Ionic requirements for the specific binding of [3H]GBR 12783 to a site associated with the dopamine uptake carrier.
At 0 degrees C, when Na+ was the only cation present in the incubation medium, increasing the Na+ concentration from 3 to 10 mM enhanced the affinity of [3H]1-[2-(++di-phenylmethoxy)ethyl]-4-(3-phenyl-2-propenyl)piperaz ine [( 3H]GBR 12783) for the specific binding site present in rat striatal membranes without affecting the Bmax. For higher Na+ concentrations, specific binding values plateaued and then slightly decreased at 130 mM Na+. In a 10 mM Na+ medium, the KD and the Bmax were, respectively, 0.23 nM and 12.9 pmol/mg of protein. In the presence of 0.4 nM [3H]GBR 12783, the half-maximal specific binding occurred at 5 mM Na+. A similar Na+ dependence was observed at 20 degrees C. Scatchard plots indicated that K+, Ca2+, Mg2+, and Tris+ acted like competitive inhibitors of the specific binding of [3H]GBR 12783. The inhibitory potency of various cations (K+, Ca2+, Mg2+, Tris+, Li+, and choline) was enhanced when the Na+ concentration was decreased from 130 to 10 mM. In a 10 mM Na+ medium, the rank order of inhibitory potency was Ca2+ (0.13 mM) greater than Mg2+ greater than Tris+ greater than K+ (15 mM). The requirement for Na+ was rather specific, because none of the other cations acted as a substitute for Na+. No anionic requirement was found: Cl-, Br-, and F- were equipotent. These results suggest that low Na+ concentrations are required for maximal binding; higher Na+ concentrations protect the specific binding site against the inhibitory effect of other cations.
Bonnet
J J
JJ
UA 1170 du CNRS, UER de Médecine et Pharmacie, Saint Etienne du Rouvray, France.
Benmansour
S
S
Vaugeois
J M
JM
Costentin
J
J
D000818:Animals; D000838:Anions / Q000494:pharmacology; D002412:Cations / Q000494:pharmacology; D008297:Male; D010879:Piperazines / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D011954:Receptors, Dopamine / Q000493:pharmacokinetics*; D012964:Sodium / Q000494:pharmacology; D014316:Tritium
D000838:Anions; D002412:Cations; D010879:Piperazines; D011954:Receptors, Dopamine; D014316:Tritium; C039737:1-(2-(diphenylmethoxy)ethyl)-4-(3-phenyl-2-propenyl)piperazine; D012964:Sodium