Extracellular Mg(2+) regulates the tight junctional localization of claudin-16 mediated by ERK-dependent phosphorylation.

Claudin-16 is involved in the paracellular reabsorption of Mg(2+) 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 Mg(2+) deprivation on the distribution and function of claudin-16 using Madin-Darby canine kidney (MDCK) cells expressing FLAG-tagged claudin-16. Mg(2+) deprivation inhibited the localization of claudin-16 at tight junctions, but did not affect the localization of other claudins. Re-addition of Mg(2+) induced the tight junctional localization of claudin-16, which was inhibited by U0126, a MEK inhibitor. Transepithelial permeability to Mg(2+) was also inhibited by U0126. The phosphorylation of ERK was reduced by Mg(2+) deprivation, and recovered by re-addition of Mg(2+). These results suggest that the MEK/ERK-dependent phosphorylation of claudin-16 affects the tight junctional localization and function of claudin-16. Mg(2+) deprivation decreased the phosphothreonine levels of claudin-16. The phosphothreonine levels of T225A and T233A claudin-16 were decreased in the presence of Mg(2+) and these mutants were widely distributed in the plasma membrane. Furthermore, TER and transepithelial Mg(2+) permeability were decreased in the mutants. We suggest that the tight junctional localization of claudin-16 requires a physiological Mg(2+) concentration and the phosphorylation of threonine residues via a MEK/ERK-dependent pathway.