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

Abstract

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+.

Study of Claudin Function by RNA Interference

“…9 -10), our studies provide strong evidence that claudin-2 functions to increase, and claudin-4 to decrease, the cation permeation. However, our data on claudin-7 function contrast with those of a recent study performed in LLC-PK1 cells using the overexpression strategy, which showed that claudin-7 concurrently decreases the paracellular Cl Ϫ conductance and increases the paracellular Na ϩ conductance (25). To resolve this discrepancy and complement our studies performed in MDCK cells, we have generated LLC-PK1 epithelial cells with suppressed gene expression of claudin-4 or -7 by RNA interference.…”

“…Loss of Claudin Function in LLC-PK1 Cells-Our data on claudin-7 function contrast with those of a recent study performed in LLC-PK1 cells using the overexpression strategy, which showed that claudin-7 simultaneously decreases the paracellular Cl Ϫ conductance and increases the paracellular Na ϩ conductance (25). The discrepancy could result from the different paracellular protein background, in that MDCK cells showed cation selectivity (P Na /P Cl , 6.14 Ϯ 0.19; diffusion potential, ϩ10.83 Ϯ 0.12 mV), whereas LLC-PK1 cells showed anion selectivity (P Na /P Cl , 0.305 Ϯ 0.002; diffusion potential, Ϫ7.97 Ϯ 0.03 mV).…”

“…The expression of siRNA-resistant isoforms of claudin-4 or -7 selectively rescued the loss of function of these claudins in LLC-PK1 cells, indicating that claudin-4 and -7 function as paracellular channels to Cl Ϫ in LLC-PK1 cells. Further studies are needed to reconcile the contrasting conclusions on claudin-7 function drawn by us and by Alexandre et al (25), although overexpression of foreign claudins in an existing tight junction context could interfere with the structural makeup of the tight junction, especially when claudins are fused to green fluorescent protein to form a large protein moiety. Moreover, the use of constitutively expressing stable cell lines generates a large amount of clonal variation in TER and dilution potentials.…”

“…The discrepancy could result from the different paracellular protein background, in that MDCK cells showed cation selectivity (P Na /P Cl , 6.14 Ϯ 0.19; diffusion potential, ϩ10.83 Ϯ 0.12 mV), whereas LLC-PK1 cells showed anion selectivity (P Na /P Cl , 0.305 Ϯ 0.002; diffusion potential, Ϫ7.97 Ϯ 0.03 mV). LLC-PK1 cells express endogenous claudins-1, -3, -4, and -7, with very little expression of claudin-2 (maintained at a very low level close to the background (25)). The lack of endogenous claudin-2 expression in LLC-PK1 cells explains the low paracellular Na ϩ permeability (7.03 Ϯ 0.03 ϫ 10 Ϫ6 cm/s) compared with that in MDCK cells (20.23 Ϯ 0.09 ϫ 10 Ϫ6 cm/s).…”

Interaction between transcellular and paracellular water transport pathways through Aquaporin 5 and the tight junction complex

“…The TJC contains transmembrane proteins such as Claudins and Occludin that form the tight junction strands, as well as scaffolding proteins such as ZO-1 (31). Claudins are thought to play a key role in determining the ion selectivity of the TJC (32,33). Because Claudin isoforms are expressed in different tissue-specific combinations, it is thought that they confer tissue specific paracellular permeability properties to epithelia (34).…”