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.
Leibniz-Institut für Molekulare Pharmakologie, Berlin, Germany.
Anna
A
Veshnyakova
Susanne M
SM
Krug
Sebastian L
SL
Mueller
Jörg
J
Piontek
Jonas
J
Protze
Michael
M
Fromm
Gerd
G
Krause
D000595:Amino Acid Sequence; D002460:Cell Line; D062445:Claudin-1 / Q000235:genetics* / Q000378:metabolism; D057167:Claudins / Q000235:genetics* / Q000378:metabolism; D004847:Epithelial Cells / Q000378:metabolism*; D006801:Humans; D007473:Ion Channels / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D010539:Permeability; D019108:Tight Junctions / Q000378:metabolism* / Q000502:physiology
C491875:CLDN2 protein, human; D062445:Claudin-1; D057167:Claudins; D007473:Ion Channels