Ionic Requirements for the Specific Binding of [3H]GBR 12783 to a Site Associated with the Dopamine Uptake Carrier

Abstract

At 0.degree.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-(diphenylmethoxy)ethyl]-4-(3-phenyl-2-propenyl)piperazine ([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 12,783, the half-maximal specific binding occurred at 5 mM Na+. A similar Na+ dependence was observed at 20.degree.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) > Mg2+ > Tris+ > 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.