Abstract
Claudins are tight junction proteins that play a key selectivity role in the paracellular conductance of ions. Numerous studies of claudin function have been carried out using the overexpression strategy to add new claudin channels to an existing paracellular protein background. Here, we report the systematic knockdown of endogenous claudin gene expression in Madin-Darby canine kidney (MDCK) cells and in LLC-PK1 cells using small interfering RNA against claudins 1-4 and 7. In MDCK cells (showing cation selectivity), claudins 2, 4, and 7 are powerful effectors of paracellular Na+ permeation. Removal of claudin-2 depressed the permeation of Na+ and resulted in the loss of cation selectivity. Loss of claudin-4 or -7 expression elevated the permeation of Na+ and enhanced the proclivity of the tight junction for cations. On the other hand, LLC-PK1 cells express little endogenous claudin-2 and show anion selectivity. In LLC-PK1 cells, claudin-4 and -7 are powerful effectors of paracellular Cl- permeation. Knockdown of claudin-4 or -7 expression depressed the permeation of Cl- and caused the tight junction to lose the anion selectivity. In conclusion, claudin-2 functions as a paracellular channel to Na+ to increase the cation selectivity of the tight junction; claudin-4 and -7 function either as paracellular barriers to Na+ or as paracellular channels to Cl-, depending upon the cellular background, to decrease the cation selectivity of the tight junction.
Highlights
Claudins have been shown to confer ion selectivity to the paracellular pathway
Using Small interfering RNA (siRNA) silencing, we found that in Madin-Darby canine kidney (MDCK) cells claudin-2, -4, and -7 are powerful effectors of paracellular cation permeation (PNa) with no effects on anion (PCl)
Removal of claudin-2 depressed the permeation of Naϩ and caused the MDCK cell to lose its cation selectivity (PNa/PCl drops from Ͼ6 to close to 1.7)
Summary
Claudins have been shown to confer ion selectivity to the paracellular pathway. In MDCK2 cells, claudin-4, -5, -8, -11, and -14 selectively decrease the permeability of cation through tight junction, whereas the permeation of anion is largely unchanged (8 –12). In LLC-PK1 cells, claudin-2, -15, -16 selectively increase the permeability of cation through the tight junction with no significant effects on anions [13,14]. When exogenous claudins are added to the tight junction, they constitute new charge-selective channels leading to a physiological phenotype that combines the contributions of both endogenous and exogenous claudins in the cell. All studies of claudin function have been carried out using the overexpression strategy, adding new claudin channels to an existing paracellular protein background. To complement these data, we have studied the function of claudin when cells become deficient in a specific claudin. We have rescued the loss of each claudin function by exogenously expressing its siRNAresistant counterpart from a different species
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