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 [2beta-carbomethoxy-3beta-(4-fluorophenyl)-[3H] tropane] and [3H]mazindol were studied at 20 degrees 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 < or =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.

Is Na + required for the binding of dopamine, amphetamine, tyramine, and octopamine to the human dopamine transporter?

“…4), and Na + removes the Tris + inhibition by the negative allosteric interaction (squiggly arrow a) that occurs between site 2 (Na + site) and the cation site in site 1, in addition to a direct effect of Na + at site 1, which depends on [Na + ]. Evidence has been advanced previously for direct occupancy of site 1 by cations, interfering with the binding of blockers (Heron et al 1996;Chen et al 1997a;Bonnet et al 1988, Li andCorera et al 2000) or substrates Reith 1999, 2000). The ultimate validation of models such as those presented in Fig.…”

“…Although this negates an absolute requirement for Na + in DA binding, a stimulatory role for Na + is still a possibility. Thus, cocaine also blocked leak currents in the absence of Na + (Sonders et al 1997), even though a stimulatory role for Na + in cocaine analog binding to DAT has been shown repeatedly (Reith and Coffey 1993;Chen et al 1997aChen et al , 1997bLi and Reith 1999;Corera et al 2000). In biochemical studies on the role of Na + in DA binding to DAT, the measuring tool is the DA-induced inhibition of binding of a radiolabeled blocker to the DAT because the dissociation of the DA-DAT complex is too rapid, ruling out successful application of commonly used techniques for separating bound from free ligand (see Li and Reith 1999).…”

“…The latter possibility can be contemplated if Tris + were to interfere only with the Na + site (site 2) postulated distal from the domain that binds blockers/substrates in the model developed based on blocker data (Chen et al 1997a;Corera et al 2000; site 1, top model in Fig. 4).…”

“…Whether the Na + site also interacts by positive allosterism with the blocker binding domain (Chen et al 1997a;Li and Reith 1999) is still under debate (Corera et al 2000). It is not yet clear that a similar model applies to the interaction between substrates and DAT.…”

“…An initial stimulatory effect upon raising [Na + ] followed by an inhibitory effect of higher Na + concentrations on the binding of blockers to DAT is accomodated by a model with (1) a cation site that overlaps with the blocker binding domain, and (2) a distal Na + site that interacts by negative allosterism with the cation site (Heron et al 1996;Chen et al 1997a;Li and Reith 1999;Corera et al 2000). Whether the Na + site also interacts by positive allosterism with the blocker binding domain (Chen et al 1997a;Li and Reith 1999) is still under debate (Corera et al 2000).…”