Participation of the second extracellular loop of claudin-5 in paracellular tightening against ions, small and large molecules

Abstract

Tight junctions control paracellular permeability. Here, we analyzed the impact of residues in the second extracellular loop (ECL2) of mouse claudin-5 on paracellular permeability. Stable expression of claudin-5(wild type) in MDCK-II cells-but not that of mutants R145A, Y148A, Y158A or E159Q-increased transepithelial electrical resistance and decreased fluorescein permeation. Expression of claudin-5(Y148A), (Y158A) or (E159Q) enhanced permeability of FITC-dextran(10 kDa), which was unchanged in cells expressing claudin-5(wild type) or claudin-5(R145A). In contrast, targeting to tight junctions, strand morphology and tight junction assembly were unchanged. It is concluded that R145 is unessential for trans-interaction of claudin-5, but necessary for tightening against small solutes and ions. The highly conserved residues Y148, Y158 and E159 in ECL2 of claudin-5 contribute to homo- and/or heterophilic trans-interaction between classic claudins and thereby tighten the paracellular space against ions, small and large molecules. These results provide novel insights into the molecular function of tight junctions.

In tight junctions, claudins regulate the interactions between occludin, tricellulin and marvelD3, which, inversely, modulate claudin oligomerization

“…In general, Tric, Md3 and to a lower extent Occl enable a more compact and integrated Cld1 strand morphology similar as occurring physiologically. Small and rectangular TJ strands are well known from epithelial cells expressing Clds and TAMPs endogenously (Piehl et al, 2010).…”

“…Cell culture, transfection and immunocytochemistry HEK-293 (Piontek et al, 2011), MDCK-II (Piehl et al, 2010) and Caco-2 (Winkler et al, 2009) cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Invitrogen, Darmstadt, Germany) containing 10% (v/v) fetal calf serum, 100 units/ ml penicillin, 100 mg/ml streptomycin, and 1% L-alanyl-L-glutamine (Invitrogen, Darmstadt, Germany). Transient mono-, co-and triple-transfections were performed using polyethylenimin (PEI) according to the supplier's recommendations (Polysciences, Eppelheim, Germany).…”

“…Tissue-specific expression of different combinations of Clds defines the tissue-specific barrier characteristics . In this respect, expression of tightening Clds, such as Cld5 (Nitta et al, 2003;Piehl et al, 2010) has been demonstrated to seal the paracellular cleft. On the other hand, pore-forming Clds, such as Cld2 (Amasheh et al, 2002) facilitate paracellular ion permeability.…”

“…On the other hand, pore-forming Clds, such as Cld2 (Amasheh et al, 2002) facilitate paracellular ion permeability. The pore-forming function of a specific Cld is preferentially determined by the first extracellular loop (Amasheh et al, 2002) while the second extracellular loop rather supports tightening of the paracellular cleft (Piehl et al, 2010). Both homophilic and heterophilic cisand trans-interactions have been observed between Clds (Basuroy et al, 2006;Furuse et al, 1999;Piontek et al, 2011).…”

Chlorpheniramine Increases Paracellular Permeability to Marker Fluorescein Lucifer Yellow Mediated by Internalization of Occludin in Murine Colonic Epithelial Cells

“…7,22) In addition, claudin-1 and -3 do not form ion-permeable pore and claudin-5 blocks ions permeability. 23) The mRNAs expression of claudin-1, -3, -4, -5, and -7 was detected in Caco-2 cells (data not shown), whereas that of claudin-1-claudin-8 except for claudin-5 was detected in MCE301 cells ( Fig. 2A) The expression of claudin subtypes is changed along the intestine during postnatal development in the mice.…”