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A Proteoliposome-Based Efflux Assay to Determine Single-molecule Properties of Cl- Channels and Transporters
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Determinants contributing to claudin ion channel formation.

Anna Veshnyakova1, Susanne M Krug, Sebastian L Mueller

  • 1Leibniz-Institut für Molekulare Pharmakologie, Berlin, Germany.

Annals of the New York Academy of Sciences
|June 8, 2012
PubMed
Summary

Claudins (Cld) form pores, with the first extracellular loop (ECL1) being key. Specific ECL1 residue substitutions in Cld-1 revealed S53E significantly impacts sealing and ion permeability, suggesting its role in paracellular barrier function.

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Area of Science:

  • Cell biology
  • Biophysics
  • Molecular biology

Background:

  • Claudins (Cld) are crucial transmembrane proteins forming the primary seal of the paracellular pathway.
  • The pore-forming properties and sealing mechanisms of claudins are not fully understood.
  • The first extracellular loop (ECL1) is hypothesized to play a significant role in claudin function.

Purpose of the Study:

  • To investigate the role of specific residues in the first extracellular loop (ECL1) of Claudin-1 (Cld-1) in regulating paracellular barrier function.
  • To identify key determinants within ECL1 responsible for sealing and/or pore formation.
  • To elucidate the mechanisms by which claudin mutations affect transepithelial resistance and ion permeability.

Main Methods:

  • Utilized Madin-Darby canine kidney (MDCK) cells stably overexpressing wild-type FLAG-Cld-1 and various mutants.
  • Quantified paracellular barrier function using transepithelial electrical resistance (TER) measurements.
  • Assessed ion permeability (Na+ and Cl-) across cell monolayers to evaluate pore characteristics.

Main Results:

  • E48K and S53E substitutions in Cld-1 significantly reduced TER and increased Na+ and Cl- permeability.
  • Single substitutions K65D and D68S, among others, did not substantially alter TER or ion permeability.
  • The S53E substitution, and to a lesser extent the S53E/D68S double substitution, decreased TER, suggesting S53 and potentially D68 are critical for sealing.
  • The induced pores showed no clear charge selectivity, indicating S53E may lead to charge-unselective pores.

Conclusions:

  • Residues within the ECL1 of Claudin-1, particularly S53 and potentially D68, are critical determinants of paracellular sealing.
  • Substitution of S53E in Cld-1 disrupts the barrier function and can induce charge-unselective pores.
  • These findings provide insights into the molecular mechanisms underlying claudin-mediated paracellular barrier integrity and ion transport.