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Molecular basis for cation selectivity in claudin-2-based paracellular pores: identification of an electrostatic interaction site.

Yu AS, Cheng MH, Angelow S, Günzel D, Kanzawa SA, Schneeberger EE, Fromm M, Coalson RD - J. Gen. Physiol. (2009)

Bottom Line: The degree of selectivity and the molecular mechanism of ion permeation through claudin pores are poorly understood.Claudin-2 pores were found to be narrow, fluid filled, and cation selective.Thus, paracellular pores use intrapore electrostatic binding sites to achieve a high conductance with a high degree of charge selectivity.

View Article: PubMed Central - PubMed

Affiliation: Division of Nephrology, Department of Medicine,University of Southern California Keck School of Medicine, Los Angeles, CA 90089, USA. alanyu@usc.edu

ABSTRACT
Paracellular ion transport in epithelia is mediated by pores formed by members of the claudin family. The degree of selectivity and the molecular mechanism of ion permeation through claudin pores are poorly understood. By expressing a high-conductance claudin isoform, claudin-2, in high-resistance Madin-Darby canine kidney cells under the control of an inducible promoter, we were able to quantitate claudin pore permeability. Claudin-2 pores were found to be narrow, fluid filled, and cation selective. Charge selectivity was mediated by the electrostatic interaction of partially dehydrated permeating cations with a negatively charged site within the pore that is formed by the side chain carboxyl group of aspartate-65. Thus, paracellular pores use intrapore electrostatic binding sites to achieve a high conductance with a high degree of charge selectivity.

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Characterization of MDCK I TetOff cell lines expressing charge-neutralizing mutants of claudin-2. (A) Immunoblot of claudin-2 expression in clones stably transfected with WT claudin-2 (WT), the indicated individual claudin-2 mutants, and a TM (TM = E53Q, D65N, and D76N) grown in the presence (+) or absence (−) of doxycycline (Dox), using antibodies to the indicated claudin isoforms. (B) Confocal images showing immunofluorescence localization of mutant claudin-2 protein in Dox− cells. (C) Effect of claudin-2 induction on expression of other tight junction membrane proteins. Cell lysates were immunoblotted with antibodies against the indicated claudin isoforms or occludin. (A and C) White lines indicate that intervening lanes have been spliced out.
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fig8: Characterization of MDCK I TetOff cell lines expressing charge-neutralizing mutants of claudin-2. (A) Immunoblot of claudin-2 expression in clones stably transfected with WT claudin-2 (WT), the indicated individual claudin-2 mutants, and a TM (TM = E53Q, D65N, and D76N) grown in the presence (+) or absence (−) of doxycycline (Dox), using antibodies to the indicated claudin isoforms. (B) Confocal images showing immunofluorescence localization of mutant claudin-2 protein in Dox− cells. (C) Effect of claudin-2 induction on expression of other tight junction membrane proteins. Cell lysates were immunoblotted with antibodies against the indicated claudin isoforms or occludin. (A and C) White lines indicate that intervening lanes have been spliced out.

Mentions: Anderson, van Itallie, and colleagues have used charge-reversing mutations that alter paracellular charge selectivity to show convincingly that the first extracellular domain of claudins lines the paracellular pore (Colegio et al., 2002, 2003; Van Itallie et al., 2003). However, it is unclear whether these mutants also inform on the location of the ion selectivity filter or merely reflect artificially created electrostatic fields within the pore that affect ion permeation. Indeed, the fact that mutations of most charged residues in the first extracellular domain have a similar effect would suggest the latter. There are three acidic residues in the first extracellular loop of claudin-2: E53, D65, and D76. To test the hypothesis that one or more of these might be responsible for the negatively charged cation-interaction site in the claudin-2 pore, we mutated each of these to its neutral, polar counterpart (E53Q, D65N, and D76N), generated a triple mutant (TM) bearing all three mutations, and generated stable, inducible MDCK I TetOff cell lines expressing each mutant. By neutralizing instead of reversing the normal charge at each site, we expect to pinpoint only those residues that are part of the normal selectivity mechanism. Clones were selected that expressed similar levels of claudin-2 protein to WT and showed good induction and suppression. Mutant cell lines exhibited similar levels of expression and subcellular localization of claudin-2 protein to the WT claudin-2 clone, and similarly had little effect on expression of endogenous claudins (Fig. 8).


