Cocaine acts as an apparent competitive inhibitor at the outward-facing conformation of the human norepinephrine transporter: Kinetic analysis of inward and outward transport

The inhibition by cocaine of inward and outward transport of dopamine (DA) at the cloned human norepinephrine transporter (hNET) and the relationship of the inhibitory patterns of cocaine to the conformational requirements of the transporter were investigated. This was done using rotating disk elect rode vol tammetry in transfected cells. The uphill uptake of external DA, t he lack of inhibition by internal substrates on DA uptake, and the accelera ted exchange of internal DA by external m-tyramine support a carrier model in which the hNET alternates between outward-facing and inward-facing confo rmations. Cocaine exhibited competitive inhibition of DA uptake, which was insensitive to intracellular substrates. In contrast, the inhibition by coc aine of the m-tyramine-induced DA efflux appeared noncompetitive relative t o intracellular DA, but competitive relative to extracellular m-tyramine. S imultaneous measurement of m-tyramine uptake and accompanying DA efflux at various concentrations of intracellular DA showed that cocaine did not alte r the ratio of DA efflux to m-tyramine uptake. Moreover, cocaine displayed similar potency for inhibiting DA uptake and efflux. Additionally, the inhi bition profile of cocaine was unrelated to the addition time of cocaine, si multaneously with or earlier than a substrate. All of the findings are cons onant with a competitive interaction between cocaine and substrates at the outward-facing conformation of the hNET. This action directly prevents the inward transport of external substrates, thereby inhibiting the outward tra nsport of internal substrates by reducing the availability of the inward-fa cing conformation. Consequently, the experimental inhibition pattern of coc aine depends on the conformation of the hNET to which the transported subst rate is exposed.