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- Title
Binding of uptake blockers to the neuronal dopamine transporter: further investigation about cationic and anionic requirements.
- Authors
Corera, Amadou Tidjane; Costentin, Jean; Bonnet, Jean-Jacques
- 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.
- Subjects
DOPAMINE; NEUROTRANSMITTERS; CATIONS; ANIONS; PROTEIN binding; BIOCHEMISTRY
- Publication
Naunyn-Schmiedeberg's Archives of Pharmacology, 2000, Vol 362, Issue 3, p213
- ISSN
0028-1298
- Publication type
Article
- DOI
10.1007/s002100000280