Claudin-2-dependent paracellular channels are dynamically gated

  1. Christopher R Weber
  2. Guo Hua Liang
  3. Yitang Wang
  4. Sudipto Das
  5. Le Shen
  6. Alan S L Yu
  7. Deborah J Nelson
  8. Jerrold R Turner  Is a corresponding author
  1. The University of Chicago, United States
  2. Lupin Research Park, India
  3. University of Kansas Medical Center, United States

Abstract

Intercellular tight junctions form selectively permeable barriers that seal the paracellular space. Trans-tight junction flux has been measured across large epithelial surfaces, but conductance across individual channels has never been measured. We report a novel trans-tight junction patch clamp technique that detects flux across individual claudin-2 channels within the tight junction of cultured canine renal tubule or human intestinal epithelial monolayers. In both cells, claudin-2 channels display conductances of ~90 pS. The channels are gated, strictly dependent on claudin-2 expression, and display size- and charge-selectivity typical of claudin-2. Kinetic analyses indicate one open and two distinct closed states. Conductance is symmetrical and reversible, characteristic of a passive, paracellular process, and blocked by reduced temperature or site-directed mutagenesis and chemical derivatization of the claudin-2 pore. We conclude that claudin-2 forms gated paracellular channels and speculate that modulation of tight junction channel gating kinetics may be an unappreciated mechanism of barrier regulation.

Article and author information

Author details

  1. Christopher R Weber

    Department of Pathology, The University of Chicago, Chicago, United States
    Competing interests
    The authors declare that no competing interests exist.
  2. Guo Hua Liang

    Department of Pathology, The University of Chicago, Chicago, United States
    Competing interests
    The authors declare that no competing interests exist.
  3. Yitang Wang

    Department of Pathology, The University of Chicago, Chicago, United States
    Competing interests
    The authors declare that no competing interests exist.
  4. Sudipto Das

    Lupin Research Park, Pune, India
    Competing interests
    The authors declare that no competing interests exist.
  5. Le Shen

    Department of Pathology, The University of Chicago, Chicago, United States
    Competing interests
    The authors declare that no competing interests exist.
  6. Alan S L Yu

    Division of Nephrology and Hypertension and the Kidney Institute, University of Kansas Medical Center, Kansas City, United States
    Competing interests
    The authors declare that no competing interests exist.
  7. Deborah J Nelson

    Department of Pharmacological and Physiological Sciences, The University of Chicago, Chicago, United States
    Competing interests
    The authors declare that no competing interests exist.
  8. Jerrold R Turner

    Department of Pathology, The University of Chicago, Chicago, United States
    For correspondence
    jturner@bsd.uchicago.edu
    Competing interests
    The authors declare that no competing interests exist.

Reviewing Editor

  1. Fiona M Watt, King's College London, United Kingdom

Publication history

  1. Received: July 6, 2015
  2. Accepted: November 12, 2015
  3. Accepted Manuscript published: November 14, 2015 (version 1)
  4. Version of Record published: February 2, 2016 (version 2)

Copyright

© 2015, Weber et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

Metrics

  • 2,523
    Page views
  • 720
    Downloads
  • 77
    Citations

Article citation count generated by polling the highest count across the following sources: Crossref, Scopus, PubMed Central.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. Christopher R Weber
  2. Guo Hua Liang
  3. Yitang Wang
  4. Sudipto Das
  5. Le Shen
  6. Alan S L Yu
  7. Deborah J Nelson
  8. Jerrold R Turner
(2015)
Claudin-2-dependent paracellular channels are dynamically gated
eLife 4:e09906.
https://doi.org/10.7554/eLife.09906

Further reading

    1. Cell Biology
    2. Structural Biology and Molecular Biophysics
    Janice M Reimer, Morgan E DeSantis ... Andres E Leschziner
    Research Advance Updated

    The lissencephaly 1 protein, LIS1, is mutated in type-1 lissencephaly and is a key regulator of cytoplasmic dynein-1. At a molecular level, current models propose that LIS1 activates dynein by relieving its autoinhibited form. Previously we reported a 3.1 Å structure of yeast dynein bound to Pac1, the yeast homologue of LIS1, which revealed the details of their interactions (Gillies et al., 2022). Based on this structure, we made mutations that disrupted these interactions and showed that they were required for dynein’s function in vivo in yeast. We also used our yeast dynein-Pac1 structure to design mutations in human dynein to probe the role of LIS1 in promoting the assembly of active dynein complexes. These mutations had relatively mild effects on dynein activation, suggesting that there may be differences in how dynein and Pac1/LIS1 interact between yeast and humans. Here, we report cryo-EM structures of human dynein-LIS1 complexes. Our new structures reveal the differences between the yeast and human systems, provide a blueprint to disrupt the human dynein-LIS1 interactions more accurately, and map type-1 lissencephaly disease mutations, as well as mutations in dynein linked to malformations of cortical development/intellectual disability, in the context of the dynein-LIS1 complex.

    1. Structural Biology and Molecular Biophysics
    Yein Christina Park, Bharat Reddy ... José D Faraldo-Gómez
    Research Article

    The force-from-lipids hypothesis of cellular mechanosensation posits that membrane channels open and close in response to changes in the physical state of the lipid bilayer, induced for example by lateral tension. Here, we investigate the molecular basis for this transduction mechanism by studying the mechanosensitive ion channel MscS from Escherichia coli and its eukaryotic homolog, MSL1 from Arabidopsis thaliana. First, we use single-particle cryo-EM to determine the structure of a novel open conformation of wild-type MscS, stabilized in a thinned lipid nanodisc. Compared with the closed state, the structure shows a reconfiguration of helices TM1, TM2 and TM3a, and widening of the central pore. Based on these structures, we examined how the morphology of the lipid bilayer is altered upon gating, using molecular dynamics simulations. The simulations reveal that closed-state MscS causes drastic protrusions in the inner leaflet of the lipid bilayer, both in the absence and presence of lateral tension, and for different lipid compositions. These deformations arise to provide adequate solvation to hydrophobic features of the protein surface in this conformation, and clearly reflect a high energy conformation for the membrane, particularly under tension. Strikingly, these protrusions are largely eradicated upon channel opening. An analogous computational study of open and closed MSL1 recapitulates these findings. The gating equilibrium of MscS channels thus appears to be dictated by two opposing conformational preferences, namely those of the lipid membrane and of the protein structure. We propose a membrane deformation model of mechanosensation, which posits that tension shifts the gating equilibrium towards the conductive state not because it alters the mode in which channel and lipids interact but because it increases the energetic cost of the morphological perturbations in the membrane induced by to the closed state.