Structure-Function Studies of Claudin Extracellular Domains by Cysteine-scanning Mutagenesis

Abstract

Claudins form size-and charge-selective pores in the tight junction that control the paracellular flux of inorganic ions and small molecules. However, the structural basis for ion selectivity of paracellular pores is poorly understood. Here we applied cysteine scanning to map the paracellular pathway of ion permeation across claudin-2-transfected Madin-Darby canine kidney type I cells. Four potential pore-lining amino acid residues in the first extracellular loop were mutated to cysteine and screened for their accessibility to thiol-reactive reagents. All mutants were functional except D65C, which formed dimers by intermolecular disulfide bonding, leading to a loss of charge and size selectivity. This suggests that claudin-2 pores are multimeric and that Asp 65 lies close to a protein-protein interface. Methanethiosulfonate reagents of different size and charge and the organic mercury derivate, p-(chloromercuri)benzenesulfonic acid, significantly decreased paracellular ion permeation across I66C-transfected cells by a mechanism that suggests steric blocking of the pore. The conductance of wild-type claudin-2 and the other cysteine mutants was only weakly affected. The rate of reaction with I66C decreased dramatically with increasing size of the reagent, suggesting that Ile 66 is buried deep within a narrow segment of the pore with its side group facing into the lumen. Furthermore, labeling with N-biotinoylaminoethyl methanethiosulfonate showed that I66C was weakly reactive, whereas Y35C was strongly reactive, suggesting that Tyr 35 is located at the protein surface outside of the pore.Sheets of polarized epithelia constitute barriers that separate fluid compartments of different chemical composition and mediate exchange of solutes and ions via transcellular and paracellular pathways. A large body of evidence suggests that transport via the paracellular pathway occurs through pores in the tight junctions that are formed by tetraspan membrane proteins, known as claudins (1-3).Our current understanding of paracellular pores is that they are size-and charge-selective water-filled channels that, in contrast to channels for transmembrane transport, are oriented parallel instead of perpendicular to the lipid layer of the cell membrane. Size exclusion experiments suggest a pore diameter of 6.4 -8 Å (4, 5). Furthermore, site-directed mutagenesis and overexpression of claudins in epithelial cells identified the first extracellular domain as playing an important role in the charge selectivity of paracellular transport (6 -8). The first extracellular domain of claudins contains various basic and acidic amino acids, some of which are conserved in different claudin isoforms, and these could be involved in the mechanism of ion permeation. Several studies have demonstrated homo-or heterotypic interaction of claudins, suggesting that paracellular pores are formed by oligomers of claudins (9 -11). Taken together, significant progress has been made in uncovering the nature of the paracellular pathway and mechanisms of selectivity ...

Comprehensive Cysteine-scanning Mutagenesis Reveals Claudin-2 Pore-lining Residues with Different Intrapore Locations

“…Thus, the results from the screening phase for G37C and S58C were false positives. We previously showed that MTS inhibition of claudin-2 I66C was due to partial steric block of the paracellular cation pore (14). We therefore reasoned that differences in the magnitude of inhibition of conductance of our cysteine mutants by thiol-reactive reagents of different size or charge ought to be informative about the geometrical location of each mutated residue within the pore.…”

“…In addition, we included I66C, a known pore-lining residue mutant (14), as a positive control. Inducible protein expression was verified by immunoblotting (Fig.…”

“…In claudin-2, it has been shown that these cysteines form an intramolecular disulfide bond and are inaccessible to thiolreactive reagents (13). Selective cysteine mutagenesis of four residues in claudin-2 identified Ile 66 as a pore-lining residue (14). Thus, the adjacent residues Asp 65 , Ile 66 , and Tyr 67 in ECL1 are all known to line the paracellular pore.…”

“…The goal was to provide new insights into the structure-function relationships of the claudin-2 pore, which might also be generalizable to other pore-forming claudins. (14), and Tyr 67 (10)) was changed to cysteine were synthesized (GenScript, Piscataway, NJ) and cloned into the pRevTREP vector (16). All plasmids are deposited in the PSI:Biology Materials Repository at Arizona State University.…”

Tight junctions: from simple barriers to multifunctional molecular gates

“…First evidence for channel formation came from structural and functional studies demonstrating that modification of the first extracellular loop of claudins affects the conductive properties of claudintransfected cells 85,86 . For example, the cation-selective claudin-2 contains a critical residue in its first extracellular loop that, if replaced with a cysteine followed by modification with the thiolreactive bulky reagent methanethiosulfonate was found to block the ion-conductive pathway 87,88 . …”