Cocaine acts as an apparent competitive inhibitor at the outward-facing conformation of the human norepinephrine transporter: Kinetic analysis of inward and outward transport
Nh. Chen et Jb. Justice, Cocaine acts as an apparent competitive inhibitor at the outward-facing conformation of the human norepinephrine transporter: Kinetic analysis of inward and outward transport, J NEUROSC, 18(24), 1998, pp. 10257-10268
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.