Molecular basis for cation selectivity in claudin-2-based paracellular pores: identification of an electrostatic interaction site.
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ABSTRACT:
SUBMITTER: Yu AS
PROVIDER: S-EPMC2606938 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The tight junction (TJ) proteins claudin-2 and -10a form paracellular cation and anion channels, respectively, and have both been reported to be expressed in the proximal tubule (PT). However, the physiological role of claudin-10a in the kidney is yet obscure. Mice deficient in claudin-10a were generated and successful knockout was confirmed by Southern Blot, Western Blot and immunofluorescence staining. The functionality of isolated PTs was investigated electrophysiologically. Compensatory regulation was studied by pharmacological intervention, RNA-Seq analysis, Western Blot and immunofluorescence staining.
2021-03-02 | GSE167928 | GEO
Project description:The tight junction protein claudin-2 is upregulated in disease. Although many studies have linked intestinal barrier loss to local and systemic disease, these have relied on macromolecular probes. In vitro analyses show, however, that these probes cannot be accommodated by size- and charge-selective claudin-2 channels. We sought to define the impact of claudin-2 channels on disease. Transgenic claudin-2 overexpression or IL-13-induced claudin-2 upregulation increased intestinal small cation permeability in vivo. IL-13 did not, however, affect permeability in claudin-2-knockout mice. Claudin-2 is therefore necessary and sufficient to effect size- and charge-selective permeability increases in vivo. In chronic disease, T cell transfer colitis severity was augmented or diminished in claudin-2-transgenic or -knockout mice, respectively. We translated the in vitro observation that casein kinase-2 (CK2) inhibition blocks claudin-2 channel function to prevent acute, IL-13-induced, claudin-2-mediated permeability increases in vivo. In chronic immune-mediated colitis, CK2 inhibition attenuated progression in claudin-2-sufficient, but not claudin-2-knockout, mice, i.e., the effect was claudin-2 dependent. Paracellular flux mediated by claudin-2 channels can therefore promote immune-mediated colitis progression. Although the mechanisms by which claudin-2 channels intensify disease remain to be defined, these data suggest that claudin-2 may be an accessible target in immune-mediated disorders, including inflammatory bowel disease.
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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.
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Project description:Claudin-2 is highly expressed in tight junctions of mouse renal proximal tubules, which possess a leaky epithelium whose unique permeability properties underlie their high rate of NaCl reabsorption. To investigate the role of claudin-2 in paracellular NaCl transport in this nephron segment, we generated knockout mice lacking claudin-2 (Cldn2(-/-)). The Cldn2(-/-) mice displayed normal appearance, activity, growth, and behavior. Light microscopy revealed no gross histological abnormalities in the Cldn2(-/-) kidney. Ultrathin section and freeze-fracture replica electron microscopy revealed that, similar to those of wild types, the proximal tubules of Cldn2(-/-) mice were characterized by poorly developed tight junctions with one or two continuous tight junction strands. In contrast, studies in isolated, perfused S2 segments of proximal tubules showed that net transepithelial reabsorption of Na(+), Cl(-), and water was significantly decreased in Cldn2(-/-) mice and that there was an increase in paracellular shunt resistance without affecting the apical or basolateral membrane resistances. Moreover, deletion of claudin-2 caused a loss of cation (Na(+)) selectivity and therefore relative anion (Cl(-)) selectivity in the proximal tubule paracellular pathway. With free access to water and food, fractional Na(+) and Cl(-) excretions in Cldn2(-/-) mice were similar to those in wild types, but both were greater in Cldn2(-/-) mice after i.v. administration of 2% NaCl. We conclude that claudin-2 constitutes leaky and cation (Na(+))-selective paracellular channels within tight junctions of mouse proximal tubules.
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Project description:Diacylglycerol pyrophosphate (DGPP) is an anionic phospholipid formed in plants, yeast, and parasites under multiple stress stimuli. It is synthesized by the phosphorylation action of phosphatidic acid (PA) kinase on phosphatidic acid, a signaling lipid with multifunctional properties. PA functions in the membrane through the interaction of its negatively charged phosphomonoester headgroup with positively charged proteins and ions. DGPP, like PA, can interact electrostatically via the electrostatic-hydrogen bond switch mechanism but differs from PA in its overall charge and shape. The formation of DGPP from PA alters the physicochemical properties as well as the structural dynamics of the membrane. This potentially impacts the molecular and ionic binding of cationic proteins and ions with the DGPP enriched membrane. However, the results of these important interactions in the stress response and in DGPP's overall intracellular function is unknown. Here, using 31P MAS NMR, we analyze the effect of the interaction of low DGPP concentrations in model membranes with the peptides KALP23 and WALP23, which are flanked by positively charged Lysine and neutral Tryptophan residues, respectively. Our results show a significant effect of KALP23 on the charge of DGPP as compared to WALP23. There was, however, no significant effect on the charge of the phosphomonoester of DGPP due to the interaction with positively charged lipids, dioleoyl trimethylammonium propane (DOTAP) and dioleoyl ethyl-phosphatidylcholine (EtPC). Divalent calcium and magnesium cations induce deprotonation of the DGPP headgroup but showed no noticeable differences on DGPP's charge. Our results lead to a novel model for DGPP-protein interaction.
