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

Effects of ions on the binding of uptake blockers to the rat dopamine transporter (rDAT) labelled with [3H]WIN 35,428 {2β-carbomethoxy-3β-(4-fluorophenyl)[3H] tropane} and [3H]mazindol were studied at 20°C. [3H]WIN 35,428 binding increased with Na+ concentrations of up to 10–60 mM and decreased at higher concentrations. At pH 7.4, incubation media containing NaCl and/or Na2HPO4/NaH2PO4 were less stimulant than an NaHCO3/NaH2PO4 medium and they shifted maximal binding values to higher ionic concentrations. In an NaHCO3/NaH2PO4-buffered medium, Na+ concentrations >10 mM decreased the binding of 0.2 nM [3H]WIN 35,428, but an increase of the radioligand concentration shifted this decrease to the right. [3H]Mazindol binding was stimulated by Na+ concentrations ≤10 mM and was rather unaffected at higher concentrations. The inhibition of [3H]WIN 35,428 binding produced by 130 mM Na+ was independent of the nature of the anion; in contrast, isothionate and H2PO4–/HCO3– produced a more pronounced inhibition of the [3H]mazindol binding than Cl– and Br–, whereas I– tended to be a stimulant.