Quantitative analysis of inward and outward transport rates in cells stably expressing the cloned human serotonin transporter: Inconsistencies with the hypothesis of facilitated exchange diffusion

Quantitative aspects of inward and outward transport of substrates by the h uman plasmalemmal serotonin transporter (hSERT) were investigated. Uptake a nd superfusion experiments were performed on human embryonic kidney 293 cel ls permanently expressing the hSERT using [H-3]serotonin (5-HT) and [H-3]1- methyl-4-phenylpyridinium (MPP+) as substrates. Saturation analyses rendere d K-m values of 0.60 and 17.0 muM for the uptake of [H-3]5-HT and [H-3]MPP, respectively. Kinetic analysis of outward transport was performed by prel abeling the cells with increasing concentrations of the two substrates and exposing them to a saturating concentration of p-chloroamphetamine (PCA; 10 muM). Apparent K-m values for PCA induced transport were 564 muM and about 7 mM intracellular [H-3] 5-HT and [H-3]MPP+, respectively. Lowering the ex tracellular Na+ concentrations in uptake and superfusion experiments reveal ed differential effects on substrate transport: at 10 mM Na+ the K-m value for [H-3]5-HT uptake increased similar to5-fold and the V-max value remaine d unchanged. The K-m value for [H-3]MPP+ uptake also increased, but the V-m ax value was reduced by 50%. When efflux was studied at saturating prelabel ing conditions of both substrates, PCA as well as unlabeled 5-HT and MPP+ ( all substances at saturating concentrations) induced the same efflux at 10 mM and 120 mM Na+. Thus, notwithstanding a 50% reduction in the V-max value of transport into the cell, MPP+ was still able to induce maximal outward transport of either substrate. Thus, hSERT-mediated inward and outward tran sport seems to be independently modulated and may indicate inconsistencies with the classical model of facilitated exchange diffusion.