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

Abstract.

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.

Ca²⁺ and Mg²⁺ more potently inhibited the [³H]WIN 35,428 binding than K⁺. All these cations recognize a site which is not mutually exclusive with that of the radioligand since they induced the dissociation of the [³H]WIN 35,428-rDAT complex, an effect which was reduced (K⁺) or modified (Ca²⁺) when the Na⁺ concentration was increased. This site is likely to be the Na⁺ site by which low Na⁺ concentrations allosterically stimulate the uptake blocker binding. However, the intensity of the cation-induced dissociations was moderate and the main component of the binding inhibition that these cations produced results from the occupancy of a cation site, mutually exclusive with that of the radioligand. Thus, the WIN 35,428 binding inhibition produced by Ca²⁺, K⁺ and Na⁺ was competitive, and Na⁺ reduced the inhibitory potency of Ca²⁺ and K⁺. This reduction was more intense for Ca²⁺ and Mg²⁺ than for K⁺, suggesting that occupancy of the cation site by a divalent cation activated a strong negative allosteric interaction between this site and the Na⁺ site.

Decrease in the Na⁺ concentration from 10 mM to 5 mM, or replacement of 5 mM HCO₃ ^–/H₂PO₄ ^– by an equimolar concentration of isethionate or Cl^– did not modify [³H]WIN 35,428 binding dissociation. Level(s) at which anions stimulate and inhibit the binding of uptake blockers remain uncertain and could be specific for each radioligand.