Extracellular Mg regulates the tight junctional localization of claudin-16 mediated by ERK-dependent phosphorylation |
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Authors: | Akira Ikari Keishi Kinjo Yohei Sasaki Junko Sugatani |
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Institution: | a Department of Pharmaco-Biochemistry, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526, Japan b Global Center of Excellence for Innovation in Human Health Sciences, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526, Japan |
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Abstract: | Claudin-16 is involved in the paracellular reabsorption of Mg2+ in the thick ascending limb of Henle. Little is known about the mechanism regulating the tight junctional localization of claudin-16. Here, we examined the effect of Mg2+ deprivation on the distribution and function of claudin-16 using Madin-Darby canine kidney (MDCK) cells expressing FLAG-tagged claudin-16. Mg2+ deprivation inhibited the localization of claudin-16 at tight junctions, but did not affect the localization of other claudins. Re-addition of Mg2+ induced the tight junctional localization of claudin-16, which was inhibited by U0126, a MEK inhibitor. Transepithelial permeability to Mg2+ was also inhibited by U0126. The phosphorylation of ERK was reduced by Mg2+ deprivation, and recovered by re-addition of Mg2+. These results suggest that the MEK/ERK-dependent phosphorylation of claudin-16 affects the tight junctional localization and function of claudin-16. Mg2+ deprivation decreased the phosphothreonine levels of claudin-16. The phosphothreonine levels of T225A and T233A claudin-16 were decreased in the presence of Mg2+ and these mutants were widely distributed in the plasma membrane. Furthermore, TER and transepithelial Mg2+ permeability were decreased in the mutants. We suggest that the tight junctional localization of claudin-16 requires a physiological Mg2+ concentration and the phosphorylation of threonine residues via a MEK/ERK-dependent pathway. |
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Keywords: | H-89 N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride MDCK Madin-Darby canine kidney PKA protein kinase A TAL thick ascending limb of Henle TER transepithelial electrical resistance |
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