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 [H-3]WIN 35,428 {2 beta-carbomethoxy-3 beta-(4-fluorophenyl)[H-3]tropane} and [H-3]mazindol were studied at 20 degrees C. [H-3]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 [H-3]WIN 35,428, but an increase of the radioligand concentration shifted this decrease to the right. [H-3]Mazindol binding was stimulated by Nat concentrations 110 mM and was rather unaffected at higher concentrations. The inhibition of [H-3]WIN 35,428 binding produced by 130 mM Naf was independent of the nature of the anion; in contrast, isothionate and H2PO4-/HCO3- produced a more pronounced inhibition of the [H-3]mazindol binding than Cl- and Br-, whereas I- tended to be a stimulant. Ca2+ and Mg2+ more potently inhibited the [H-3]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 [H-3]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 ex-elusive 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 Nai 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 [H-3]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.