Complex ionic control of [ 3H]GBR 12783 binding to the dopamine neuronal carrier

At 20°C, [ 3H]GBR 12783, {1-[2-(diphenylmethoxy)ethyl]4-(3-phenyl-2-([1- 3H]propenyl)-piperazine} dissociated from the dopamine neuronal carrier present in rat striatal membranes with a t 1/2 value of 27 min. At this temperature, KCl, CaCl 2 and MgCl 2 increased the binding dissociation, revealing that they recognize a binding site which is not mutually exclusive with that of [ 3H]GBR 12783. The comparison of the ability of KCl to increase the binding dissociation (by 160% at 30 mM KCl) with its potency as a binding inhibitor ( K i = 2.6 ± 0.3 mM) suggests an involvement of two recognition sites for K + in binding inhibition, a not mutually exclusive site and another, mutually exclusive, site. Divalent cations mainly inhibited the binding via a mutually exclusive site since 3 mM Ca 2+ and 10 mM Mg 2+ increased the binding dissociation by 90% at 20°C whereas their K i values were 0.049 ± 0.006 and 0.141 ± 0.035 mM, respectively. Involvement of this mutually exclusive site was also supported by the persistence of the binding inhibition elicited by Ca 2+ and Mg 2+ at 0°C, a temperature at which they reduced the binding dissociation. At 20°C, 100 mM NaCl did not modify [ 3H]GBR 12783 binding but it antagonized the binding dissociation elicited by inhibitory cations. Ca 2+ reduced the off-rate of [ 3H]GBR 12783 binding at 0°C and in the presence of 100 mM Na +. Finally, [ 3H]GBR 12783-binding dissociation was increased by high ‘cytosolic’ K + while ‘synaptic’ concentrations of Na +, K +, Ca 2+, Mg 2+ and Cl − were ineffective. A reduction of H 2 PO 4 − HCO 3 − from 10 to 5 mM and a substitution of 5 mM H 2 PO 4 − HCO 3 − by 5 mM Cl − increased the binding dissociation, suggesting that an anion-binding site could also regulate the binding