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Mapping the claudin‐2 pore: comprehensive cysteine‐scanning mutagenesis and thiol modification of claudin‐2 first extracellular loop
Author(s) -
Li Jiahua,
Nakamura Tamako,
Zhuo Min,
Pei Lei,
Yu Alan S.L.
Publication year - 2012
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.26.1_supplement.885.20
Subject(s) - claudin , cysteine , chemistry , paracellular transport , extracellular , mutant , mutagenesis , biophysics , biochemistry , tight junction , permeability (electromagnetism) , biology , membrane , gene , enzyme
Claudins are tight junction proteins that determine paracellular permeability in epithelia. The first extracellular loop of claudin‐2 forms a paracellular cation pore. To understand the structure and function of this pore, we aim to conduct a comprehensive cysteine‐scanning mutagenesis of the first extracellular loop and map amino acids that line the cation pore or are located at the intermolecular interface. Up to now, we have generated 20 cysteine mutants and made stably transfected, polyclonal MDCK I Tet‐Off cells. Protein expression was first confirmed by Western blot. Mutants S39C, T42C, T56C, G60C, T62C and L70C showed a high molecular weight band consistent with dimer formation, suggesting that these mutated residues are located near the intermolecular interface. Next, the accessibility of the introduced cysteines was assessed by adding the thiol‐reactive reagent, 2‐(trimethylammonium) ethyl methanethiosulfonate (MTSET), to the cells. Block of the pore by MTSET was monitored by recording any change of transepithelial resistance (TER). Mutants S33C, T56C, S68C and T69C showed 14%, 26%, 35% and 16% increase of TER, respectively, suggesting that these residues are located near the narrow part of the pore. We conclude that we have identified 6 new residues located near the intermolecular interface and 4 new residues located near the cation pore. Supported by NIH grants R01DK062283, R01DK062283‐07S1 and U01GM094627.