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J. Biol. Chem., Vol. 281, Issue 47, 36117-36123, November 24, 2006

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Study of Claudin Function by RNA Interference^*

Jianghui Hou, Antonio S. Gomes, David L. Paul, and Daniel A. Goodenough¹

From the Departments of Cell Biology and Neurobiology, Harvard Medical School, Boston, Massachusetts 02115

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.

Received for publication, September 14, 2006 , and in revised form, September 29, 2006.

^* This work was supported by National Institutes of Health Grants GM18974 and GM37751. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1-S7.

¹ To whom correspondence should be addressed: Dept. of Cell Biology, Harvard Medical School, 240 Longwood Ave., Boston, MA 02115. Tel.: 617-432-1652; Fax: 617-432-2955; E-mail: Daniel_goodenough{at}hms.harvard.edu.

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