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First published online 21 April 2009
doi: 10.1242/jcs.040113

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Claudin-10 exists in six alternatively spliced isoforms that exhibit distinct localization and function

¹ Institute of Clinical Physiology, Charité, 12200 Berlin, Germany
² Department of Pediatric Nephrology, Charité, 13535 Berlin, Germany
³ Institute of Molecular and Cell Biology (IMCB), Singapore 138673
⁴ Max-Delbrueck-Center for Molecular Medicine, 13125 Berlin, Germany

View larger version (29K): [in this window] [in a new window]   Fig. 1. Predicted membrane topology of claudin-10. Membrane topology of claudin-10a (A) and claudin-10b (B) variants as predicted by the UniProt database. Cysteines of the first extracellular loop (black circles) and electrically charged amino acids of the two extracellular loops (+, –) are indicated. Claudin-10a_i1 and claudin-10a_i3 lack 19 amino acids (57 base pairs) in the first extracellular loop as indicated by 57, claudin-10a_i2, claudin-10a_i3 and claudin-10b_i1 lack the 36 amino acids encoded by exon 4 (as indicated by exon 4), encompassing the fourth transmembrane region. In P mutants the two C-terminal amino acids (YV) of the PDZ-binding motif are mutated (AA).

View larger version (50K): [in this window] [in a new window]   Fig. 2. Differential murine Cldn10 expression. (A) RT-PCR analysis of mouse kidney for Cldn10. Both Cldn10a (left) and Cldn10b (right) are expressed here. Additionally, a 57 bp shorter transcript, indicated with an asterisk (*), was found for Cldn10a. The size of the marker fragments in bp is indicated on the right. (B) Semi-quantitative RT-PCR for Cldn10 variants on mouse multiple tissue cDNA (MTC) panels (Clontech). These panels are normalized for four housekeeping genes. The PCR results after 35 cycles are shown. As a positive control, manually obtained kidney cDNA was used. Whereas Cldn10a variants are found in kidney and uterus, Cldn10b is expressed ubiquitously with the highest and lowest expression levels in kidney and liver, respectively. The size of the marker fragments in bp is indicated on the right. (C) PCRs on cDNA from dissected nephron segments. PCT, proximal convoluted tubule; mTAL, medullary thick ascending limb of Henle; CCD, cortical collecting duct; OMCD and IMCD, outer and inner medullary collecting duct. Whole kidney cDNA was used as a positive control. The size of the marker fragments, in bp, is indicated on the right.

View larger version (17K): [in this window] [in a new window]   Fig. 3. Alternative splice variants in mouse. Diagram of the genomic organization of the Cldn10 gene and below it, the alternative transcripts that were found. Six Cldn10 messengers were found in mouse kidney, resulting from alternative first exons, an alternative splice donor site in exon 1a, indicated with an asterisk (*), and in some cases a deletion of exon 4. The genomic sequence is depicted as a line. Exons, depicted as filled rectangles, are given above the genomic structure. UTRs are depicted as narrower filled rectangles in front of the ATG start codons and after the TAA stop codon.

View larger version (80K): [in this window] [in a new window]   Fig. 4. Confocal images demonstrating subcellular localization of Cldn10 isoforms in MDCK-II cells. (A) Subcellular localization of Cldn10 isoforms in MDCK-II cells. MDCK-II cells were transiently transfected with all six mouse Cldn10 variant constructs, fixed with 2% PFA and stained with anti-ZO-1 (red) and anti-HA (green) antibodies. (B) ER-retained Cldn10 isoforms. MDCK-II cells transiently transfected with the three Cldn10 variants lacking exon 4 were fixed with 2% PFA and stained to visualize ER (anti-calrecticulin; green) and Cldn10 isoforms (anti-HA; red). (C) Subcellular localization of isoforms with mutated PDZ-binding motif. MDCK-II cells were transiently transfected with Cldn10aP, Cldn10a_v1P and Cldn10bP mutants, fixed with 2% PFA and stained with anti-ZO-1 (red) and anti-HA (green) antibodies. (D) Z-scans of Cldn10-expressing cell layers fixed with methanol and stained for claudin-10 (red) and occludin (green). Yellow staining indicates colocalization of claudin-10 and occludin within the tight junction. Nuclei are stained with DAPI (blue). Basolateral side is to the left, apical side to the right. Detector gain was adjusted to yield similar tight junctional claudin-10 signals in all cells. Under these conditions, a strong claudin-10 background signal is visible for all PDZ mutants, indicating that most of the protein does not reach the TJ. (E) Binding of C-terminal Cldn10 to the ZO-1 PDZ domain. In vitro transcribed and translated ZO-1 PDZ domain was incubated with peptides corresponding to the C-terminal of wild-type or mutant Cld10 coupled to beads. Proteins bound to beads were analyzed by SDS-PAGE, western blot and probed with Myc antibody. (One of three similar experiments.)

