Assembly of Tight Junction Strands: Claudin-10b and Claudin-3 Form Homo-Tetrameric Building Blocks that Polymerise in a Channel-Independent Manner.

Tight junctions regulate paracellular permeability size and charge selectively. Models have been proposed for the molecular architecture of tight junction strands and paracellular channels. However, they are not fully consistent with experimental and structural data. Here, we analysed the architecture of claudin-based tight junction strands and channels by cellular reconstitution of strands, structure-guided mutagenesis, in silico protein docking and oligomer modelling. Prototypic channel- (Cldn10b) and barrier-forming (Cldn3) claudins were analysed. Förster resonance energy transfer (FRET) assays indicated multistep claudin polymerisation, starting with cis-oligomerization specific to the claudin subtype, followed by trans-interaction-triggered cis-polymerisation. Alternative protomer interfaces were modelled in silico and tested by cysteine-mediated crosslinking, confocal- and freeze fracture EM-based analysis of strand formation. The analysed claudin mutants included also mutations causing the HELIX syndrome. The results indicated that protomers in Cldn10b and Cldn3 strands form similar antiparallel double rows, as has been suggested for Cldn15. Mutually stabilising -hydrophilic and hydrophobic - cis- and trans-interfaces were identified that contained novel key residues of extracellular segments ECS1 and ECS2. Hydrophobic clustering of the flexible ECS1 ß1ß2 loops together with ECS2-ECS2 trans-interaction is suggested to be the driving force for conjunction of tetrameric building blocks into claudin polymers. Cldn10b and Cldn3 are indicated to share this polymerisation mechanism. However, in the paracellular centre of tetramers, electrostatic repulsion may lead to formation of pores (Cldn10b) and electrostatic attraction to barriers (Cldn3). Combining in vitro data and in silico modelling, this study improves mechanistic understanding of paracellular permeability regulation by elucidating claudin assembly and its pathologic alteration as in HELIX syndrome.

Intestinal insulin/IGF1 signalling through FoxO1 regulates epithelial integrity and susceptibility to colon cancer.

Obesity promotes the development of insulin resistance and increases the incidence of colitis-associated cancer (CAC), but whether a blunted insulin action specifically in intestinal epithelial cells (IECs) affects CAC is unknown. Here, we show that obesity impairs insulin sensitivity in IECs and that mice with IEC-specific inactivation of the insulin and IGF1 receptors exhibit enhanced CAC development as a consequence of impaired restoration of gut barrier function. Blunted insulin signalling retains the transcription factor FOXO1 in the nucleus to inhibit expression of Dsc3, thereby impairing desmosome formation and epithelial integrity. Both IEC-specific nuclear FoxO1ADA expression and IEC-specific Dsc3 inactivation recapitulate the impaired intestinal integrity and increased CAC burden. Spontaneous colonic tumour formation and compromised intestinal integrity are also observed upon IEC-specific coexpression of FoxO1ADA and a stable Myc variant, thus suggesting a molecular mechanism through which impaired insulin action and nuclear FOXO1 in IECs promotes CAC.

Claudin-2-mediated cation and water transport share a common pore.

AIM: Claudin-2 is a tight junction protein typically located in 'leaky' epithelia exhibiting large paracellular permeabilities like small intestine and proximal kidney tubule. Former studies revealed that claudin-2 forms paracellular channels for small cations like sodium and potassium and also paracellular channels for water. This study analyses whether the diffusive transport of sodium and water occurs through a common pore of the claudin-2 channel. METHODS: Wild-type claudin-2 and different claudin-2 mutants were expressed in MDCK I kidney tubule cells using an inducible system. Ion and water permeability and the effect of blocking reagents on both were investigated on different clones of the mutants. RESULTS: Neutralization of a negatively charged cation interaction site in the pore with the mutation, D65N, decreased both sodium permeability and water permeability. Claudin-2 mutants (I66C and S68C) with substitution of the pore-lining amino acids with cysteine were used to test the effect of steric blocking of the claudin-2 pore by thiol-reactive reagents. Addition of thiol-reactive reagents to these mutants simultaneously decreased conductance and water permeability. Remarkably, all experimental perturbations caused parallel changes in ion conductance and water permeability, disproving different or independent passage pathways. CONCLUSION: Our results indicate that claudin-2-mediated cation and water transport are frictionally coupled and share a common pore. This pore is lined and determined in permeability by amino acid residues of the first extracellular loop of claudin-2.

TcpC protein from E. coli Nissle improves epithelial barrier function involving PKCζ and ERK1/2 signaling in HT-29/B6 cells.

