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Updated: Aug 25, 2026

Functional Assessment of Intestinal Tight Junction Barrier and Ion Permeability in Native Tissue by Ussing Chamber Technique
Published on: May 26, 2021
Thr203 of claudin-1, a putative phosphorylation site for MAP kinase, is required to promote the barrier function of
Masato Fujibe1, Hideki Chiba, Takashi Kojima
1Department of Pathology, Sapporo Medical University School of Medicine, Sapporo 060-8556, Japan.
Abstract:
Mitogen-activated protein kinase (MAPK) modulates the barrier function of tight junctions. We identified a putative phosphorylation site for MAPK at around Thr203 (PKPTP) in claudin-1, and determined the biological significance of this site. To this end, using the rat lung endothelial cell line RLE, we generated cells expressing doxycycline (Dox)-inducible wild-type claudin-1 and its mutant with substitution of Thr203 to Ala, and named them RLE:rtTA:CL1 and RLE:rtTA:CL1T203A, respectively. We herein show, by measurement of transendothelial electrical resistance and paracellular flux of mannitol and inulin, that functional tight junctions were reconstituted in both cells by Dox-induced expression of claudin-1. Interestingly, the barrier functions of tight junctions were less developed in RLE:rtTA:CL1T203A cells compared with RLE:rtTA:CL1 cells. Consistently, levels of both detergent-insoluble claudin-1 protein and its threonine-phosphorylation after Dox treatment were low in RLE:rtTA:CL1T203A cells compared to RLE:rtTA:CL1 cells. Furthermore, pretreatment with the MAPK inhibitor PD98059 markedly suppressed the barrier function and amount of detergent-insoluble claudin-1 in Dox-exposed RLE:rtTA:CL1 cells, whereas it marginally influenced those in RLE:rtTA:CL1T203A cells. These findings indicate that Thr203 of claudin-1 is required to enhance the barrier function of claudin-1-based tight junctions, probably via its phosphorylation and subsequent integration into tight junctions.
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