April 2014
Volume 55, Issue 13
Free
ARVO Annual Meeting Abstract  |   April 2014
Inhibition of Kir4.1 (KCNJ10) produces EGF-like effect in corneal epithelial cells
Author Affiliations & Notes
  • Daohong Lin
    Pharmacology, New York Medical College, Valhalla, NY
  • Chengbaio Zhang
    Pharmacology, New York Medical College, Valhalla, NY
  • Lijun Wang
    Pharmacology, New York Medical College, Valhalla, NY
  • Xiaotong Su
    Pharmacology, New York Medical College, Valhalla, NY
  • Footnotes
    Commercial Relationships Daohong Lin, None; Chengbaio Zhang, None; Lijun Wang, None; Xiaotong Su, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science April 2014, Vol.55, 5500. doi:
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    • Get Citation

      Daohong Lin, Chengbaio Zhang, Lijun Wang, Xiaotong Su; Inhibition of Kir4.1 (KCNJ10) produces EGF-like effect in corneal epithelial cells. Invest. Ophthalmol. Vis. Sci. 2014;55(13):5500.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract
 
Purpose
 

To examine the role of Kcnj10, a member of inwardly rectifier of K+ channels (Kirs), in regeneration of corneal epithelial cells by modulating the membrane potential. Furthermore, to test the hypothesis that Kcnj10 expression plays a key role in stimulation of growth factors release, TGFA and EGF, through elevating membrane potential, thereby increasing active Rac1 (GTP-Rac1, an important GTPases in corneal epithelial cells) and enhancing EGFR/AKT phosphorylation pathway.

 
Methods
 

A primary mouse corneal epithelial cells (pMCE) was established using the explant culture method. Expression level of Kcnj10 and other Kir channels was examined by qRT-PCR. An immortalized human corneal epithelial cell line (HCE) was also used to transfect siRNA-Kcnj10 or siRNA-control. Levels of TGFA and EGF in medium and cell lysate were measured by ELISA. Co-immunoprecipitation and pull-down assay were used to quantify the levels of phosphorylated EGFR and GTP bound form of Rac1. The patch-clamp technique was employed to measure K+ currents and membrane potentials in corneal cells from Kcnj10 knock-out (KO) mice and their wild-type (wt) littermate.

 
Results
 

Patch-clamp experiments demonstrated that the deletion of Kcnj10 reduced barium sensitive inwardly K currents by more than 80% in pMCE from KCNJ10 KO mice. Moreover, cell membrane potential was depolarized in pMCE of KO mice compared to that of wt mice, suggesting that Kcnj10 plays a dominant role in determining K conductance and cell membrane potential in corneal cells. Down-regulation of Kcnj10 with siRNA decreased whole-cell K currents, activated Rac1 and enhanced EGF/TGFA release, leading to stimulation of EGFR signaling pathway.

 
Conclusions
 

Kcnj10 is the major Kir channels expressed in corneal epithelial cells and it plays a key role in determining the membrane potential. Inhibition of Kcnj10-induced depolarization of corneal epithelial cells and produced EGF-like effect. This study suggest the pivotal and novel role of Kcnj10 in controlling corneal epithelial cells regeneration after injury.

 
 
Kcnj10 is the major Kir channel in mouse corneal cells.
 
Kcnj10 is the major Kir channel in mouse corneal cells.
 
 
Inhibition of Kcnj10 enhanced active GTP-Rac1 in corneal epithelial cells.
 
Inhibition of Kcnj10 enhanced active GTP-Rac1 in corneal epithelial cells.
 
Keywords: 482 cornea: epithelium • 569 ion channels • 714 signal transduction  
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