SPECIFIC BINDING OF [H-3] GBR-12783 TO THE DOPAMINE NEURONAL CARRIER INCLUDED IN POLARIZED MEMBRANES

We have compared the properties of the binding to the neuronal dopamin e carrier located either in polarized membranes of synaptosomes or in non polarized, classical membranes. Non-polarized membranes were prepa red by sonication of the partially purified synaptosomal fraction obta ined from rat striatum which was used as the source of polarized membr anes. Binding experiments were carried out at 37-degrees-C in Krebs Ri nger related media. [H-3]GBR 12783 xy)ethyl]4-(3-phenyl-2-[1-H-3]prope nyl)piperazine) specifically bound with a nanomolar affinity to a homo geneous population of site (maximal binding site concentration: 8-10 p mol/mg protein). Pure uptake inhibitors, but not substrates, competed for the [H-3]GBR 12783 binding site located in polarized membranes of synaptosomes at concentrations effective against dopamine neuronal tra nsport. Except for [H-3]GBR 12783, the replacement of Cl- by isethiona te- did not result in significant change in the ability of pure uptake inhibitors to compete for the specific binding site. A reduction in t he Na+ concentration from 135 to 10 mM induced a significant decrease in the inhibitory potency of GBR 12783, mazindol, nomifensine and meth ylphenidate. This decrease was likely to result from the presence of K +, Mg2+ and Ca2+, whose inhibitory effects were modified and/or increa sed by decreasing the Na+ concentration. These data indicate that the membrane polarity is not clearly involved in the binding of pure uptak e inhibitors to the dopamine neuronal carrier; furthermore they underl ine the critical role of Na+ and K+ transmembrane gradients in both th e recognition of the carrier by dopamine and its inward transport.