Claudin-21 Has a Paracellular Channel Role at Tight Junctions

Abstract

Claudin protein family members, of which there are at least 27 in humans and mice, polymerize to form tight junctions (TJs) between epithelial cells, in a tissue- and developmental stage-specific manner. Claudins have a paracellular barrier function. In addition, certain claudins function as paracellular channels for small ions and/or solutes by forming selective pores at the TJs, although the specific claudins involved and their functional mechanisms are still in question. Here we show for the first time that claudin-21, which is more highly expressed in the embryonic than the postnatal stages, acts as a paracellular channel for small cations, such as Na(+), similar to the typical channel-type claudins claudin-2 and -15. Claudin-21 also allows the paracellular passage of larger solutes. Our findings suggest that claudin-21-based TJs allow the passage of small and larger solutes by both paracellular channel-based and some additional mechanisms.

FIG 1

Mouse claudin-21 localization in MDCK I transfectant clones. (A) MDCK I cells or Venus-claudin-21-expressing transfected MDCK I cells (MDCK I-Venus-claudin-21 cells) were cultured to confluence on glass coverslips and examined by confocal laser scanning microscopy. The cells were triple stained with an anti-GFP pAb, an anti-claudin-21 pAb, and an anti-ZO-1 MAb. The anti-GFP-positive signals overlapped the anti-claudin-21-positive signals and the anti-ZO-1-positive signals. Stacked images of the apical or basal side of the epithelial cells are shown. Green, GFP; red, claudin-21; blue, ZO-1. Bars, 10 μm. (B) MDCK I cells or Venus-claudin-15-expressing transfected MDCK I cells (MDCK I-Venus-claudin-15 cells) were cultured to confluence on glass coverslips and examined by confocal laser scanning microscopy. The cells were stained with an anti-GFP pAb and an anti-ZO-1 MAb. The anti-GFP-positive signals overlapped the anti-ZO-1-positive signals. Stacked images of the apical or basal side of the epithelial cells are shown. Green, GFP; red, ZO-1. Bars, 10 μm.

FIG 2

Physiological analyses of exogenous claudin-21-expressing MDCK I cells. (A) (a) Measurement of transepithelial electric resistance in mock-transfected MDCK I cells (MDCK I-Venus), Venus-claudin-21-expressing transfected MDCK I cells (MDCK I-Venus-claudin-21), and Venus-claudin-15-expressing transfected MDCK I cells (MDCK I-Venus-claudin-15) (n = 3/group). (b) Transepithelial ion permeabilities for Na⁺ and Cl⁻ of MDCK I-Venus, MDCK I-Venus-claudin-21, and MDCK I-Venus-claudin-15 cells (n = 4/group). n.s., not significant; **, P < 0.01; ***, P < 0.001. (c) Monovalent cation transepithelial permeabilities of MDCK I-Venus and MDCK I-Venus-claudin-21 cells (n = 4/group). **, P < 0.01. (d) Transepithelial ion permeabilities for Na⁺, MA⁺, EA⁺, TMA⁺, TEA⁺, Arg⁺, and NMDG⁺ of MDCK I-Venus, MDCK I-Venus-claudin-21, and MDCK I-Venus-claudin-15 cell sheets at 37°C (n = 4/group). Statistical analysis was performed on 2 sets of data from MDCK I-Venus and MDCK I-Venus-claudin-21 cells or MDCK I-Venus and MDCK I-Venus-claudin-15 cells. ***, P < 0.001. (B) (a) Transepithelial ion permeabilities for Na⁺, MA⁺, EA⁺, TMA⁺, TEA⁺, Arg⁺, and NMDG⁺ of MDCK I-Venus, MDCK I-Venus-claudin-21, and MDCK I-Venus-claudin-15 cell sheets at 4°C (n = 4/group). Statistical analysis was performed on 2 sets of data from MDCK I-Venus and MDCK I-Venus-claudin-21 cells or MDCK I-Venus and MDCK I-Venus-claudin-15 cells. **, P < 0.01; ***, P < 0.001. (b) Estimation of the approximate diameter of claudin-21's pore (arrow). (c) Temperature-dependent paracellular permeabilities (permeabilities at 37°C minus permeabilities at 4°C) for Na⁺, MA⁺, EA⁺, TMA⁺, TEA⁺, Arg⁺, and NMDG⁺ of MDCK I-Venus, MDCK I-Venus-claudin-21, and MDCK I-Venus-claudin-15 cell sheets (n = 4/group). Statistical analysis was performed on 2 sets of data from MDCK I-Venus and MDCK I-Venus-claudin-21 cells or MDCK I-Venus and MDCK I-Venus-claudin-15 cells. ***, P < 0.001. (d) Estimation of the size exclusion threshold of the temperature-dependent paracellular permeability due to claudin-21 (arrow). (e) Estimation of the approximate diameter of claudin-15's pore (arrow). (f) Estimation of the size exclusion threshold of the temperature-dependent paracellular permeability due to claudin-15 (arrow).

FIG 3

Structural analysis of the claudin-21-based paracellular pore. (A) Alignment of the amino acid sequences of paracellular channel-type mouse claudins, including claudin-21. Numbers at the top indicate the amino acid positions of mouse claudin-15. Negatively and positively charged amino acids in the first extracellular segment are shown in red and blue, respectively. Representative claudin motifs are shown in gray. (B) (a) Electrostatic potential of the claudin-15 surface contoured from −2 kT/e (red) to +2 kT/e (blue). (b) Ribbon representation of claudin-15 with a transparent surface and viewed from the same direction as in panel a, with color changes from the N terminus (blue) to the C terminus (red). (C) Electrostatic potential surfaces of the extracellular domains of homology models (claudin-2, -21, and -10a), viewed as in panel B. Dashed ovals indicate the β3-β4 region, where residues important for charge selectivity of the paracellular channel are located.

FIG 4

Claudin-21 expression in mouse tissues. Immunofluorescence micrographs show epididymides from an E18 mouse (A) and an adult mouse (B) costained with an anti-claudin-21 pAb and an antioccludin MAb or with antiserum and an antioccludin MAb. DAPI (4′,6-diamidino-2-phenylindole) was used to detect nuclei. The anti-claudin-21- or antiserum-positive signals (green), the antioccludin-positive signals (red), and nuclei (blue) are shown. Bars, 50 μm.