Chloride channel ClC- 2 enhances intestinal epithelial tight junction barrier function via regulation of caveolin-1 and caveolar trafficking of occludin.
Previous studies have demonstrated that the chloride channel ClC-2 plays a critical role in intestinal epithelial tight junction (TJ) barrier function via intracellular trafficking of TJ protein occludin. To study the mechanism of ClC-2-mediated TJ barrier function and intracellular trafficking of occludin, we established ClC-2 over-expressing Caco-2 cell line (Caco-2CLCN2) by full length ClC-2 ORF transfection. ClC-2 over-expression (Caco-2CLCN2) significantly enhanced TJ barrier (increased TER by ≥2 times and reduced inulin flux by 50%) compared to control Caco-2pEZcells. ClC-2 over-expression (Caco-2CLCN2) increased occludin protein level compared to control Caco-2pEZcells. Surface biotinylation assay revealed reduced steady state endocytosis of occludin in Caco-2CLCN2cells. Furthermore, ClC-2 over-expression led to reduction in caveolin-1 protein level and diminishment of caveolae assembly. Caveolae disruption increased TJ permeability in control but not ClC-2 over-expressing Caco-2CLCN2cells. Selective ClC-2 channel blocker GaTx2 caused an increase in caveolin-1 protein level and reduced occludin level. Delivery of cell permeable caveolin-1 scaffolding domain reduced the occludin protein level. Over all, these results suggest that ClC- 2 enhances TJ barrier function in intestinal epithelial cells via regulation of caveolin-1 and caveolae-mediated trafficking of occludin.
Department of Internal Medicine, University of New Mexico School of Medicine, Albuquerque, NM 87131, USA. Electronic address: pnighot@salud.unm.edu.
Prashant K
PK
Nighot
Department of Internal Medicine, University of New Mexico School of Medicine, Albuquerque, NM 87131, USA.
Lana
L
Leung
Department of Internal Medicine, University of New Mexico School of Medicine, Albuquerque, NM 87131, USA.
Thomas Y
TY
Ma
D015153:Blotting, Western; D000090007:CLC-2 Chloride Channels; D021941:Caveolae / Q000378:metabolism*; D051242:Caveolin 1 / Q000378:metabolism*; D002463:Cell Membrane Permeability; D049109:Cell Proliferation; D002478:Cells, Cultured; D018118:Chloride Channels / Q000378:metabolism*; D004705:Endocytosis / Q000502:physiology; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D006801:Humans; D007413:Intestinal Mucosa / Q000378:metabolism*; D007422:Intestines / Q000166:cytology; D062793:Occludin / Q000378:metabolism*; D021381:Protein Transport; D019108:Tight Junctions / Q000378:metabolism*
C495540:CAV1 protein, human; D000090007:CLC-2 Chloride Channels; D051242:Caveolin 1; D018118:Chloride Channels; D062793:Occludin
Caveolae; Caveolin-1; Endocytosis; Lysosomes; Occludin; Tight Junction