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doi: 10.1242/10.1242/jcs.00165

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Claudin-2 expression induces cation-selective channels in tight junctions of epithelial cells

¹ Department of Clinical Physiology, Benjamin Franklin Medical School, Freie Universität Berlin, Hindenburgdamm 30, 12200 Berlin, Germany
² Department of Gastroenterology, Infectiology and Rheumatology, Benjamin Franklin Medical School, Freie Universität Berlin, Hindenburgdamm 30, 12200 Berlin, Germany
³ Department of Pharmacology, Benjamin Franklin Medical School, Freie Universität Berlin, Hindenburgdamm 30, 12200 Berlin, Germany

View larger version (11K): [in a new window]   Fig. 1. Transepithelial impedance analysis. (A) Electrical equivalent circuit of the epithelium. See Materials and Methods for explanation. (B) Typical impedance data (open circles) of an C7-cld-2 monolayer (transfected with claudin-2 cDNA) in regular Ringer's solution and in low Ca2+ Ringer's. The Nyquist plot shows the real and the imaginary parts of the transepithelial impedance as a function of frequency. Dots on a solid line depict the least-squares fit of the data to the electrical model.

View larger version (23K): [in a new window]   Fig. 2. Expression of tight junction proteins in MDCK-C7 and C11. Western blots and densitometry. 2.5 µg of samples were loaded on SDS gel, electrophoresed, blotted and genuine expression of occludin, claudin-1, -2 and -3 was detected using polyclonal antibodies. Densitometric analysis of western blots was performed using quantification software (see Materials and Methods), Expression of occludin (A) and claudin-3 (D) did not differ significantly, whereas claudin-1 (B) and -2 (C) were lower in C7 (n=4-6).

View larger version (108K): [in a new window]   Fig. 3. Analyses of the subcellular distribution of TJ proteins. Immunofluorescence studies of claudins-1, -2 and -3 (red) and occludin (green) in MDCK-C7 (A), and -C11 (B) cells were performed using the confocal microscope technique (see Materials and Methods). Colocalization (yellow) was detected for all claudins, except for claudin-2 in MDCK-C7. Z-scans (C,D) as shown on the right or above the top views were performed in 200 nm steps over a range of 10 µm. The Z-scans of claudin-1 in C7 (C) and C11 (D) revealed that a marked expression of claudin-1 is detectable in subjunctional membrane areas in C11 (circles).

View larger version (82K): [in a new window]   Fig. 4. (A) Transfection of C7 with claudin-2 cDNA. Western blots detecting claudin-1 and claudin-2 in MDCK clones stably transfected with claudin-2 cDNA (C7-cld-2), compared with control (transfected with vector alone, C7-vec). (B) Decrease of Repi by introducing claudin-2 into MDCK-C7 cells. (C) Permeability of 4K FITC dextran in C7-vec and C7-cld-2. (D,E) Comparison of claudin-2 expression in the different clones using a confocal microscope. Detection of claudin-2 (red) and occludin (green) in C7-vec (D) and in C7-cld-2 (E). Transfectants did not show detectable changes in claudin-1 and -3 (not shown). F) Z-scans of C7-cld-2 detecting occludin (green) and claudin-2 (red). Scans as shown on the right or above the top views were performed in 200 nm steps over a range of 10 µm. A tight junctional colocalization of claudin-2 with occludin is observed.

View larger version (21K): [in a new window]   Fig. 5. Paracellular resistance (Rpara) of MDCK monolayers as derived from transepithelial impedance analysis. In monolayers of C7-cld-2 (transfected with claudin-2 cDNA), Rpara was lower (P<0.001) than in clone C7-vec (transfected with vector only), although higher (P<0.001) than in MDCK clone C11-vec. With low Ca2+ Ringer solution, Rpara decreased in the C7 clones. In C11-vec exposed to high Ca2+ Ringer's, Rpara increased.

View larger version (20K): [in a new window]   Fig. 6. (A) Relative permeabilities with reference to Na+ (defined as 1) for K+, NMDG+, choline+, Cl- and Br- in C7-vec, C7-cld2, and C11-vec, as derived from measurement of biionic (Na+/K+, Na+/NMDG+, Na+/choline+) and dilution potentials (Na+/Cl-, Na+/Br-, n=6). Lower values compared with C7-vec were detected for NMDG+, choline+, Cl- and Br- in both clones C7-cld-2 and C11-vec (P<0.001). (B) Permeabilities for mannitol and lactulose were calculated from 3H-mannitol and 3H-lactulose fluxes and were not significantly different in monolayers of MDCK clone C7 without or with transfection with claudin-2 cDNA (n=4 and 6, respectively), although they were lower than in clone C11-vec (n=4 and 6, respectively, P<0.05). (C) Partial ion conductivities calculated for Na+, K+ and Cl- in C7-vec, C7-cld-2 and C11-vec (n=6). Because Na+ and K+ were almost equally permeable but Na+ is 26-fold higher concentrated than K+, the contribution of Na+ to overall conductivity is about 25-fold of that of K+.

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