title: Association of paracellin-1 with ZO-1 augments the reabsorption of divalent cations in renal epithelial cells.
abstract: Paracellin-1 (PCLN-1) belongs to the claudin family of tight junction proteins and possibly plays a critical role in the reabsorption of magnesium and calcium. So far, the physiological properties of PCLN-1 have not been clarified. In the present study, we investigated whether PCLN-1 is associated with ZO-1. We also investigated whether (45)Ca(2+) transport across the paracellular barrier is affected by this association. In vitro binding analysis using glutathione S-transferase fusion protein showed that the C-terminal TRV sequence, especially Thr and Val residues, of PCLN-1 interacts with ZO-1. Next, PCLN-1 was stably expressed in Madin-Darby canine kidney cells using a FLAG tagging vector. ZO-1 was co-immunoprecipitated with the wild-type PCLN-1 and the alanine substitution (TAV) mutant. However, mutants of the deletion (Delta TRV) and the alanine substitution (ARV and TRA) inhibited the association of PCLN-1 with ZO-1. Confocal immunofluorescence demonstrated that the wild-type PCLN-1 and the TAV mutant localized in the tight junction along with ZO-1, but the Delta TRV, ARV, and TRA mutants were widely distributed in the lateral membrane including the tight junction area. Interestingly, monolayers of cells expressing the wild-type PCLN-1 and the TAV mutant showed higher activities of (45)Ca(2+) transport from apical to basal compartments, compared with those expressing the Delta TRV, ARV, and TRA mutants and the mock cells. (45)Ca(2+) transport was inhibited by increased magnesium concentration suggesting that magnesium and calcium were competitively transported by PCLN-1. It was noted that a positive electrical potential gradient enhanced (45)Ca(2+) transport from apical to basal compartments without affecting the opposite direction of transport. Thus, PCLN-1 localizes to the tight junction followed by association with ZO-1, and the PCLN-1.ZO-1 complex may play an essential role in the reabsorption of divalent cations in renal epithelial cells.
authors.affiliation: Department of Environmental Biochemistry and Toxicology, University of Shizuoka, 52-1 Yada, Shizuoka 422-8526, Japan. ikari@u-shizuoka-ken.ac.jp
authors.forename: Akira
authors.initials: A
authors.lastname: Ikari
authors.forename: Naho
authors.initials: N
authors.lastname: Hirai
authors.forename: Morihiko
authors.initials: M
authors.lastname: Shiroma
authors.forename: Hitoshi
authors.initials: H
authors.lastname: Harada
authors.forename: Hideki
authors.initials: H
authors.lastname: Sakai
authors.forename: Hisayoshi
authors.initials: H
authors.lastname: Hayashi
authors.forename: Yuichi
authors.initials: Y
authors.lastname: Suzuki
authors.forename: Masakuni
authors.initials: M
authors.lastname: Degawa
authors.forename: Kuniaki
authors.initials: K
authors.lastname: Takagi
mesh_terms: D000042:Absorption; D000818:Animals; D001692:Biological Transport; D002118:Calcium / Q000378:metabolism; D002132:Calcium Radioisotopes; D002413:Cations, Divalent / Q000378:metabolism*; D002460:Cell Line; D057167:Claudins; D004285:Dogs; D017097:Electric Impedance; D004847:Epithelial Cells / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D017353:Gene Deletion; D015870:Gene Expression; D005982:Glutathione Transferase / Q000235:genetics; D007163:Immunosorbent Techniques; D007668:Kidney / Q000378:metabolism*; D008274:Magnesium / Q000378:metabolism; D008565:Membrane Proteins / Q000032:analysis / Q000235:genetics / Q000502:physiology*; D051379:Mice; D018613:Microscopy, Confocal; D016296:Mutagenesis; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D010750:Phosphoproteins / Q000502:physiology*; D011485:Protein Binding; D011817:Rabbits; D051381:Rats; D011993:Recombinant Fusion Proteins; D019108:Tight Junctions / Q000737:chemistry; D014162:Transfection; D062826:Zonula Occludens-1 Protein
chemical_list: D002132:Calcium Radioisotopes; D002413:Cations, Divalent; D057167:Claudins; D008565:Membrane Proteins; D010446:Peptide Fragments; D010750:Phosphoproteins; D011993:Recombinant Fusion Proteins; C569043:Tjp1 protein, mouse; C569044:Tjp1 protein, rat; D062826:Zonula Occludens-1 Protein; C502010:claudin 16; D005982:Glutathione Transferase; D008274:Magnesium; D002118:Calcium