Activation of a polyvalent cation-sensing receptor decreases magnesium transport via claudin-16 |
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Authors: | Akira Ikari Chiaki Okude Yohei Sasaki Junko Sugatani Masakuni Degawa Masao Miwa |
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Affiliation: | a Department of Pharmaco-Biochemistry, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526, Japan b Department of Molecular Toxicology, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526, Japan c COE Program in the 21st Century, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526, Japan |
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Abstract: | Renal magnesium is mainly reabsorbed by a paracellular pathway in the thick ascending limb of Henle. The expression of claudin-16 increased magnesium transport in Madin-Darby canine kidney (MDCK) cells. Little is known about the regulatory mechanism of magnesium transport via claudin-16. Here we examined the effect of a polyvalent cation-sensing receptor (CaSR) on the intracellular distribution of and transport of magnesium by claudin-16. FLAG-tagged claudin-16 was stably expressed in MDCK cells using a Tet-OFF system. The activation of CaSR by magnesium, calcium, neomycin, and gadolinium did not affect the expression of FLAG-tagged claudin-16, CaSR, or ZO-1, a tight junctional scaffolding protein. These activators decreased the phosphoserine level of FLAG-tagged claudin-16 and the association of FLAG-tagged claudin-16 with ZO-1. The activation of CaSR induced a decrease in PKA activity. Immunofluorescence microscopy revealed that FLAG-tagged claudin-16 is distributed at the cell-cell border under unstimulated conditions, whereas it translocates to the intracellular compartment, mainly lysosome, with the activation of CaSR. In contrast, the distribution of ZO-1 was unaffected by the activation. The expression of FLAG-tagged claudin-16 increased transepithelial electrical resistance (TER) and transepithelial magnesium transport without affecting FITC-dextran (MW 4000) flux. The activation of CaSR decreased TER and magnesium transport, which were recovered by co-treatment with dibutyryl cAMP, a membrane-permeable cAMP analogue. Taken together, CaSR activation may decrease PKA activity, resulting in a decrease in phosphorylated claudin-16, the translocation of claudin-16 to lysosome and a decrease in magnesium reabsorption. |
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Keywords: | CaSR, polyvalent cation-sensing receptor MDCK, Madin-Darby canine kidney PKA, protein kinase A TAL, thick ascending limb TER, transepithelial electrical resistance |
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