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Molecular Pharmacology

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Research ArticleArticle

Five Amino Acids in the Innermost Cavity of the Substrate Binding Cleft of Organic Cation Transporter 1 Interact with Extracellular and Intracellular Corticosterone

Christopher Volk, Valentin Gorboulev, Alexander Kotzsch, Thomas D. Müller and Hermann Koepsell
Molecular Pharmacology August 2009, 76 (2) 275-289; DOI: https://doi.org/10.1124/mol.109.054783
Christopher Volk
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Valentin Gorboulev
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Alexander Kotzsch
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Thomas D. Müller
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Hermann Koepsell
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Abstract

We have shown previously that Leu447 and Gln448 in the transmembrane helix (TMH) 10 of rat organic cation transporter rOCT1 are critical for inhibition of cation uptake by corticosterone. Here, we tested whether the affinity of corticosterone is different when applied from the extracellular or intracellular side. The affinity of corticosterone was determined by measuring the inhibition of currents induced by tetraethylammonium+ (TEA+) in Xenopus laevis oocytes expressing rOCT1. Either corticosterone and TEA+ were added to the bath simultaneously or the oocytes were preincubated with corticosterone, washed, and TEA+-induced currents were determined subsequently. In mutant L447Y, Ki values for extracellular and intracellular corticosterone were decreased, whereas in mutant Q448E, only the Ki for intracellular corticosterone was changed. Modeling of the interaction of corticosterone with rOCT1 in the inward- or outward-facing conformation predicted direct binding to Leu447, Phe160 (TMH2), Trp218 (TMH4), Arg440 (TMH10), and Asp475 (TM11) from both sides. In mutant F160A, affinities for extracellular and intracellular corticosterone were increased, whereas maximal inhibition was reduced in W218F and R440K. In stably transfected epithelial cells, the affinities for inhibition of 1-methyl-4-phenyl-pyridinium+ (MPP+) uptake by extracellular and intracellular corticosterone were decreased when Asp475 was replaced by glutamate. In mutants F160A, W218Y, R440K, and L447F, the affinities for MPP+ uptake were changed, and in mutant D475E, the affinity for TEA+ uptake was changed. The data suggest that Phe160, Trp218, Arg440, Leu447, and Asp475 are located within an innermost cavity of the binding cleft that is alternatingly exposed to the extracellular or intracellular side during substrate transport.

Footnotes

  • ↵1 Note that the Ki(cort.) value for inhibition of wild-type rOCT1 by extracellular corticosterone measured in HEK293 cells was lower compared with the Ki(cort.) value measured in X. laevis oocytes (Table 1). Recent data suggest that these differences as well as different affinities of some organic cations observed after expression of rOCT1 in HEK293 cells versus oocytes, are due to different regulatory states of rOCT1 in the two expression systems (H. Koepsell, V. Gorboulev, and U. Roth, unpublished data).

  • This work was supported by the Deutsche Forschungsgemeinschaft Grant SFB 487/A4.

  • ABBREVIATIONS: OCT, organic cation transporter; r, rat; TMH, transmembrane helix; TEA, tetraethylammonium; MPP, 1-methyl-4-phenylpyridinium; MOPS, 3-(N-morpholino)propanesulfonic acid; TBuA, tetrabutylammonium; HEK, human embryonic kidney; PBS, phosphate-buffered saline; LacY, lactose permease from Escherichia coli; ANOVA, analysis of variance.

    • Accepted May 12, 2009.
    • Received January 15, 2009.
  • The American Society for Pharmacology and Experimental Therapeutics
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Molecular Pharmacology: 76 (2)
Molecular Pharmacology
Vol. 76, Issue 2
1 Aug 2009
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Research ArticleArticle

Five Amino Acids in the Innermost Cavity of the Substrate Binding Cleft of Organic Cation Transporter 1 Interact with Extracellular and Intracellular Corticosterone

Christopher Volk, Valentin Gorboulev, Alexander Kotzsch, Thomas D. Müller and Hermann Koepsell
Molecular Pharmacology August 1, 2009, 76 (2) 275-289; DOI: https://doi.org/10.1124/mol.109.054783

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Research ArticleArticle

Five Amino Acids in the Innermost Cavity of the Substrate Binding Cleft of Organic Cation Transporter 1 Interact with Extracellular and Intracellular Corticosterone

Christopher Volk, Valentin Gorboulev, Alexander Kotzsch, Thomas D. Müller and Hermann Koepsell
Molecular Pharmacology August 1, 2009, 76 (2) 275-289; DOI: https://doi.org/10.1124/mol.109.054783
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