A multi‐pore model of the blood–brain barrier tight junction strands recapitulates the permeability features of wild‐type and mutant claudin‐5

In the blood-brain barrier (BBB), endothelial cells are joined by tight junctions (TJs), multi-protein assemblies that seal the paracellular space and restrict molecular transport. Among the BBB TJ proteins, Claudin-5 (Cldn15) is the most abundant one. Structural models for claudin complexes, first introduced for channel-forming, selectively permeable claudins, comprise protomers arranged to form paracellular pores that regulate transport by electrostatic and/or steric effects arising from pore-lining residues. With limited exceptions, computational studies explored oligomers of only a few subunits, while TJs are formed by extended polymeric strands. Here, we employ multi-microsecond all-atom molecular dynamics and free-energy (FE) calculations to study two distinct models of TJ-forming Cldn15 complexes, called multi-Pore I and multi-Pore II, each comprising 16 protomers arranged around three adjacent pores. FE calculations of water and ions permeation reveal that, in both models, ion transport is hindered by FE barriers higher than in single pores. Moreover, only the multi-Pore I model captures the Cldn15 G60R variant's effect, making it anion-permeable. The results provide insights into Cldn15 structure and function and validate a structural model of BBB TJs useful for studying barrier impairment in brain diseases and for developing therapeutic approaches.