COMPLEX IONIC CONTROL OF [H-3] GBR-12783 BINDING TO THE DOPAMINE NEURONAL CARRIER

At 20 degrees C, [H-3]GBR 12783, )ethyl]4-(3-phenyl-2-([1-H-3]propenyl )-piperazine} dissociated from the dopamine neuronal carrier present i n rat striatal membranes with a t(1/2) value of 27 min. At this temper ature, KCl, CaCl2, and MgCl2 increased the binding dissociation, revea ling that they recognize a binding site which is not mutually exclusiv e with that of [H-3]GBR 12783. The comparison of the ability of KCl to increase the binding dissociation (by 160% at 30 mM KCI) with its pot ency as a binding inhibitor (K-i = 2.6 +/- 0.3 mM) suggests an involve ment of two recognition sites for K+ in binding inhibition, a not mutu ally exclusive site and another, mutually exclusive, site. Divalent ca tions mainly inhibited the binding via a mutually exclusive site since 3 mM Ca2+ and 10 mM M(2+) increased the binding dissociation by 90% a t 20 degrees C whereas their K-i values were 0.049 +/- 0.006 and 0.141 +/- 0.035 mM, respectively. Involvement of this mutually exclusive si te was also supported by the persistence of the binding inhibition eli cited by Ca2+ and Mg2+ at 0 degrees C, a temperature at which they red uced the binding dissociation. At 20 degrees C, 100 mM NaCl did not mo dify [H-3]GBR 12783 binding but it antagonized the binding dissociatio n elicited by inhibitory cations. Ca2+ reduced the off-rate of [H-3]GB R 12783 binding at 0 degrees C and in the presence of 100 mM Na+. Fina lly, [H-3]GBR 12783-binding dissociation was increased by high 'cytoso lic' K+ while 'synaptic' concentrations of Na+ K+, Ca2+, Mg2+ and Cl- were ineffective. A reduction of H2PO4-/HCO3- from 10 to 5 mM and a su bstitution of 5 mM H2PO4-/HCO3- by 5 mM Cl- increased the binding diss ociation, suggesting that an anion-binding site could also regulate th e binding.