Determinants contributing to claudin ion channel formation

Pore-forming properties of claudins (Cld) are likely defined by residues of their first extracellular loop (ECL1). Detailed mechanisms are unclear. MDCK cells overexpressing FLAG-Cld-1 wild-type and mutants were characterized by transepithelial resistance (TER) and ion permeability measurements. Replacing ECL1 residues of sealing Cld-1 by corresponding Cld-2 residues we aimed to identify new determinants responsible for sealing and/or pore formation. We found that E48K and S53E substitutions in human Cld-1 strongly reduced TER and increased permeability for Na+ and Cl-. In contrast, K65D, D68S, and other single substitutions showed no significant change of TER and permeability for Na+ and Cl-. Double substitution S53E/K65D did not change TER and ion permeability, whereas S53E/D68S decreased TER, albeit weaker than S53E. Ratio of permeabilities for Na+ and Cl- revealed no clear charge specificity of the pore induced by S53E or S53E/D68S in Cld-1, suggesting that primarily S53 and potentially D68 in Cld-1 are involved in sealing of the paracellular cleft and that charge-unselective pores may be induced by substituting S53E.