Molecular basis for cation selectivity in claudin-2-based paracellular pores: identification of an electrostatic interaction site.

Yu AS, Cheng MH, Angelow S, Günzel D, Kanzawa SA, Schneeberger EE, Fromm M, Coalson RD - J. Gen. Physiol. (2009)

Characterization of MDCK I TetOff cell lines expressing charge-neutralizing mutants of claudin-2. (A) Immunoblot of claudin-2 expression in clones stably transfected with WT claudin-2 (WT), the indicated individual claudin-2 mutants, and a TM (TM = E53Q, D65N, and D76N) grown in the presence (+) or absence (−) of doxycycline (Dox), using antibodies to the indicated claudin isoforms. (B) Confocal images showing immunofluorescence localization of mutant claudin-2 protein in Dox− cells. (C) Effect of claudin-2 induction on expression of other tight junction membrane proteins. Cell lysates were immunoblotted with antibodies against the indicated claudin isoforms or occludin. (A and C) White lines indicate that intervening lanes have been spliced out.
© Copyright Policy
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2606938&req=5

fig8: Characterization of MDCK I TetOff cell lines expressing charge-neutralizing mutants of claudin-2. (A) Immunoblot of claudin-2 expression in clones stably transfected with WT claudin-2 (WT), the indicated individual claudin-2 mutants, and a TM (TM = E53Q, D65N, and D76N) grown in the presence (+) or absence (−) of doxycycline (Dox), using antibodies to the indicated claudin isoforms. (B) Confocal images showing immunofluorescence localization of mutant claudin-2 protein in Dox− cells. (C) Effect of claudin-2 induction on expression of other tight junction membrane proteins. Cell lysates were immunoblotted with antibodies against the indicated claudin isoforms or occludin. (A and C) White lines indicate that intervening lanes have been spliced out.
Mentions: Anderson, van Itallie, and colleagues have used charge-reversing mutations that alter paracellular charge selectivity to show convincingly that the first extracellular domain of claudins lines the paracellular pore (Colegio et al., 2002, 2003; Van Itallie et al., 2003). However, it is unclear whether these mutants also inform on the location of the ion selectivity filter or merely reflect artificially created electrostatic fields within the pore that affect ion permeation. Indeed, the fact that mutations of most charged residues in the first extracellular domain have a similar effect would suggest the latter. There are three acidic residues in the first extracellular loop of claudin-2: E53, D65, and D76. To test the hypothesis that one or more of these might be responsible for the negatively charged cation-interaction site in the claudin-2 pore, we mutated each of these to its neutral, polar counterpart (E53Q, D65N, and D76N), generated a triple mutant (TM) bearing all three mutations, and generated stable, inducible MDCK I TetOff cell lines expressing each mutant. By neutralizing instead of reversing the normal charge at each site, we expect to pinpoint only those residues that are part of the normal selectivity mechanism. Clones were selected that expressed similar levels of claudin-2 protein to WT and showed good induction and suppression. Mutant cell lines exhibited similar levels of expression and subcellular localization of claudin-2 protein to the WT claudin-2 clone, and similarly had little effect on expression of endogenous claudins (Fig. 8).

Bottom Line: The degree of selectivity and the molecular mechanism of ion permeation through claudin pores are poorly understood.Claudin-2 pores were found to be narrow, fluid filled, and cation selective.Thus, paracellular pores use intrapore electrostatic binding sites to achieve a high conductance with a high degree of charge selectivity.

View Article: PubMed Central - PubMed

Affiliation: Division of Nephrology, Department of Medicine,University of Southern California Keck School of Medicine, Los Angeles, CA 90089, USA. alanyu@usc.edu

ABSTRACT
Paracellular ion transport in epithelia is mediated by pores formed by members of the claudin family. The degree of selectivity and the molecular mechanism of ion permeation through claudin pores are poorly understood. By expressing a high-conductance claudin isoform, claudin-2, in high-resistance Madin-Darby canine kidney cells under the control of an inducible promoter, we were able to quantitate claudin pore permeability. Claudin-2 pores were found to be narrow, fluid filled, and cation selective. Charge selectivity was mediated by the electrostatic interaction of partially dehydrated permeating cations with a negatively charged site within the pore that is formed by the side chain carboxyl group of aspartate-65. Thus, paracellular pores use intrapore electrostatic binding sites to achieve a high conductance with a high degree of charge selectivity.

Show MeSH
Related in: MedlinePlus