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.
Keywords: 482 cornea: epithelium •
569 ion channels •
714 signal transduction