Molecular regulation of intestinal tight junction strand formation and stability

Epithelial homeostasis and the formation of selectively permeable tissue barriers depend on claudins. In the intestine, claudin-2 and claudin-15 both form paracellular water and cation, i.e., Na + , selective channels and are differentially development and in disease. The observation that knockout of both claudins results in perinatal death but that deletion of only one are viable demonstrates functional redundancy of claudin-2 and claudin-15. HYPOTHESIS: We hypothesized that differences in the molecular interactions underlying claudin-2 and claudin-15 polymerization may explain the distinct patterns of their expression in development and disease. METHODS: We used sequence alignments, structural predictions, and molecular dynamics simulations to compare claudins 2 and 15. Polymerization, i.e., strand formation, was imaged by super-resolution (STED) microscopy. Transepithelial electrical resistance was measured to assess channel conductivity. RESULTS: Sequence analysis showed that glutamine-81 (Q81) within the second transmembrane helix is unique: other claudins have arginine at the corresponding site, e.g., claudin-15 R79 . Evolutionary conservation of claudin-2 Q81 suggests that it may confer unique structural or functional properties. Consistent with this, molecular dynamics simulations showed that claudin-2 Q81 mutation altered the numbers and sites of potential homomeric interactions. Comprehensive screening of claudin-2 Q81 and claudin-15 R79 mutants confirmed the essential contributions of these residues to assembly and architecture of polymeric strand networks in nonepithelial cells, indicating distinct claudin-2 and claudin-15 polymerization interfaces. When expressed in epithelial cells, claudin-2 Q81 or claudin-15 R79 mutagenesis altered strand network complexity and paracellular channel function. Complementary mutations of non-conserved residues within the third transmembrane helix rescued claudin function, indicating that a novel intramembranous interaction modifies claudin helix bundling, polymerization, and function. CONCLUSION: These data define a previously unrecognized interface between the second and third transmembrane helices that stabilizes claudin structure to support polymerization and channel function. Moreover, the distinct impact of corresponding mutations on claudin-2 and claudin-15 structure and function suggest that the role and importance of this intramembranous binding interface differs within the claudin protein family. We speculate that this interface may modify strand dynamics that dictate unique functional or regulatory aspects of otherwise similar claudins. DFG Walter Benjamin Fellowship (531212047), HDDC Pilot and Feasibility Grant (2024A004855), NIH individual grants (R01DK061931, R01DK068271, R21GM144801, R01DK048106) This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.