title: Tight junctional localization of claudin-16 is regulated by syntaxin 8 in renal tubular epithelial cells.
abstract: Claudin-16 (CLDN16) regulates the paracellular reabsorption of Mg(2+) in the thick ascending limb of Henle's loop. However, the mechanism regulating the tight junctional localization of CLDN16 remains unknown. In yeast two-hybrid systems, we found that CLDN16 bound to syntaxin 8 (STX8), a target soluble N-ethylmaleimide-sensitive factor attachment protein receptor. We have examined the effect of STX8 on the localization and function of CLDN16 using Madin-Darby canine kidney cells expressing FLAG-tagged CLDN16. A pulldown assay showed that the carboxyl cytoplasmic region of human CLDN16 bound to STX8. CLDN16 was localized in the thick ascending limb, whereas STX8 was widely distributed throughout the rat kidney. An association between CLDN16 and STX8 was observed in rat renal homogenates and Madin-Darby canine kidney cells. STX8 siRNA decreased the cell surface localization of CLDN16 and transepithelial electrical resistance and permeability to Mg(2+) but increased the co-localization of CLDN16 with early endosome and lysosome markers. Dephosphorylation of CLDN16 by protein kinase A inhibitors and S217A mutant, a dephosphorylated form, decreased the association with STX8 and the cell surface localization of CLDN16. Recycling assays indicated that STX8 siRNA decreased the trafficking of CLDN16 to the plasma membrane without affecting endocytosis. Dominant negative Rab11 and recycling inhibitor primaquine decreased the cell surface localization of CLDN16, which was similar to that in STX8 siRNA-transfected cells. These results suggest that STX8 mediates the recycling of CLDN16 and constitutes an important component of the CLDN16 trafficking machinery in the kidney.
authors.affiliation: From the Laboratory of Biochemistry, Department of Biopharmaceutical Sciences, Gifu Pharmaceutical University, Gifu 501-1196, Japan.
authors.forename: Akira
authors.initials: A
authors.lastname: Ikari
authors.forename: Chie
authors.initials: C
authors.lastname: Tonegawa
authors.forename: Ayumi
authors.initials: A
authors.lastname: Sanada
authors.forename: Toru
authors.initials: T
authors.lastname: Kimura
authors.forename: Hideki
authors.initials: H
authors.lastname: Sakai
authors.forename: Hisayoshi
authors.initials: H
authors.lastname: Hayashi
authors.forename: Hajime
authors.initials: H
authors.lastname: Hasegawa
authors.forename: Masahiko
authors.initials: M
authors.lastname: Yamaguchi
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Yamazaki
authors.forename: Satoshi
authors.initials: S
authors.lastname: Endo
authors.forename: Toshiyuki
authors.initials: T
authors.lastname: Matsunaga
authors.forename: Junko
authors.initials: J
authors.lastname: Sugatani
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D057167:Claudins / Q000235:genetics / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000235:genetics / Q000378:metabolism; D004285:Dogs; D011992:Endosomes / Q000235:genetics / Q000378:metabolism; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D007687:Kidney Tubules, Proximal / Q000166:cytology / Q000378:metabolism*; D008247:Lysosomes / Q000235:genetics / Q000378:metabolism; D061985:Madin Darby Canine Kidney Cells; D008297:Male; D020125:Mutation, Missense; D010766:Phosphorylation / Q000502:physiology; D011485:Protein Binding / Q000502:physiology; D021381:Protein Transport / Q000502:physiology; D050765:Qa-SNARE Proteins / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D019108:Tight Junctions / Q000235:genetics / Q000378:metabolism*
chemical_list: D057167:Claudins; D050765:Qa-SNARE Proteins; C502010:claudin 16; D017868:Cyclic AMP-Dependent Protein Kinases
keywords: Claudin; Confocal Microscopy; Kidney; Magnesium Homeostasis; Syntaxin; Tight Junctions; Trafficking