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 electrode voltammetry in transfected cells. The uphill uptake of external DA, the lack of inhibition by internal substrates on DA uptake, and the accelerated exchange of internal DA by external m-tyramine support a carrier model in which the hNET alternates between outward-facing and inward-facing conformations. Cocaine exhibited competitive inhibition of DA uptake, which was insensitive to intracellular substrates. In contrast, the inhibition by cocaine of the m-tyramine-induced DA efflux appeared noncompetitive relative to intracellular DA, but competitive relative to extracellular m-tyramine. Simultaneous measurement of m-tyramine uptake and accompanying DA efflux at various concentrations of intracellular DA showed that cocaine did not alter the ratio of DA efflux to m-tyramine uptake. Moreover, cocaine displayed similar potency for inhibiting DA uptake and efflux. Additionally, the inhibition profile of cocaine was unrelated to the addition time of cocaine, simultaneously with or earlier than a substrate. All of the findings are consonant 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 transport of internal substrates by reducing the availability of the inward-facing conformation. Consequently, the experimental inhibition pattern of cocaine depends on the conformation of the hNET to which the transported substrate is exposed.