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Homodimers of the Claudin‐2 Polymerize to Tight Junctions of Canine Kidney Epithelial Cells

Claudin‐2 (CLDN‐2) is a member of a family of 27 tight junction transmembrane proteins. Claudins regulate paracellular ion transport in mammalian epithelial cells by forming either a barrier or a size‐ and charge‐selective pore. CLDN‐2 forms a high‐conductance cation pore, and controls sodium ion permeability. Little is known about the structural basis for ion selectivity of claudins. Their molecular organization into multiprotein complexes at tight junctions is also poorly understood. Recent biochemical studies have suggested that CLDN‐2 can form stable dimers in Madin‐Darby canine kidney (MDCK) II cells, while our mutagenesis studies indicate that CLDN‐2 pores exist as high order multimers. In the current work, we overexpressed tandem homodimers of CLDN‐2 in high‐resistance MDCK I TetOff cells. By Western blotting, cells exhibited inducible expression of CLDN‐2 dimers (~33 kDa) in the absence of doxycycline (−dox), which was almost completely suppressed by doxycycline. By immunofluorescence, the dimeric species of CLDN‐2 were colocalized with ZO‐1 at the apical junctional complex, with some additional intracellular staining at perinuclear area. The transepithelial resistance of cells overexpressing dimers of CLDN‐2 (−dox) was 1.5–2 times lower than in the presence of doxycycline. Our results indicate that tandem dimers of CLDN‐2 polymerize to tight junctions and function normally as paracellular pores suggesting that the native stoichiometry of CLDN‐2 is a multiple of two subunits. Our study may help to understand how claudins are organized into oligomers at tight junctions. This study is supported by NIH grant U01‐GM094627