The probiotic Escherichia coli Nissle 1917 (EcN) is widely used to maintain remission in ulcerative colitis. This is thought to be mediated by various immunomodulatory and barrier-stabilizing effects in the intestine. In this study, the mechanisms of barrier modulation by EcN were studied in the human epithelial HT-29/B6 cell culture model.EcN supernatant increased transepithelial resistance (TER) and reduced permeability to mannitol because of sealing of the paracellular passage pathway as revealed by two-path impedance spectroscopy. This increase in TER was attributed to the TcpC protein of EcN. TcpC induced protein kinase C-ζ (PKCζ) and extracellular-signal-regulated kinase 1/2 (ERK1/2) phosphorylation, which in turn resulted in upregulation of the barrier-forming tight junction protein claudin-14. By specific silencing of protein expression by small interfering RNA (siRNA), the sealing function of claudin-14 was confirmed. In conclusion, the TcpC protein of EcN affects innate immunity by improving intestinal barrier function through upregulation of claudin-14 via PKCζ and ERK1/2 signaling.

Claudins of intestine and nephron - a correlation of molecular tight junction structure and barrier function.

A prerequisite of epithelial transport is a paracellular barrier function, which seals the tissue against an uncontrolled leak flux. Moreover, selective paracellular permeability has been shown to be crucial for physiological epithelial transport function. Claudins are tetraspan tight junction proteins which play a major role in paracellular ion permeability across epithelia. The multigene family consists of 24 members and several splice variants which show distinct tissue-specific expression profiles. Moreover, in diseases associated with a loss of barrier function such as forms of inflammatory bowel disease, the expression of claudins is altered. Functional characterization of single claudins revealed specific contribution to barrier properties in epithelia. This review gives an overview on the exploration of molecular structure and barrier function along the intestine and nephron, which not only share mechanisms of selective restriction of the paracellular pathway but also exhibit distinct organ-specific characteristics.

Changes in expression and distribution of claudin 2, 5 and 8 lead to discontinuous tight junctions and barrier dysfunction in active Crohn's disease.

BACKGROUND: Epithelial barrier function is impaired in Crohn's disease. AIM: To define the underlying cellular mechanisms with special attention to tight junctions. METHODS: Biopsy specimens from the sigmoid colon of patients with mild to moderately active or inactive Crohn's disease were studied in Ussing chambers, and barrier function was determined by impedance analysis and conductance scanning. Tight junction structure was analysed by freeze fracture electron microscopy, and tight junction proteins were investigated immunohistochemically by confocal laser scanning microscopy and quantified in immunoblots. Epithelial apoptosis was analysed in terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labelling and 4',6-diamidino-2-phenylindole staining. RESULTS: Patients with active Crohn's disease showed an impaired intestinal barrier function as indicated by a distinct reduction in epithelial resistance. As distribution of conductivity was even, focal epithelial lesions (eg, microerosions) did not contribute to barrier dysfunction. Instead, freeze fracture electron microscopy analysis showed reduced and discontinuous tight junction strands. Occludin and the sealing tight junction proteins claudin 5 and claudin 8 were downregulated and redistributed off the tight junction, whereas the pore-forming tight junctions protein claudin 2 was strongly upregulated, which constitute the molecular basis of tight junction changes. Other claudins were unchanged (claudins 1, 4 and 7) or not detectable in sigmoid colon (claudins 11, 12, 14, 15 and 16). Claudin 2 upregulation was less pronounced in active Crohn's disease compared with active ulcerative colitis and was inducible by tumour necrosis factor alpha. As a second source of impaired barrier function, epithelial apoptosis was distinctly increased in active Crohn's disease (mean (SD) 5.2 (0.5)% v 1.9 (0.2)% in control). By contrast, barrier function, tight junction proteins and apoptosis were unaffected in Crohn's disease in remission. CONCLUSION: Upregulation of pore-forming claudin 2 and downregulation and redistribution of sealing claudins 5 and 8 lead to altered tight junction structure and pronounced barrier dysfunction already in mild to moderately active Crohn's disease.

Restitution of single-cell defects in the mouse colon epithelium differs from that of cultured cells.

