Tight junctional localization of claudin-16 is regulated by syntaxin 8 in renal tubular epithelial cells.
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.
['Ikari|Akira|A|', 'Tonegawa|Chie|C|', 'Sanada|Ayumi|A|', 'Kimura|Toru|T|', 'Sakai|Hideki|H|', 'Hayashi|Hisayoshi|H|', 'Hasegawa|Hajime|H|', 'Yamaguchi|Masahiko|M|', 'Yamazaki|Yasuhiro|Y|', 'Endo|Satoshi|S|', 'Matsunaga|Toshiyuki|T|', 'Sugatani|Junko|J|']
D019943:Amino Acid Substitution D000818:Animals D057167:Claudins D017868:Cyclic AMP-Dependent Protein Kinases D004285:Dogs D011992:Endosomes D004847:Epithelial Cells D006801:Humans D007687:Kidney Tubules, Proximal D008247:Lysosomes D061985:Madin Darby Canine Kidney Cells D008297:Male D020125:Mutation, Missense D010766:Phosphorylation D011485:Protein Binding D021381:Protein Transport D050765:Qa-SNARE Proteins D051381:Rats D017208:Rats, Wistar D019108:Tight Junctions
Tight Junctions Syntaxin Kidney Trafficking Claudin Confocal Microscopy Magnesium Homeostasis