Cysteine mutagenesis to study the structure of claudin‐2 paracellular pores

Abstract

Claudins are tight junction proteins that control passive paracellular transport of small ions across epithelia. Claudin‐2 is expressed in proximal renal tubules which partly re‐absorb Na(+) via the paracellular pathway and it forms cation‐selective paracellular pores in high resistance cell lines. To test the hypothesis that amino acid residues in the 1st extracellular loop line the paracellular pore, we used cysteine mutagenesis of Y35, H57, D65 and I66 in claudin‐2 and expressed these mutants in stable transfected MDCK I cells. H57C and D65C showed a partial loss of cation permeability (50% of P(Na+) of wild‐type claudin‐2), suggesting that these residues may participate in cation permeation. D65C also exhibited marked loss of charge and size selectivity and appeared as a dimer on non‐reducing polyacrylamide gels, indicating that conformational changes resulting from intermolecular disulfide bonding disrupted the pore structure. Permeability and selectivity of Y35C and I66C were preserved. Addition of cationic methanethiosulfonate reagents to I66C caused a prompt decrease in conductance of up to 35%, suggesting that this residue may line the pore lumen and be accessible to extracellular cations. By applying methods used to investigate transcellular ion channels, we are beginning to map the position of key residues within the paracellular pore and develop MTS reagents as potential pore blocking tools.