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Fig. 6. Dilution potential measurements revealed that overexpression of claudin-7 decreased paracellular permeability to Cl– and increased paracellular permeability to Na+. (A) LLC-PK1 cells stably transfected with GFP or claudin-7-GFP were grown on Snapwell filters. Once monolayers reached confluence, the filter rings containing cell monolayers were mounted into EasyMount chambers. Both apical and basal chambers were filled with P1 buffer containing 140 mM NaCl. Subsequently, P1 buffer in the basal chamber was replaced by P2 buffer with 70 mM NaCl (1 and 2) or P3 buffer with 35 mM NaCl (3 and 4). Dilution potential values were significantly lower (P<0.001) in claudin-7-overexpressing cells (2 and 4) compared to that of controls (1 and 3). The reduction in dilution potentials was a combined effect of Na+ and Cl–. (B) The experimental procedure was the same as in A except that NaCl in P1, P2 and P3 buffers was replaced by arginine-HCl to remove the contribution of Na+ on the reduction of dilution potentials in cells overexpressing claudin-7. In this case, dilution potential values were reduced 100% (P<0.001) in claudin-7-overexpressing cells (2 and 4) compared to that of controls (1 and 3). (C) NaCl was replaced by lysine-HCl in the P1, P2 and P3 buffers. The percentage decrease in dilution potentials was almost the same as with arginine-HCl (P<0.001). (D) To eliminate the contribution of Cl– to the dilution potential, sodium aspartate was used in all three buffers. In the absence of a Cl– concentration gradient, the dilution potentials in both control and claudin-7-overexpressing cells changed from positive to negative. The dilution potentials did not decrease; instead, they significantly increased (P<0.001) in cells overexpressing claudin-7 (2 and 4) compared to controls (1 and 3). Data are represented as means ± s.e.m. from at least three mixed clones (n=12 for each experiment).
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