Claudins are the critical transmembrane proteins in tight junctions. Claudin-5, for instance, prevents paracellular permeation of small molecules. However, the molecular interaction mechanism is unknown. Hence, the claudin-claudin interaction and tight junction strand formation were investigated using systematic single mutations. Claudin-5 mutants transfected into tight junction-free cells demonstrated that the extracellular loop 2 is involved in strand formation via trans-interaction, but not via polymerization, along the plasma membrane of one cell. Three phenotypes were obtained: the tight junction type (wild-type-like trans- and cis-interaction; the disjunction type (blocked trans-interaction); the intracellular type (disturbed folding). Combining site-directed mutagenesis, live-cell imaging-, electron microscopy-, and molecular modeling data led to an antiparallel homodimer homology model of the loop. These data for the first time explain how two claudins hold onto each other and constrict the paracellular space. The intermolecular interface includes aromatic (F147, Y148, Y158) and hydrophilic (Q156, E159) residues. The aromatic residues form a strong binding core between two loops from opposing cells. Since nearly all these residues are conserved in most claudins, our findings are of general relevance for all classical claudins. On the basis of the data we have established a novel molecular concept for tight junction formation.

Together, these findings demonstrate that CPE binds to the hydrophobic turn and flanking polar residues in the loop 2 of claudin-3 outside tight junctions. The data can be used for the specific design of CPE-based modulators of tight junctions, to improve drug delivery, and as chemotherapeutics for tumors overexpressing claudins.

Lumen expansion driven by hydrostatic pressure occurs during many morphogenetic processes. Although it is well established that members of the Claudin family of transmembrane tight junction proteins determine paracellular tightness within epithelial/endothelial barrier systems, functional evidence for their role in the morphogenesis of lumenized organs has been scarce. Here, we identify Claudin5a as a core component of an early cerebral-ventricular barrier system that is required for ventricular lumen expansion in the zebrafish embryonic brain before the establishment of the embryonic blood-brain barrier. Loss of Claudin5a or expression of a tight junction-opening Claudin5a mutant reduces brain ventricular volume expansion without disrupting the polarized organization of the neuroepithelium. Perfusion experiments with the electron-dense small molecule lanthanum nitrate reveal that paracellular tightness of the cerebral-ventricular barrier decreases upon loss of Claudin5a. Genetic analyses show that the apical neuroepithelial localization of Claudin5a depends on epithelial cell polarity and provide evidence for concerted activities between Claudin5a and Na B rain morphogenesis in the zebrafish embryo involves a wellstudied ventricular lumen expansion process which occurs at early stages of development between 17 and 21 h after fertilization (hpf) (1). Genetic analyses showed that the osmoregulatory ion pump ATPase, Na + /K + transporting, alpha 1 polypeptide (Atp1a1) is critically important for lumen expansion which suggests a role in the generation of hydrostatic pressure (1). However, the embryonic cerebral barrier expected to maintain luminal fluids and ions within the brain ventricles remains unknown. It is known that the bloodbrain barrier forms 2 days after brain ventricle expansion (2). Also, the choroid plexus, which develops from the ependymal layer lining the ventral floor of the cerebral ventricle, is not functional at these stages and forms the blood-cerebrospinal fluid barrier only at later stages and in the adult fish (3, 4). To elucidate the nature of the cerebral-ventricular barrier system required for initial lumen expansion, we focused our attention on the neuroepithelium lining the cerebral cavities. We hypothesized that Claudins (Cldn) may contribute to the cerebral-ventricular barrier based on their established roles in the regulation of tight junction (TJ) barriers (5, 6). Claudins are characterized as either barrier-or pore-forming. In a recent study, Bagnat and colleagues demonstrated an involvement of the pore-forming Cldn15 in gut lumen expansion in the zebrafish embryo (7). Moreover, the C-terminal half of Clostridium perfringens enterotoxin (C-CPE), a polypeptide with inhibitory activity to several barrier-forming Claudins including Cldn3, Cldn4, and Cldn6, affected murine blastocoel cavity expansion, which is another lumen expansion process that requires hydrostatic pressure (8). Together, these studies implied an important function of Claudins in brain ventricular lumen expansion.Her...

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.

Most claudins are tight junction (TJ)-forming proteins. However, their interaction on the molecular level remains unresolved. It is hypothesized that the extracellular loops specify these claudin functions. It is assumed that the first extracellular loop (ECL1) is critical for determining the paracellular tightness and the selective paracellular ion permeability, and that the second extracellular loop may cause narrowing of the paracellular cleft. Using a combination of site-directed mutagenesis and homology modeling for the second extracellular loop (ECL2) of claudin-5, we found several amino acids important for claudin folding and/or trans-interaction to claudins in neighboring cells. These sensitive residues are highly conserved within one group of claudins, whereas the corresponding positions in the remaining claudins show a large sequence variety. Further functional data and analysis of sequence similarity for all claudins has led to their differentiation into two groups, designated as classic claudins (1-10, 14, 15, 17, 19) and nonclassic claudins (11-13, 16, 18, 20-24). This also corresponds to conserved structural features at ECL1 for classic claudins. Based on this, we propose a hypothesis for different pore-forming claudins. Pore formation or tightness is supported by the spatial encounter of a surplus of repulsing or attracting amino acid types at ECL1. A pore is likely opened by repulsion of equally charged residues, while an encounter of unequally charged residues leads to tight interaction. These considerations may reveal the ECLs of claudins as decisive submolecular determinants that specify the function of a claudin.