Chloride channels and transporters in human corneal epithelium

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.

Figure 1. RT-PCR products for CLCs from human corneal epithelial cells

Transformed human corneal epithelial cell samples (tHCE) were used. DNA ladder is on the left. Tissue specific markers are on the right.

Figure 2. Western blot confirmed expression of proteins of CLC family members in human corneal epithelial cells

Human corneal epithelial tissue, primary cultured human corneal epithelial cells and transformed human corneal epithelial cells were probed with specific antibodies against CLC-2, CLC-4, and CFTR. Mouse heart tissue was used as positive control for CLC-2, mouse brain tissue was used as positive control for CLC-4 and CFTR. Tubulin was used as a loading control. Because of the variation in the expression level of target proteins, we varied loading quantity significantly.

Figure 3. Confocal images of immunofluorescence staining for CLC family members in human corneal epithelium

CLC family members CLC-2, CLC-3, CLC-4, CLC-6 and CFTR show distinct distribution in human corneal epithelium. Actin (not show here) and DAPI (for nuclear staining) were used as controls. (A), CLC-2 is distributed at the apical and basal layers of corneal epithelium. (B) and (C), CLC-3, and CLC-4 are expressed at the apical layer. (D) and (E), CLC-6 and CFTR expressed preferentially at the apical layers with expression level gradually decreasing towards the basal layer. Tear side is orientated to the top and basal side towards the bottom.

Figure 4. Gradients of CLC6 and CFTR staining in human corneal epithelium

Quantitative analyses were performed with Metamorph software. (A) Fluorescence intensity along the shaded rectangles is shown in the corresponding line graph below. Actin filament staining is used as an internal control. (B) The fluorescence intensity of CFTR and CLC-6 in the apical, middle and basal layers of corneal epithelium was significantly different from each other. Apical layers show strongest staining, which gradually decreased towards the basal layers. The fluorescence intensity of CFTR and CLC-6 staining was normalized using the fluorescence value of actin filaments. ** P<0.01.

Figure 5. Confocal images of immunofluorescence staining for CLC family members in human corneal endothelium

Human corneal endothelial cells are positive for immunostaining of CLC family members CLC-2, CLC-3, CLC-4, CLC-6 and CFTR. DAPI (for nuclear) staining was used to show the endothelium. Control staining using isoform IgG, or omission of the primary antibodies showed no staining (not show here). Stromal side is orientated to the top and apical side is orientated towards the bottom.

Figure 6. Chloride currents recorded in human corneal epithelial cells

Whole-cell chloride currents recorded from primary cultured human corneal epithelial cells (A), transformed HCE cells (B) and tsA201 cells as negative controls (C). The currents were recorded in bath solutions contained 140 mM Cl^- (left) or 30 mM Cl^- (middle). The current-voltage relationships are shown on the right. The zero current in the recordings are indicated by the dotted lines.