The transport efficiency (TE) describes the performance of a transport protein for a specific substrate. To compare the TE of different transporters, the number of active transporters in the plasma membrane must be monitored, as it may vary for each transporter and experiment. Available methods, like LC–MS quantification of tryptic peptides, fail to discriminate inactive intracellular transporters or, like cell-surface biotinylation followed by affinity chromatography and Western blotting, are imprecise and very laborious. We wanted to normalize active transporters by the activity of a second transporter. A transporter tandem, generated by joining two transporter cDNAs into a single open reading frame, should guarantee a 1 : 1 stoichiometry. Here we created a series of tandems with different linkers between the human ergothioneine (ET) transporter ETT (gene symbol SLC22A4) and organic cation transporter OCT2 (SLC22A2). The linker sequence strongly affected the expression strength. The stoichiometry was validated by absolute peptide quantification and untargeted peptide analysis. Compared with wild-type ETT, the normalized ET clearance of the natural variant L503F was higher (f = 1.34); G462E was completely inactive. The general usefulness of the tandem strategy was demonstrated by linking several transporters with ETT; every construct was active in both parts. Transporter tandems can be used - without membrane isolation or protein quantification — as precise tools for transporter number normalization, to identify, for example, relevant transporters for a drug. It is necessary, however, to find suitable linkers, to check the order of transporters, and to verify the absence of functional interference by saturation kinetics.
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November 2020
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Cover Image
Cover Image
Differential binding of citrate and BI01383298 at the substrate-binding site of human and mouse NaCTs (sodium-coupled citrate transporter). This model provides insight into the molecular basis of selective inhibition of human NaCT by BI01383298. For further information on this model, see the article by Higuchi and colleagues (pp. 4149–4165). Image provided by Vadivel Ganapathy.
Research Article|
November 09 2020
Transporter tandems: precise tools for normalizing active transporter in the plasma membrane
Julia Tschirka;
Julia Tschirka
Investigation
1Department of Pharmacology, Faculty of Medicine and University Hospital Cologne, University of Cologne, Gleueler Straße 24, 50931 Cologne, Germany
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Markus Bach;
Markus Bach
Investigation
1Department of Pharmacology, Faculty of Medicine and University Hospital Cologne, University of Cologne, Gleueler Straße 24, 50931 Cologne, Germany
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Ilmars Kisis;
Ilmars Kisis
Investigation
1Department of Pharmacology, Faculty of Medicine and University Hospital Cologne, University of Cologne, Gleueler Straße 24, 50931 Cologne, Germany
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Julia Lemmen;
Julia Lemmen
Investigation
2Clinical Development, Bayer AG, Aprather Weg 18a, 42113 Wuppertal, Germany
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Mark Jean Gnoth;
Mark Jean Gnoth
Investigation
3DMPK, Bayer AG, Aprather Weg 18a, 42113 Wuppertal, Germany
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Dirk Gründemann
1Department of Pharmacology, Faculty of Medicine and University Hospital Cologne, University of Cologne, Gleueler Straße 24, 50931 Cologne, Germany
Correspondence: Dirk Gründemann (dirk.gruendemann@uni-koeln.de)
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Publisher: Portland Press Ltd
Received:
August 20 2020
Revision Received:
October 12 2020
Accepted:
October 19 2020
Accepted Manuscript online:
October 19 2020
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© 2020 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society
2020
Biochem J (2020) 477 (21): 4191–4206.
Article history
Received:
August 20 2020
Revision Received:
October 12 2020
Accepted:
October 19 2020
Accepted Manuscript online:
October 19 2020
Citation
Julia Tschirka, Markus Bach, Ilmars Kisis, Julia Lemmen, Mark Jean Gnoth, Dirk Gründemann; Transporter tandems: precise tools for normalizing active transporter in the plasma membrane. Biochem J 13 November 2020; 477 (21): 4191–4206. doi: https://doi.org/10.1042/BCJ20200666
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