Integrity of colon epithelium is of crucial importance and, as small defects occur constantly, rapid repair (restitution) is essential. To investigate the mechanism of restitution, single-cell lesions were induced in mouse colonic surface epithelia by iontophoretic injection of Ca2+. Closure of the resulting defects was monitored using confocal laser scanning microscopy (CLSM), and functional sealing by electrophysiological techniques. Restitution was evaluated as the time constant tau of the exponential decrease in conductance of an induced leak and amounted to 0.28 min under control conditions. After 4 min, the leak was completely sealed. Repair was thus considerably faster than in previously investigated HT-29/B6 cells (tau=5.73 min). As in cultured cells, cytochalasin D delayed restitution in native colon epithelia (tau=0.69 min), indicating the involvement of actin in the healing process; however, no accumulation of actin surrounding the lesion was detected. Long-term incubation of epithelia with IFN-gamma alone or in combination with TNF-alpha increased tau to 0.49 and 0.59 min, respectively. In contrast to cultured cells, TNF-alpha alone did not affect restitution. A brief (<10 min) exposure to the sterile filtered supernatant of hemolytic E. coli O4 cultures did not affect the morphology of the epithelium, but delayed restitution. In CLSM studies, defects were still clearly visible 4 min after the onset of lesion induction. The supernatant of a nonhemolytic E. coli O4 mutant did not exhibit this effect. In conclusion, single-cell defects in native colon cause functional leaks that seal faster than in cell cultures. Proinflammatory cytokines and pathogenic bacteria delay restitution. This suggests a key role of very small lesions at the onset of pathogenic processes in the intestine.

Calcium-magnesium interactions in pancreatic acinar cells.

Although the role of calcium (Ca2+) in the signal transduction and pathobiology of the exocrine pancreas is firmly established, the role of magnesium (Mg2+) remains unclear. We have characterized the intracellular distribution of Mg2+ in response to hormone stimulation in isolated mouse pancreatic acinar cells and studied the role of Mg2+ in modulating Ca2+ signaling using microspectrofluorometry and digital imaging of Ca2+- or Mg2+-sensitive fluorescent dyes as well as Mg2+-sensitive intracellular microelectrodes. Our results indicate that an increase in intracellular Mg2+ concentrations reduced the cholecystokinin (CCK) -induced Ca2+ oscillations by inhibiting the capacitive Ca2+ influx. An intracellular Ca2+ mobilization, on the other hand, was paralleled by a decrease in [Mg2+]i, which was reversible upon hormone withdrawal independent of the electrochemical gradients for Mg2+, Ca2+, Na+, and K+, and not caused by Mg2+ efflux from acinar cells. In an attempt to characterize possible Mg2+ stores that would explain the reversible, hormone-induced intracellular Mg2+ movements, we ruled out mitochondria or ATP as potential Mg2+ buffers and found that the CCK-induced [Mg2+]i decrease was initiated at the basolateral part of the acinar cells, where most of the endoplasmic reticulum (ER) is located, and progressed from there toward the apical pole of the acinar cells in an antiparallel fashion to Ca2+ waves. These experiments represent the first characterization of intracellular Mg2+ movements in the exocrine pancreas, provide evidence for possible Mg2+ stores in the ER, and indicate that the spatial and temporal distribution of intracellular Mg concentrations profoundly affects acinar cell Ca2+ signaling.

Apparent intracellular Mg2+ buffering in neurons of the leech Hirudo medicinalis.

The apparent intracellular Mg2+ buffering, or muffling (sum of processes that damp changes in the free intracellular Mg2+ concentration, [Mg2+](i), e.g., buffering, extrusion, and sequestration), was investigated in Retzius neurons of the leech Hirudo medicinalis by iontophoretic injection of H+, OH-, or Mg2+. Simultaneously, changes in intracellular pH and the intracellular Mg2+, Na+, or K+ concentration were recorded with triple-barreled ion-selective microelectrodes. Cell volume changes were monitored measuring the tetramethylammonium (TMA) concentration in TMA-loaded neurons. Control measurements were carried out in electrolyte droplets (diameter 100-200 microm) placed on a silver wire under paraffin oil. Droplets with or without ATP, the presumed major intracellular Mg2+ buffer, were used to quantify the pH dependence of Mg2+ buffering and to determine the transport index of Mg2+ during iontophoretic injection. The observed pH dependence of [Mg2+](i) corresponded to what would be expected from Mg2+ buffering through ATP. The quantity of Mg2+ muffling, however, was considerably larger than what would be expected if ATP were the sole Mg2+ buffer. From the decrease in Mg2+ muffling in the nominal absence of extracellular Na+ it was estimated that almost 50% of the ATP-independent muffling is due to the action of Na+/Mg2+ antiport.

Intracellular pH of crab and crayfish muscle fibre types during GABA application and inhibitory nerve stimulation.

The inhibitory motoneurons of crustaceans form synapses both with the sarcolemma of muscle fibres and with the very distal branchings of the excitatory motoneurons. The transmitter of these synapses is GABA (gamma-aminobutyric acid) which is known to open Cl- channels. Studies on the dactyl opener muscle of crayfish suggest that application of GABA not only leads to an increase in the Cl- permeability but also to a considerable HCO3- conductance that causes an intracellular acidification. To investigate possible physiological implications, we measured the intracellular pH of various muscle fibre types of crayfish and crab using pH-sensitive microelectrodes. Independent of the presence or absence of inhibitory innervation, bath application of 10(-5) mol l(-1) GABA led to acidification in all fibre types (pH change: 0.14 +/- 0.08, n = 11). In no preparation was a change in intracellular pH observed upon stimulation of specific or common inhibitory motoneurons with 1040 pulses s(-1) for 2-5 min. The results suggest that HCO3- conductance cannot be activated through synaptic GABA receptors. However, all crustacean muscle fibre types seem to possess extrasynaptic GABA-sensitive channels that exhibit a considerable HCO3- conductance. The physiological importance of these channels remains to be elucidated.

