASHION

The present study addresses the possibility that there are different c ocaine-related and mazindol-related binding domains on the dopamine tr ansporter (DAT) that show differential sensitivity to cations. The eff ects of Zn2+, Mg2+, Hg2+, Li+, K+, and Na+ were assessed on the bindin g of [H-3]mazindol and [H-3]WIN 35,428 to the human (h) DAT expressed in C6 glioma cells under identical conditions for intact cell and memb rane assays, The latter were performed at both 0 and 21 degrees C. Zn2 + (30-100 mu M) stimulated binding of both radioligands to membranes, with a relatively smaller effect for [H-3]mazindol; Mg2+ (0.1-100 mu M ) had no effect; Hg2+ at similar to 3 mu M stimulated binding to membr anes, with a relatively smaller effect for [H-3]mazindol than [H-3]WIN 35,428 at 0 degrees C, and at 30-100 mu M inhibited both intact cell and membrane binding; Li+ and K+ substitution (30-100 mM) inhibited bi nding to membranes more severely than to intact cells; and Na+ substit ution was strongly stimulatory. With only a few exceptions, the patter ns of ion effects were remarkably similar for both radioligands at bot h 0 and 21 degrees C, suggesting the involvement of common binding dom ains on the hDAT impacted similarly by cations, Therefore, if there ar e different binding domains for WIN 35,428 and mazindol, these are not affected differentially by the cations studied in the present experim ents, except for the stimulatory effect of Zn2+ at 0 and 21 degrees C and Hg2+ at 0 degrees C.