Hou Jianghui, Gomes Antonio S, Paul David L, Goodenough Daniel A
Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115, USA.
J Biol Chem. 2006 Nov 24;281(47):36117-23. doi: 10.1074/jbc.M608853200. Epub 2006 Oct 3.
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.
紧密连接蛋白是在细胞旁离子传导中起关键选择性作用的蛋白质。许多关于紧密连接蛋白功能的研究采用过表达策略，在现有的细胞旁蛋白背景中添加新的紧密连接蛋白通道。在此，我们报告了使用针对紧密连接蛋白1 - 4和7的小干扰RNA，在犬肾上皮细胞（MDCK）和猪肾近端小管上皮细胞（LLC - PK1）中系统性敲低内源性紧密连接蛋白基因表达的情况。在MDCK细胞（表现出阳离子选择性）中，紧密连接蛋白2、4和7是细胞旁Na⁺渗透的有力调节因子。去除紧密连接蛋白2会降低Na⁺的渗透，并导致阳离子选择性丧失。紧密连接蛋白4或7表达缺失会提高Na⁺的渗透，并增强紧密连接对阳离子的偏好性。另一方面，LLC - PK1细胞几乎不表达内源性紧密连接蛋白2，表现出阴离子选择性。在LLC - PK1细胞中，紧密连接蛋白4和7是细胞旁Cl⁻渗透的有力调节因子。敲低紧密连接蛋白4或7的表达会降低Cl⁻的渗透，并使紧密连接失去阴离子选择性。总之，紧密连接蛋白2作为细胞旁Na⁺通道，增加紧密连接的阳离子选择性；紧密连接蛋白4和7根据细胞背景，要么作为Na⁺的细胞旁屏障，要么作为Cl⁻的细胞旁通道，降低紧密连接的阳离子选择性。