View larger version (21K): [in this window] [in a new window]   Fig. 5. Transepithelial resistance and Na+ to Cl– permeability ratio of transfected MDCK-C7 cell layers. (A) Transfection with Cldn10 caused transepithelial resistance (Rt) to decrease in all MDCK-C7 cell layers except those transfected with mouse Cldn10a (**P<0.01). By contrast, Rt of co-cultures of MDCK-C7 cells transfected with mouse Cldn10a and Cldn10a_v1 was increased (**P<0.01, mean ± s.e.m., n=13-41). (B) Na+ over Cl– permeability ratio (PNa/PCl) was greatly increased in Cldn10b-transfected MDCK-C7 cell layers and in co-cultures of Cldn10a- and Cldn10b-transfected cells. Cldn10bP mutant-transfected MDCK-C7 cells also showed increased PNa/PCl values, however, the increase was considerably reduced, compared with cells transfected with the intact PDZ-binding motif (**P<0.01, mean ± s.e.m., n=3-35).

View larger version (26K): [in this window] [in a new window]   Fig. 6. Anion permeabilities (relative to PNa) induced by claudin-10 isoforms in MDCK-II and MDCK-C7 cells. (A) Transepithelial resistance (left, Rt) and relative anion permeability (right; black bars, NO3–; grey bars, Cl–) of mouse Cldn10a- and Cldn10a_v1-transfected, low resistance MDCK-II cells. Rt was not significantly affected by Cldn10a and Cldn10a_v1 transfection (n=8-12), whereas relative anion permeabilities were increased in Cldn10a-transfected MDCK-II cell layers (**P<0.01, mean ± s.e.m., n=3-7). (B) Relative anion permeability (black bars, NO3–; grey bars, Cl–; white bars, pyruvate) of Cldn10a-, Cldn10a_v1- and Cldn10b-transfected, high resistance MDCK-C7 cells (mouse or human Cldn10 as indicated). Cldn10a transfection increased relative PNO3 and decreased relative PPyruvate, whereas Cldn10a_v1 transfection had no effect. Cldn10b transfection caused an apparent decrease in relative anion permeability, because of the increase in PNa, as shown in Fig. 5B. Co-culture of Cldn10a- and Cldn10a_v1-transfected cell layers retained the increased relative PNO3 but not the decreased relative PPyruvate (*P<0.05, **P<0.01, mean ± s.e.m., n=4-27).

View larger version (31K): [in this window] [in a new window]   Fig. 7. Cation permeabilities (relative to PCl) induced by claudin-10 isoforms in MDCK-C7 cells. (A) Relative monovalent cation permeabilities of MDCK-C7 cells transfected with Cldn10a, Cldn10a_v1 and Cldn10b, YFP::Cldn10b, and P mutants (mouse or human Cldn10 as indicated). Cldn10b transfection of MDCK-C7 cells caused monovalent cation permeabilities to increase. Eisenman sequence changed from IV in controls (inset) to X. Both effects were abolished by N-terminal YFP. Transfection with Cldn10b-P caused a minor increase in cation permeabilities but no change in Eisenman sequence. Upon co-culture of Cldn10a- and Cldn10b-transfected cells, cation permeability was high, but permeability sequence changed to Eisenman sequence I (**P<0.01, mean ± s.e.m., n=3-35). (B) Relative divalent cation permeabilities of MDCK-C7 cells transfected with Cldn10a, Cldn10a_v1 and Cldn10b (mouse or human Cldn10 as indicated). Cldn10b transfection of MDCK-C7 cells caused divalent cation permeabilities to increase. Cldn10a and Cldn10a_v1 transfection had no effect on divalent cation permeability, whereas co-culture of these cells caused a reduction in divalent cation permeability. (*P<0.05, **P<0.01, mean ± s.e.m., n=3-18).

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