Evidence for the Sequential Formation of Two Complexes Between an Uptake Inhibitor, GBR 12783 [1‐[2‐(Diphenylmethoxy)ethyl]‐4‐(3‐Phenyl‐2‐Propenyl)piperazine], and the Neuronal Transporter of Dopamine

: Incubation of a crude synaptosomal fraction from ratstriatum with GBR 12783 at 37°C produced an inhibition of the specificuptake of [ 3 H]dopamine that increased with time. The inhibitionincreased when GBR 12783 was present during preincubation and incubation(IC 50 = 1.85 ± 0.1 n M ) instead of incubation alone(IC 50 = 25 ± 3.5 n M ). Time‐course studies of uptakeinhibition demonstrated that a first collision transporter‐inhibitor complex(TI) was formed immediately after addition of GBR 12783 so that the initialuptake velocity ( V o ) decreased for increasingconcentrations of inhibitor ( K i ≥ 20 n M ). TIslowly isomerized to a more stable complex TI * ( K * i ≤ 5 n M ) with a value of t 1/2 = 20‐270 s. Fits of data to model 2 in which thesteady‐state uptake ( V S ) is set to zero were generallypreferred, suggesting that formation of TI * could tend toirreversibility, as a consequence of a very low reverse isomerization. Asexpected, k, V o , and V S tended tosteady‐state values in an asymptotic manner for high concentrations of GBR12783. GBR 12783 at 2.5 n M produced a mixed inhibition of the uptake,with an increase in K M and a decrease in V max ; these effects were improved for 10 n M GBR12783 and at 20°C. These results are discussed in relation to previousdata concerning [ 3 H]GBR 12783 binding. The present work gives the first experimental demonstration that dopamine uptake blockers can act according to a two‐step mechanism of inhibition ; this is of great interest, because these inhibitors can oppose the effects of cocaine or amphetamine on the transporter according to a reaction that is partly nondependent on the concentration of the abused agent.