The Coculture Method to Examine Interactions Between Claudin Isoforms in Tight Junction-Free HEK293

The paracellular transport of water, ions, and small solutes is regulated by tight junctions (TJs) in �epithelial, endothelial, and mesothelial cells. Both the prolonged increase and decrease of the paracellular permeability are involved in various diseases. Claudins, a family of at least 24 integral membrane proteins in TJs, are major components of TJs and usually more than two claudin isoforms are found in TJs. The combination and mixing ratios of claudin isoforms determine the paracellular permeability in TJs. To create the paracellular permeability barrier, claudins must interact laterally in one membrane (cis -interaction) and also must interact by head-to-head binding between adjacent cells (trans -interaction). Therefore, examination of claudin–claudin interactions provides insights into the mechanism of regulation of the paracellular permeability. This study introduced coculture systems using TJ-bearing MDCK II cells and TJ-free HEK293 cells to examine claudin–claudin interactions.