RT Journal Article SR Electronic T1 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 JF Molecular Pharmacology JO Mol Pharmacol FD American Society for Pharmacology and Experimental Therapeutics SP 1129 OP 1137 DO 10.1124/mol.59.5.1129 VO 59 IS 5 A1 Harald H. Sitte A1 Birgit Hiptmair A1 Julia Zwach A1 Christian Pifl A1 Ernst A. Singer A1 Petra Scholze YR 2001 UL http://molpharm.aspetjournals.org/content/59/5/1129.abstract AB Quantitative aspects of inward and outward transport of substrates by the human plasmalemmal serotonin transporter (hSERT) were investigated. Uptake and superfusion experiments were performed on human embryonic kidney 293 cells permanently expressing the hSERT using [3H]serotonin (5-HT) and [3H]1-methyl-4-phenylpyridinium (MPP+) as substrates. Saturation analyses rendered K mvalues of 0.60 and 17.0 μM for the uptake of [3H]5-HT and [3H]MPP+, respectively. Kinetic analysis of outward transport was performed by prelabeling the cells with increasing concentrations of the two substrates and exposing them to a saturating concentration of p-chloroamphetamine (PCA; 10 μM). Apparent K m values for PCA induced transport were 564 μM and about 7 mM intracellular [3H]5-HT and [3H]MPP+, respectively. Lowering the extracellular Na+ concentrations in uptake and superfusion experiments revealed differential effects on substrate transport: at 10 mM Na+ theK m value for [3H]5-HT uptake increased ∼5-fold and the V max value remained unchanged. The K m value for [3H]MPP+ uptake also increased, but theV max value was reduced by 50%. When efflux was studied at saturating prelabeling 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 theV 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 transport seems to be independently modulated and may indicate inconsistencies with the classical model of facilitated exchange diffusion.