Abstract
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.
Footnotes
- Received September 9, 2000.
- Accepted January 25, 2001.
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Send reprint requests to: Ernst A. Singer, M.D., Department of Pharmacology, University of Vienna, Waehringerstr. 13A, A-1090 Vienna, Austria. E-mail: ernst.singer{at}univie.ac.at
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This work was supported by the Austrian Science Foundation, project P13183. Part of the work was presented at the 1) 41st Spring meeting of the German Society of Pharmacology and Toxicology, 2000 March 21–23, Mainz, Germany; and 2) Forum of European Neuroscience, 2000 June 24–28, Brighton, UK. (i) Sitte, HH, Scholze P, Hiptmair B, Pifl C and Singer EA (2000) Transport rates in cells stably expressing the cloned human serotonin transporter: differences in uptake and release. Naunyn-Schmiedeberg's Arch Pharmacol 361(Suppl 4):R29. (ii) Scholze P, Sitte HH, Hiptmair B, Zwach J and Singer EA (2000) Inward and outward transport rates for serotonin and MPP+(N-methyl-phenylpyridinium) in cells stably expressing the cloned human serotonin transporter. Eur J Neurosci 12(Suppl 11):20.04.
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H.H.S. and P.S. contributed equally to this work.
- The American Society for Pharmacology and Experimental Therapeutics
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