The first extracellular domain of claudin-7 affects paracellular Cl- permeability.

Tight junctions (TJ) constitute paracellular diffusion channels regulating the passage of ions and solutes across epithelia. We recently demonstrated that overexpression of the TJ membrane protein claudin-7 in LLC-PK1 cells decreases paracellular permeability to Cl(-) and increases paracellular permeability to Na(+). To investigate the effect of charged amino acid residues in extracellular domains (ED) of claudin-7 on paracellular charge selectivity, we created claudin-7 mutants by replacing negatively charged amino acids on ED with positively charged amino acids. Immunofluorescence light microscopy showed that these mutant proteins were correctly targeted to the cell junction. Ultrastructure examination of TJ morphology did not reveal any difference between cells expressing wildtype (WT) and mutant claudin-7. However, electrophysiological studies showed increased Cl(-) permeability in cells expressing first extracellular domain (ED1) mutants, but not second extracellular domain (ED2) mutants, compared to that of WT claudin-7. Our results demonstrate that negatively charged amino acids in ED1 of claudin-7 are involved in modulating paracellular Cl(-) permeability.

Overexpression of claudin-7 decreases the paracellular Cl- conductance and increases the paracellular Na+ conductance in LLC-PK1 cells.

Tight junctions form the primary barrier regulating the diffusion of fluid, electrolytes and macromolecules through the paracellular pathway. Claudins are the major structural and functional components of tight junction strands and are considered as the best candidates for forming paracellular channels. They are a family of integral membrane proteins with more than 20 members and show distinct tissue distribution patterns. In this study, we found that claudin-7 is expressed in the distal and collecting tubules and the thick ascending limb of Henle of porcine and rat kidneys. To investigate the role of claudin-7 in paracellular transport, we have overexpressed a mouse claudin-7 construct in LLC-PK1 cells. Overexpression of claudin-7 did not affect the expression and localization of endogenous claudin-1, -3, -4, -7, and ZO-1. However, transepithelial electrical resistance in claudin-7-overexpressing cells was greatly increased. In addition, electrophysiological measurements revealed a dramatic reduction of dilution potentials in claudin-7-overexpressing cells compared to that of control cells. To determine which ions are responsible for the effects of claudin-7 overexpression on transepithelial electrical resistance and dilution potentials, we applied an ion substitution strategy. When NaCl was replaced with sodium aspartate, transepithelial electrical resistance was significantly decreased and dilution potentials were increased in claudin-7-overexpressing cells as compared to controls, the opposite effects from that of using NaCl. Furthermore, when NaCl was substituted by arginine-HCl or lysine-HCl, the increase in transepithelial electrical resistance was greater and the reduction in dilution potentials was smaller. Taken together, our studies demonstrated for the first time that the effect of claudin-7 overexpression in LLC-PK1 cells on paracellular transport is mediated through a concurrent decrease in the paracellular conductance to Cl(-) and an increase in the paracellular conductance to Na(+). These results support the model that claudin-7 may form a paracellular barrier to Cl(-) while acting as a paracellular channel to Na(+).