Pediatr Nephrol (2011) 26:2133–2142 2135 Fig. 1 Biochemical components of tight junction: transmembrane protein: claudin, occludin, junctional adhesion molecule (JAM) seal the paracellular space between adjacent epithelial cells, separating the cell into apical compartment and basolateral compartment. Claudin constitutes the paracellular barrier and pore by homogenic and heterogenic interaction. C-termini of claudin, occludin, and JAM have the paracellular pore/barrier itself came from chimera and mutagenesis studies. As mentioned above, claudins have two extracellular loops. Colegio et al. [19] swapped the extracellular loops between claudin-2 (‘pore claudin’) and claudin-4 (‘barrier claudin’) and showed that the first extracellular loop (ECL) of claudin-2 with the second ECL of claudin-4 still functioned as a pore, whereas the first ECL of claudin-4 with the second ECL of claudin-2 functioned as a barrier. Thus, it directly supports the theory that claudins form either a paracellular pore or a barrier and that this is determined by the first ECL. Taking this a step further, mutagenesis studies of the first ECL of claudins revealed the existence of sites conferring permeability and selectivity. Using charge-reversing mutations, Colegio [20] first showed that K65D (positive charge mutated to negative charge) in claudin-4 increased Na+ permeability, while D55R, E64K (negative charge mutated to positive charge) both individually and synergistically reversed the claudin-15 ion preference from Na+ to Cl-. However, the effect of reversing the charge at a site may not necessarily reveal the normal physiological role of the residue at that site but could instead reflect non-physiological effects of introducing the opposite charge. Yu et al. [21] therefore used charge-neutralizing mutations in claudin 2. They found that D65 appears to be the major contributor to an intra pore Na+ binding site. In aggregate, these data support PDZ binding domain linking to scaffold ZO protein. ZO protein can bind directly [60] to cytoskeleton actin filament. Protein kinase, protein phosphatase, and transcription factors (not shown in the figure) can interact with cytosolic part of claudin, occludin and JAM and ZO protein to regulate tight junction assembly a model in which the first ECL of claudins forms the lining of the paracellular pore and determines the magnitude and selectivity of its permeability to small ions. Properties of the claudin pore In general, the claudin pore is a narrow, fluid-filled, charge- and size-selective pore formed by the first extracellular loops of adjacent claudin. Ions are transported passively, parallel to the cell membrane within the paracellular space, driven by the chemical and electrical gradient. Van Itallie [22] applied molecules of different sizes to the apical side of the epithelial cell layer and measured the concentration of the small molecules from the basolateral site. They reported that the radius of claudin pores was ∼4 Å in MDCK cell lines. Similarly, Yu et al. [21] measured the permeability of organic cations and estimated the pore size of claudin 2 as 6.5 Å in diameter. The molecular basis of the charge selectivity of the claudin pore is electrostatic interaction of partially dehydrated permeating ions with an oppositely charged site within the pore (e.g., D65 in cation- selective claudin-2) [21]. More recently, the structure of the first ECL fold has begun to be explored by cysteine mutagenesis [23]. By mutating several amino acids of the first ECL of claudin-2 to cysteine, it was possible to study the accessibility of the mutated residue to extracellularly