title: Activation of a polyvalent cation-sensing receptor decreases magnesium transport via claudin-16.
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.
authors.affiliation: Department of Pharmaco-Biochemistry, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526, Japan. ikari@u-shizuoka-ken.ac.jp
authors.forename: Akira
authors.initials: A
authors.lastname: Ikari
authors.forename: Chiaki
authors.initials: C
authors.lastname: Okude
authors.forename: Hayato
authors.initials: H
authors.lastname: Sawada
authors.forename: Yohei
authors.initials: Y
authors.lastname: Sasaki
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Yamazaki
authors.forename: Junko
authors.initials: J
authors.lastname: Sugatani
authors.forename: Masakuni
authors.initials: M
authors.lastname: Degawa
authors.forename: Masao
authors.initials: M
authors.lastname: Miwa
mesh_terms: D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D003994:Bucladesine / Q000494:pharmacology; D002460:Cell Line; D057167:Claudins; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004285:Dogs; D017097:Electric Impedance; D008247:Lysosomes / Q000187:drug effects / Q000378:metabolism; D008274:Magnesium / Q000378:metabolism*; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D021381:Protein Transport / Q000187:drug effects; D044169:Receptors, Calcium-Sensing / Q000819:agonists / Q000378:metabolism*; D013752:Tetracycline; D019108:Tight Junctions / Q000187:drug effects / Q000378:metabolism
chemical_list: D057167:Claudins; D008565:Membrane Proteins; D044169:Receptors, Calcium-Sensing; C502010:claudin 16; D003994:Bucladesine; D017868:Cyclic AMP-Dependent Protein Kinases; D013752:Tetracycline; D008274:Magnesium