Chloride channels and transporters in human corneal epithelium

Lin Cao, Xiao-Dong Zhang, Xiaobo Liu, Tsung-Yu Chen, Min Zhao

Research output: Contribution to journalArticle

19 Citations (Scopus)

Abstract

Transport of water and electrolytes is critical for corneal clarity. Recent studies indicate another important function of transport of ions and electrolytes - establishing wound electric fields that guide cell migration. We found chloride (Cl(-)) flux is a major component of the corneal wound electric current. In order to elucidate the mechanisms of Cl(-) transport, we studied Cl(-) channels and transporters in human corneal epithelial (HCE) cells. We tested a transformed human corneal epithelial cell line (tHCE), primary cultures of human corneal epithelial cells (pHCE), and human donor corneas. We first used RT-PCR to determine expression levels of mRNA of CLC (Cl(-) channels/transporters of CLC gene family) family members and CFTR (cystic fibrosis transmembrane conductance regulator) in HCE cells. We then confirmed protein expression and distribution of selected CLC family members and CFTR with Western blot and immunofluorescence confocal microscopy. Finally, Cl(-) currents were recorded with electrophysiological techniques. The mRNAs of CLC-2, CLC-3, CLC-4, CLC-5, CLC-6, and CFTR were detected in the HCE cell line. CLC-1 and CLC-7 were not detectable. Western blot and immunostaining confirmed protein expression and distribution of CLC-2, CLC-3, CLC-4, CLC-6 and CFTR in human corneal epithelium. CLC-2 preferentially labeled the apical and basal layers, while CLC-3 and CLC-4 labeled only the superficial layer. CLC-6 and CFTR labeling showed a unique gradient with strong staining in apical layers which gradually decreased towards the basal layers. Corneal endothelium was positive for CLC-2, CLC-3, CLC-4, CLC-6 and possibly CFTR. Human corneal epithelial cells demonstrated voltage dependent Cl(-) currents. HCE cells express functional Cl(-) channels and transporters. CLC-2, CLC-3, CLC-4, CLC-6, and CFTR had distinct expression patterns in human corneal epithelium. Those molecules and their distribution may play important roles in maintaining resting Cl(-) fluxes and in regulating Cl(-) flux at corneal wounds, which may be a major contributor to wound electrical signaling.

Original languageEnglish
Pages (from-to)771-779
Number of pages9
JournalExperimental Eye Research
Volume90
Issue number6
Early online date24 Mar 2010
DOIs
Publication statusPublished - Jun 2010

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Corneal Epithelium
Chloride Channels
Cystic Fibrosis Transmembrane Conductance Regulator
Epithelial Cells
Chlorides
Wounds and Injuries
Electrolytes
Western Blotting
Corneal Endothelium
Cell Line
Messenger RNA
Ion Transport
Fluorescence Microscopy
Confocal Microscopy
Cornea
Cell Movement
Proteins
Staining and Labeling
Polymerase Chain Reaction

Keywords

  • anion transport proteins
  • blotting, Western
  • cell line, transformed
  • chloride channels
  • chlorides
  • cystic fibrosis transmembrane conductance regulator
  • electrophysiology
  • endothelium, corneal
  • epithelium, corneal
  • fluorescent antibody technique, indirect
  • gene expression regulation
  • humans
  • ion transport
  • microscopy, confocal
  • RNA, messenger
  • reverse transcriptase polymerase chain reaction
  • voltage-dependent anion channels

Cite this

Chloride channels and transporters in human corneal epithelium. / Cao, Lin; Zhang, Xiao-Dong; Liu, Xiaobo; Chen, Tsung-Yu; Zhao, Min.

In: Experimental Eye Research, Vol. 90, No. 6, 06.2010, p. 771-779.

Research output: Contribution to journalArticle

Cao, Lin ; Zhang, Xiao-Dong ; Liu, Xiaobo ; Chen, Tsung-Yu ; Zhao, Min. / Chloride channels and transporters in human corneal epithelium. In: Experimental Eye Research. 2010 ; Vol. 90, No. 6. pp. 771-779.
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AB - Transport of water and electrolytes is critical for corneal clarity. Recent studies indicate another important function of transport of ions and electrolytes - establishing wound electric fields that guide cell migration. We found chloride (Cl(-)) flux is a major component of the corneal wound electric current. In order to elucidate the mechanisms of Cl(-) transport, we studied Cl(-) channels and transporters in human corneal epithelial (HCE) cells. We tested a transformed human corneal epithelial cell line (tHCE), primary cultures of human corneal epithelial cells (pHCE), and human donor corneas. We first used RT-PCR to determine expression levels of mRNA of CLC (Cl(-) channels/transporters of CLC gene family) family members and CFTR (cystic fibrosis transmembrane conductance regulator) in HCE cells. We then confirmed protein expression and distribution of selected CLC family members and CFTR with Western blot and immunofluorescence confocal microscopy. Finally, Cl(-) currents were recorded with electrophysiological techniques. The mRNAs of CLC-2, CLC-3, CLC-4, CLC-5, CLC-6, and CFTR were detected in the HCE cell line. CLC-1 and CLC-7 were not detectable. Western blot and immunostaining confirmed protein expression and distribution of CLC-2, CLC-3, CLC-4, CLC-6 and CFTR in human corneal epithelium. CLC-2 preferentially labeled the apical and basal layers, while CLC-3 and CLC-4 labeled only the superficial layer. CLC-6 and CFTR labeling showed a unique gradient with strong staining in apical layers which gradually decreased towards the basal layers. Corneal endothelium was positive for CLC-2, CLC-3, CLC-4, CLC-6 and possibly CFTR. Human corneal epithelial cells demonstrated voltage dependent Cl(-) currents. HCE cells express functional Cl(-) channels and transporters. CLC-2, CLC-3, CLC-4, CLC-6, and CFTR had distinct expression patterns in human corneal epithelium. Those molecules and their distribution may play important roles in maintaining resting Cl(-) fluxes and in regulating Cl(-) flux at corneal wounds, which may be a major contributor to wound electrical signaling.

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KW - blotting, Western

KW - cell line, transformed

KW - chloride channels

KW - chlorides

KW - cystic fibrosis transmembrane conductance regulator

KW - electrophysiology

KW - endothelium, corneal

KW - epithelium, corneal

KW - fluorescent antibody technique, indirect

KW - gene expression regulation

KW - humans

KW - ion transport

KW - microscopy, confocal

KW - RNA, messenger

KW - reverse transcriptase polymerase chain reaction

KW - voltage-dependent anion channels

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