TY - JOUR 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 SP - 1129 LP - 1137 DO - 10.1124/mol.59.5.1129 VL - 59 IS - 5 AU - Harald H. Sitte AU - Birgit Hiptmair AU - Julia Zwach AU - Christian Pifl AU - Ernst A. Singer AU - Petra Scholze Y1 - 2001/05/01 UR - http://molpharm.aspetjournals.org/content/59/5/1129.abstract N2 - 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. ER -