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Author Notes:

To whom correspondence may be addressed: Vascular Biology Center, Medical College of Georgia, Augusta University, 1459 Laney-Walker Blvd., Augusta, GA 30912-2500. Tel.: 706-721-9470; E-mail: rlucas@augusta.edu.

To whom correspondence may be addressed: Dept. of Physiology, Emory University, Atlanta, GA 30322-3110; Tel.: 404-727-7421; E-mail: deaton@emory.edu.

To whom correspondence may be addressed: Vascular Biology Center, Medical College of Georgia, Augusta University, 1459 Laney-Walker Blvd., Augusta, GA 30912-2500. Tel.: 706-721-9470; E-mail: iczikora@augusta.edu.

Author contributions: R. L., A. A., J. H., T. C., D. C. E., and I. C. conceived and coordinated the study.

R. L., D. C. E., and I. C. wrote the paper.

R. L., Q. Y., A. A., B. J. D., T. L. T., S. S., I. L., H. S., B. F., A. F. G., S. T., M. L., T. C., M. A., W. S., R. L.-G., D. C. E., and I. C. designed, performed, and analyzed the experiments.

Q. Y., B. J. D., O. A.-K., S. T., and T. L. T. provided technical assistance and contributed to the preparation of the figures.

All authors reviewed the results and approved the final version of the manuscript.

The authors declare that they have no conflicts of interest with the contents of this article.

The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

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Research Funding:

This work was supported, in whole or in part, by American Heart Association Postdoctoral Award 15POST22820021, by a Bridge Funding Award from the vice-president for research at Augusta University, by a Transregio 84/2 SFB Grant from the Deutsche Forschungsgemeinschaft “Innate Immunity of the Lung,” and by NIDDK, National Institutes of Health Grants R37 DK037963, R01 DK100564, and K01 DK099617.

Keywords:

  • Science & Technology
  • Life Sciences & Biomedicine
  • Biochemistry & Molecular Biology
  • epithelial sodium channel (ENaC)
  • peptides
  • Streptococcus
  • tumor necrosis factor (TNF)
  • ubiquitylation (ubiquitination)
  • EPITHELIAL SODIUM-CHANNEL
  • TUMOR-NECROSIS-FACTOR
  • XENOPUS 2F3 CELLS
  • ACUTE LUNG INJURY
  • FACTOR-ALPHA
  • NA+ CHANNEL
  • EDEMA REABSORPTION
  • FLUID TRANSPORT
  • PULMONARY-EDEMA
  • TRAFFICKING

The Lectin-like Domain of TNF Increases ENaC Open Probability through a Novel Site at the Interface between the Second Transmembrane and C-terminal Domains of the -Subunit

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Journal Title:

Journal of Biological Chemistry

Volume:

Volume 291, Number 45

Publisher:

, Pages 23440-23451

Type of Work:

Article | Final Publisher PDF

Abstract:

Regulation of the epithelial sodium channel (ENaC), which regulates fluid homeostasis and blood pressure, is complex and remains incompletely understood. The TIP peptide, a mimic of the lectin-like domain of TNF, activates ENaC by binding to glycosylated residues in the extracellular loop of ENaC-α as well as to a hitherto uncharacterized internal site. Molecular docking studies suggested three residues, Val 567 , Glu 568 , and Glu 571 , located at the interface between the second transmembrane and C-terminal domains of ENaC-α, as a critical site for binding of the TIP peptide. We generated Ala replacement mutants in this region of ENaC-α and examined its interaction with TIP peptide (3M, V567A/E568A/E571A; 2M, V567A/E568A; and 1M, E571A). 3M and 2M ENaC-α but not 1M ENaC-α, displayed significantly reduced binding capacity to TIP peptide and to TNF. When overexpressed in H441 cells,3M mutant ENaC-α formed functional channels with similar gating and density characteristics as the WT subunit and efficiently associated with the β and γ subunits in the plasma membrane. We subsequently assayed for increased open probability time and membrane expression, both of which define ENaC activity, following addition of TIP peptide. TIP peptide increased open probability time in H441 cells overexpressing wild type and 1M ENaC-α channels, but not 3M or 2M ENaC-α channels. On the other hand, TIP peptide-mediated reduction in ENaC ubiquitination was similar in cells overexpressing either WT or 3M ENaC-α subunits. In summary, this study has identified a novel site in ENaC-α that is crucial for activation of the open probability of the channel, but not membrane expression, by the lectin-like domain of TNF.

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© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

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