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

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.

SUBMITTER: Yu AS

PROVIDER: S-EPMC2606938 | biostudies-literature | 2009 Jan

REPOSITORIES: biostudies-literature

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

Yu Alan S L AS Cheng Mary H MH Angelow Susanne S Günzel Dorothee D Kanzawa Sanae A SA Schneeberger Eveline E EE Fromm Michael M Coalson Rob D RD

The Journal of general physiology 20090101 1

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 selec ...[more]

PMID: 19114638

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Project description:Claudin proteins constitute the backbone of tight junctions (TJs) regulating paracellular permeability for solutes and water. The molecular mechanism of claudin polymerization and paracellular channel formation is unclear. However, a joined double-rows architecture of claudin strands has been supported by experimental and modeling data. Here, we compared two variants of this architectural model for the related but functionally distinct cation channel-forming claudin-10b and claudin-15: tetrameric-locked-barrel vs octameric-interlocked-barrels model. Homology modeling and molecular dynamics simulations of double-membrane embedded dodecamers indicate that claudin-10b and claudin-15 share the same joined double-rows architecture of TJ-strands. For both, the results indicate octameric-interlocked-barrels: Sidewise unsealed tetrameric pore scaffolds interlocked with adjacent pores via the β1β2 loop of the extracellular segment (ECS) 1. This loop mediates hydrophobic clustering and, together with ECS2, cis- and trans-interaction between claudins of the adjacent tetrameric pore scaffolds. In addition, the β1β2 loop contributes to lining of the ion conduction pathway. The charge-distribution along the pore differs between claudin-10b and claudin-15 and is suggested to be a key determinant for the cation- and water permeabilities that differ between the two claudins. In the claudin-10b simulations, similar as for claudin-15, the conserved D56 in the pore center is the main cation interaction site. In contrast to claudin-15 channels, the claudin-10b-specific D36, K64 and E153 are suggested to cause jamming of cations that prevents efficient water passage. In sum, we provide novel mechanistic information about polymerization of classic claudins, formation of embedded channels and thus regulation of paracellular transport across epithelia. Graphical Abstract ga1 Highlights • Polymerization and paracellular channel formation of claudin-10b and of claudin-15 were analyzed.• MD simulations of double-membrane-embedded claudin dodecamers were performed.• Octameric-interlocked pore barrels are indicated to constitute cation channels in tight junction strands.• Charge-distribution along pore differs between claudin-10b and claudin-15 channels.• This is suggested to be a key determinant for the cation- and water permeabilities that differ between both channels.

Project description:BackgroundThe tight junction proteins claudin-2 and claudin-10a form paracellular cation and anion channels, respectively, and are expressed in the proximal tubule. However, the physiologic role of claudin-10a in the kidney has been unclear.MethodsTo investigate the physiologic role of claudin-10a, we generated claudin-10a-deficient mice, confirmed successful knockout by Southern blot, Western blot, and immunofluorescence staining, and analyzed urine and serum of knockout and wild-type animals. We also used electrophysiologic studies to investigate the functionality of isolated proximal tubules, and studied compensatory regulation by pharmacologic intervention, RNA sequencing analysis, Western blot, immunofluorescence staining, and respirometry.ResultsMice deficient in claudin-10a were fertile and without overt phenotypes. On knockout, claudin-10a was replaced by claudin-2 in all proximal tubule segments. Electrophysiology showed conversion from paracellular anion preference to cation preference and a loss of paracellular Cl- over HCO3- preference. As a result, there was tubular retention of calcium and magnesium, higher urine pH, and mild hypermagnesemia. A comparison with other urine and serum parameters under control conditions and sequential pharmacologic transport inhibition, and unchanged fractional lithium excretion, suggested compensative measures in proximal and distal tubular segments. Changes in proximal tubular oxygen handling and differential expression of genes regulating fatty acid metabolism indicated proximal tubular adaptation. Western blot and immunofluorescence revealed alterations in distal tubular transport.ConclusionsClaudin-10a is the major paracellular anion channel in the proximal tubule and its deletion causes calcium and magnesium hyper-reabsorption by claudin-2 redistribution. Transcellular transport in proximal and distal segments and proximal tubular metabolic adaptation compensate for loss of paracellular anion permeability.

Dataset Information

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

Publications

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

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