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Vol. 59, Issue 5, 1129-1137, May 2001

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

Harald H. Sitte, Birgit Hiptmair, Julia Zwach, Christian Pifl, Ernst A. Singer, and Petra Scholze

Institute of Pharmacology (H.H.S., P.S., B.H., E.A.S.) and Brain Research Institute (C.P.), University of Vienna, Vienna, Austria

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 Km values 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 Km 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+ the Km value for [3H]5-HT uptake increased ~5-fold and the Vmax value remained unchanged. The Km value for [3H]MPP+ uptake also increased, but the Vmax 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 the Vmax 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.

Copyright © 2001 by The American Society for Pharmacology and Experimental Therapeutics


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