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.
Highlights
Magnesium is an important cofactor for various enzymes
The alanine substitution mutant for arginine (TAV) of the PSD95/DglA/ZO-1-like domain (PDZ)-binding motif bound to ZO-1, to the wildtype, but the deletion mutant (⌬TRV) and the alanine substitution mutants for threonine (ARV) or valine (TRA) did not
We revealed that the Thr and Val residues, but not the Arg residue, of the PDZ-binding motif are essential for the association of PCLN-1 with ZO-1 using the glutathione S-transferase (GST) pull-down assay and immunoprecipitation analysis
Summary
Magnesium is an important cofactor for various enzymes. The bodies magnesium balance is regulated by the kidney,. Monolayers of cells expressing the wild-type PCLN-1 and the TAV mutant showed higher activities of 45Ca2؉ transport from apical to basal compartments, compared with those expressing the ⌬TRV, ARV, and TRA mutants and the mock cells. The wild-type and the mutants of FLAG-tagged PCLN-1 were stably expressed in MDCK cells.
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