PT  - JOURNAL ARTICLE
AU  - Harald H. Sitte
AU  - Birgit Hiptmair
AU  - Julia Zwach
AU  - Christian Pifl
AU  - Ernst A. Singer
AU  - Petra Scholze
TI  - 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
AID  - 10.1124/mol.59.5.1129
DP  - 2001 May 01
TA  - Molecular Pharmacology
PG  - 1129--1137
VI  - 59
IP  - 5
4099  - http://molpharm.aspetjournals.org/content/59/5/1129.short
4100  - http://molpharm.aspetjournals.org/content/59/5/1129.full
SO  - Mol Pharmacol2001 May 01; 59
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.