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Project description:The large intestine plays a pivotal role in water and electrolyte balance. Paracellular transport may play a role in ion transport mechanisms in the cecum and large intestine; however, these molecular mechanisms and their physiological roles have not been fully studied. Claudin-15 forms a cation channel in tight junctions in the small intestine, but its role in the cecum and large intestine has not been investigated. This study aimed to explore the physiological role of claudin-15 in the cecum and large intestine using claudin-15 (Cldn15) KO mice. Electrical conductance, short-circuit current, Na+ flux, and dilution potential were assessed in isolated tissue preparations mounted in Ussing chambers. The induced short-circuit current of short-chain fatty acids, which are fermentative products in the intestinal tract, was also measured. Compared to wild type mice, the electrical conductance and paracellular Na+ flux was decreased in the cecum, but not the middle large intestine, while in both the cecum and the middle large intestine, paracellular Na+ permeability was decreased in Cldn15 KO mice. These results suggest that claudin-15 is responsible for Na+ permeability in the tight junctions of the cecum and large intestine and decreased Na+ permeability in the cecum may cause impaired absorption function.
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Project description:Cation selectivity and coupling are important attributes of cation-coupled symporters. Salmonella typhimurium melibiose permease (MelBSt) catalyzes the co-transport of galactosides with cations (H+, Li+, or Na+). 3-D crystal structures of MelBSt have revealed the molecular recognition for sugar substrates, but the cation binding and coupling mechanisms have not been defined to atomic levels. In its human homolog MFSD2A, a lethal mutation was mapped at its Na+-binding pocket; however, none of the structures in this subfamily resolved its cation binding. In this study, molecular dynamics simulations reveal the binding interactions of Na+ and Li+ with MelBSt. Interestingly, Thr121, the lethal mutation position in MFSD2A, forms stable interaction with Na+ but is at a distance from Li+. Most mutations among 11 single-site Thr121 mutants of MelBSt exhibited little effects on the galactoside binding, but largely altered the cation selectivity with severe inhibitions on Na+ binding. Few mutants (Pro and Ala) completely lost the Na+ binding and Na+-coupled transport, but their Li+ or H+ modes of activity were largely retained. It can be concluded that Thr121 is necessary for Na+ binding, but not required for the binding of H+ or Li+, so a subset of the Na+-binding pocket is enough for Li+ binding. In addition, the protein stability for some mutants can be only retained in the presence of Li+, but not by Na+ due to the lack of affinity. This finding, together with other identified thermostable mutants, supports that the charge balance of the cation-binding site plays an important role in MelBSt protein stability.
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Project description:Claudin-10a deficiency shifts proximal tubular Cl- permeability to cation selectivity via claudin-2 translocation
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Project description:Claudin protein family members, of which there are at least 27 in humans and mice, polymerize to form tight junctions (TJs) between epithelial cells, in a tissue- and developmental stage-specific manner. Claudins have a paracellular barrier function. In addition, certain claudins function as paracellular channels for small ions and/or solutes by forming selective pores at the TJs, although the specific claudins involved and their functional mechanisms are still in question. Here we show for the first time that claudin-21, which is more highly expressed in the embryonic than the postnatal stages, acts as a paracellular channel for small cations, such as Na(+), similar to the typical channel-type claudins claudin-2 and -15. Claudin-21 also allows the paracellular passage of larger solutes. Our findings suggest that claudin-21-based TJs allow the passage of small and larger solutes by both paracellular channel-based and some additional mechanisms.
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Project description:Claudins (Cldns) form a large family of protein homologs that are essential for the assembly of paracellular tight junctions (TJs), where they form channels or barriers with tissue-specific selectivity for permeants. In contrast to several family members whose physiological role has been identified, the function of claudin 4 (Cldn4) remains elusive, despite experimental evidence suggesting that it can form anion-selective TJ channels in the renal epithelium. Computational approaches have recently been employed to elucidate the molecular basis of Cldns' function, and hence could help in clarifying the role of Cldn4. In this work, we use structural modeling and all-atom molecular dynamics simulations to transfer two previously introduced structural models of Cldn-based paracellular complexes to Cldn4 to reproduce a paracellular anion channel. Free energy calculations for ionic transport through the pores allow us to establish the thermodynamic properties driving the ion-selectivity of the structures. While one model shows a cavity permeable to chloride and repulsive to cations, the other forms barrier to the passage of all the major physiological ions. Furthermore, our results confirm the charge selectivity role of the residue Lys65 in the first extracellular loop of the protein, rationalizing Cldn4 control of paracellular permeability.
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