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Participation of the second extracellular loop of claudin-5 in paracellular tightening against ions, small and large molecules

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Abstract

Tight junctions control paracellular permeability. Here, we analyzed the impact of residues in the second extracellular loop (ECL2) of mouse claudin-5 on paracellular permeability. Stable expression of claudin-5wild type in MDCK-II cells—but not that of mutants R145A, Y148A, Y158A or E159Q—increased transepithelial electrical resistance and decreased fluorescein permeation. Expression of claudin-5Y148A, Y158A or E159Q enhanced permeability of FITC-dextran10 kDa, which was unchanged in cells expressing claudin-5wild type or claudin-5R145A. In contrast, targeting to tight junctions, strand morphology and tight junction assembly were unchanged. It is concluded that R145 is unessential for trans-interaction of claudin-5, but necessary for tightening against small solutes and ions. The highly conserved residues Y148, Y158 and E159 in ECL2 of claudin-5 contribute to homo- and/or heterophilic trans-interaction between classic claudins and thereby tighten the paracellular space against ions, small and large molecules. These results provide novel insights into the molecular function of tight junctions.

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Abbreviations

Cld:

Claudin

Da:

Dalton

DMEM:

Dulbecco’s modified Eagle’s medium

ECL:

Extracellular loop

FITC:

Fluorescein isothiocyanate

HBSS:

Hank’s buffered salt solution

HRP:

Horseradish peroxidase

MDCK:

Madin-Darby canine kidney

PBS:

Phosphate buffered saline

Pfluorescein :

Permeability coefficient of fluorescein

PFD10 :

Permeability coefficient of 10 kDa FITC-dextran

SEM:

Standard error of the mean

TEER:

Transepithelial electrical resistance

TJ:

Tight junctions

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Acknowledgments

We thank Barbara Eilemann for technical assistance. We gratefully acknowledge the help of Ria Knittel in freeze-fracturing. This work was funded by DFG BL308/7-3, 7-4 and PI 837/2-1.

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Authors and Affiliations

  1. Leibniz-Institut für Molekulare Pharmakologie, FMP, Robert-Rössle-Str. 10, 13125, Berlin, Germany

    Christian Piehl, Jörg Piontek, Jimmi Cording & Ingolf E. Blasig

  2. Institute of Pathology, University of Tübingen, Liebermeisterstraße 8, 72076, Tübingen, Germany

    Hartwig Wolburg

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  1. Christian Piehl
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  2. Jörg Piontek
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  3. Jimmi Cording
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  5. Ingolf E. Blasig
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Corresponding author

Correspondence to Ingolf E. Blasig.

Additional information

C. Piehl and J. Piontek contributed equally to this work.

Electronic supplementary material

Fig. S1.

The amino acid substitution Y148A in claudin-5 does not affect disassembly nor reassembly of tight junctions. (A) MDCK-II cells, stably transfected with Cld5wt or Cld5Y148A were cultured until confluent before standard medium was replaced by medium containing 2mM EGTA for Ca2+-delpetion. The subcellular localization of endogenous Cld1 (green), exogenous Cld5 (red) and the nuclei (blue) were detected before, 0.5, 1, 2 and 3 h after removal of Ca2+ by immunostaining and subsequent confocal microscopy. With increasing time of Ca2+-depletion the detected amount of Cld5 and Cld1 in the plasma membrane decreased and that in intracellular compartments increased for Cld5wt- as well as for Cld5Y148A-expressing cells. At no time point internalization of Cld5 or Cld1 was more pronounced for Cld5Y148A- than for Cld5wt-expressing cells (B) The subcellular localization of endogenous Cld1 (green), exogenous Cld5 (red) and nuclei (blue) were detected at 0, 0.25, 0.5, 1, 2, 3 and 4 h after media exchange to Ca2+ containing standard medium. Between Cld5wt- and Cld5Y148A-expressing cells, no difference in the reappearance at the plasma membrane was detectable for either Cld1 or Cld5. (JPEG 3.61 MB)

Fig. S1.

The amino acid substitution Y148A in claudin-5 does not affect disassembly nor reassembly of tight junctions. (A) MDCK-II cells, stably transfected with Cld5wt or Cld5Y148A were cultured until confluent before standard medium was replaced by medium containing 2mM EGTA for Ca2+-delpetion. The subcellular localization of endogenous Cld1 (green), exogenous Cld5 (red) and the nuclei (blue) were detected before, 0.5, 1, 2 and 3 h after removal of Ca2+ by immunostaining and subsequent confocal microscopy. With increasing time of Ca2+-depletion the detected amount of Cld5 and Cld1 in the plasma membrane decreased and that in intracellular compartments increased for Cld5wt- as well as for Cld5Y148A-expressing cells. At no time point internalization of Cld5 or Cld1 was more pronounced for Cld5Y148A- than for Cld5wt-expressing cells (B) The subcellular localization of endogenous Cld1 (green), exogenous Cld5 (red) and nuclei (blue) were detected at 0, 0.25, 0.5, 1, 2, 3 and 4 h after media exchange to Ca2+ containing standard medium. Between Cld5wt- and Cld5Y148A-expressing cells, no difference in the reappearance at the plasma membrane was detectable for either Cld1 or Cld5. (JPEG 4.70 MB)

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Piehl, C., Piontek, J., Cording, J. et al. Participation of the second extracellular loop of claudin-5 in paracellular tightening against ions, small and large molecules. Cell. Mol. Life Sci. 67, 2131–2140 (2010). https://doi.org/10.1007/s00018-010-0332-8

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