title: Claudin-2 pore function requires an intramolecular disulfide bond between two conserved extracellular cysteines.
abstract: Claudins constitute a family of tight junction transmembrane proteins whose first extracellular loop (ECL1) determines the paracellular permeability and ion selectivity in epithelia. There are two cysteines in the ECL1 that are conserved among all claudins. We hypothesized that these extracellular cysteines are linked by an intramolecular disulfide bond that is necessary for correct pore folding and function. To test this, we mutated C54 and C64 in claudin-2, either individually or together to alanine or serine, and generated stable Madin-Darby canine kidney (MDCK) I Tet-off cell lines. Immunoblotting showed a higher molecular mass band in the mutants with a single cysteine mutation, consistent with a claudin-2 dimer, suggesting that the two conserved cysteines normally form an intramolecular disulfide bond in wild-type claudin-2. By immunofluorescent staining, the alanine mutants were mislocalized intracellularly, while the serine mutants were expressed at the tight junction. Thus dimerization of both C54A and C64A did not require tight junction expression, suggesting that C54 and C64 are located near an intermolecular interface involved in cis-interaction. The conductance and Na(+) permeability of the serine mutants were markedly lower than the wild type, but there was no difference between the single mutants and the double mutant. We conclude that the disulfide bond between the conserved extracellular cysteines in claudin-2 is necessary for pore formation, probably by stabilizing the ECL1 fold, but is not required for correct protein trafficking. We further speculate that this role is generalizable to other claudin family members.
authors.affiliation: Division of Nephrology and Hypertension, University of Kansas Medical Center, Kansas City, Kansas 66160, USA.
authors.forename: Jiahua
authors.initials: J
authors.lastname: Li
authors.forename: Susanne
authors.initials: S
authors.lastname: Angelow
authors.forename: Anna
authors.initials: A
authors.lastname: Linge
authors.forename: Min
authors.initials: M
authors.lastname: Zhuo
authors.forename: Alan S L
authors.initials: AS
authors.lastname: Yu
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D002460:Cell Line; D062446:Claudin-2 / Q000235:genetics / Q000378:metabolism*; D003545:Cysteine / Q000737:chemistry*; D004220:Disulfides / Q000737:chemistry*; D004285:Dogs; D005786:Gene Expression Regulation / Q000502:physiology; D061985:Madin Darby Canine Kidney Cells; D009154:Mutation; D018272:Porins / Q000737:chemistry
chemical_list: D062446:Claudin-2; D004220:Disulfides; D018272:Porins; D003545:Cysteine
keywords: cysteines; disulfide bond; ion channel; paracellular transport; tight junction