Claudin-4 forms paracellular chloride channel in the kidney and requires claudin-8 for tight junction localization

Abstract

Tight junctions (TJs) play a key role in mediating paracellular ion reabsorption in the kidney. The paracellular pathway in the collecting duct of the kidney is a predominant route for transepithelial chloride reabsorption that determines the extracellular NaCl content and the blood pressure. However, the molecular mechanisms underlying the paracellular chloride reabsorption in the collecting duct are not understood. Here we showed that in mouse kidney collecting duct cells, claudin-4 functioned as a Cl(-) channel. A positively charged lysine residue at position 65 of claudin-4 was critical for its anion selectivity. Claudin-4 was observed to interact with claudin-8 using several criteria. In the collecting duct cells, the assembly of claudin-4 into TJ strands required its interaction with claudin-8. Depletion of claudin-8 resulted in the loss of paracellular chloride conductance, through a mechanism involving its recruitment of claudin-4 during TJ assembly. Together, our data show that claudin-4 interacts with claudin-8 and that their association is required for the anion-selective paracellular pathway in the collecting duct, suggesting a mechanism for coupling chloride reabsorption with sodium reabsorption in the collecting duct.

Fig. 1.

Effects of CLDN4 and its charge-neutralizing mutants in mIMCD3 cells on paracellular ion conductance. (A) Amino acid sequence alignment of the first extracellular loops of CLDN2 and CLDN4. Negatively charged amino acids are labeled in red; positively charged amino acids are in blue. Note the charge of amino acid differs at the position 65 of CLDN4 from CLDN2. Dilution potential values (B) and P_Cl/P_Na (C) across mIMCD3 cell monolayers expressing mouse CLDN4-siRNA (282), human WT CLDN4 and its mutants, individually or in pairs, are shown. *P < 0.01 relative to CLDN4-siRNA, n = 3; **P < 0.01 relative to CLDN4-WT, n = 3.

Fig. 2.

CLDN4 interacts with CLDN8. (A) Y2H assays showing interaction of CLDN4 with CLDN8, determined by using three reporter genes (HIS3, lacZ, and ADE2) in the yeast NMY51 strain. CLDN8 also interacts strongly with itself and with CLDN3 and CLDN7. Shown are plates with selective medium lacking leucine and tryptophan (SD-LW), indicating the transforming of both bait and prey vectors; with SD-LWHA, indicating the expression of reporter genes HIS3 and ADE2; and the β-galactosidase assay. (B) Coimmunoprecipitation of CLDN4 and CLDN8 cotransfected in HEK293T cells. Antibodies used for coimmunoprecipitation are shown above the lanes; antibody for blot visualization is shown at left.

Fig. 3.

CLDN4 delocalization in CLDN8-siRNA cells. (A) In polarized M-1 cells (shown as representative), CLDN4 is mislocalized to the cytoplasm (arrow) in the absence of CLDN8. CLDN3 or CLDN7 TJ localization is not dependent on CLDN8. (B) In subconfluent, not fully polarized, M-1 cells, CLDN4 is localized predominantly at sites of cell-cell interaction (green arrow), variably at the nonjunctional plasma membrane (green arrowhead) and occasionally in traveling intracellular vesicles (red arrow). In the absence of CLDN8, CLDN4 is confined to the ER (counterstained with anti-BiP antibody) and the Golgi apparatus (counterstained with anti-GM130 antibody).