Mechanisms of Mg2+ influx, efflux and intracellular 'muffling' in leech neurones and glial cells.

Mg2+ is known to influence conductance and gating properties of a multitude of ion channels and is thus able to modulate synaptic transmission. Therefore, a tight regulation of the intracellular free Mg2+ concentration ([Mg2+]i) in neurones and glial cells is crucial for maintaining the functions of central nervous systems. [Mg2+]i is regulated through the balance of Mg2+ influx and Mg2+ efflux, together with heavy damping of [Mg2+]i changes through intracellular buffering and sequestration. To investigate the mechanisms involved in [Mg2+]i regulation, neurones and glial cells from the central nervous system of the leech Hirudo medicinalis proved to be an ideal model system. The present article summarizes the evidence for a Mg2+ influx pathway which is distinct from that for Ca2+, for a dual regulation of Mg2+ efflux (a 1 Na+/1 Mg2+ antiport and a Na(+)-independent Mg2+ efflux mechanism), for pH-dependent Mg2+ buffering through ATP and other intracellular Mg2+ binding components and for the involvement of mitochondria in intracellular Mg2+ sequestration.

Na+-dependent regulation of the free Mg2+ concentration in neuropile glial cells and P neurones of the leech Hirudo medicinalis.

To investigate the Mg2+ regulation in neuropile glial (NG) cells and pressure (P) neurones of the leech Hirudo medicinalis the intracellular free Mg2+ ([Mg2+]i) and Na+ ([Na+]i) concentrations, as well as the membrane potential (Em), were measured using Mg2+- and Na+-selective microelectrodes. The mean steady-state values of [Mg2+]i were found to be 0.91 mM (mean Em=-63.6 mV) in NG cells and 0.20 mM (mean Em=-40.6 mV) in P neurones with a [Na+]i of 6.92 mM (mean Em=-61.6 mV) and 7.76 mM (mean Em=-38.5 mV), respectively. When the extracellular Mg2+ concentration ([Mg2+]o) was elevated, [Mg2+]i in P neurones increased within 5-20 min whereas in NG cells a [Mg2+]i increase occurred only after long-term exposure (6 h). After [Mg2+]o was reduced back to 1 mM, a reduction of the extracellular Na+ concentration ([Na+]o) decreased the inwardly directed Na+ gradient and reduced the rate of Mg2+ extrusion considerably in both NG cells and P neurones. In P neurones Mg2+ extrusion was reduced to 15.4% in Na+-free solutions and to 6.0% in the presence of 2 mM amiloride. Mg2+ extrusion from NG cells was reduced to 6.2% in Na+-free solutions. The results suggest that the major [Mg2+]i-regulating mechanism in both cell types is Na+/ Mg2+ antiport. In P neurones a second, Na+-independent Mg2+ extrusion system may exist.

Intracellular alkalinization causes Mg2+ release from intracellular binding sites in leech Retzius neurones.

Four-barrelled ion-sensitive microelectrodes were developed to enable simultaneous measurement of intracellular free Mg2+ and Na+ concentrations ([Mg2+]i, [Na+]i), intracellular pH and the membrane potential. The electrodes were used to investigate pH-induced [Mg2+]i changes in Retzius neurones of the leech Hirudo medicinalis. The application of propionate or CO2/HCO3--buffered bath solutions caused a transient intracellular acidification, an initial [Mg2+]i decrease and a continuous [Na+]i increase. In the presence of CO2/HCO3- this [Na+]i increase was more pronounced and might be the reason for the slow increase in [Mg2+]i following the initial decrease. The withdrawal of propionate or CO2/HCO3--buffered bath solutions caused a transient alkalinization which was accompanied by a slight but significant [Mg2+]i increase, even in the nominal absence of extracellular Mg2+, while [Na+]i returned to its original value. The alkalinization-induced [Mg2+]i increase could be reduced to about 50% by the application of 1-10 microM cyclosporin A, an inhibitor of the mitochondrial permeability transition pore (MTP). Phenylarsine oxide, an MTP activator, caused a [Mg2+]i increase with characteristics similar to those of the alkalinization-induced increase, which could not be attributed to any changes in [Na+]i or pHi. It is concluded that an intracellular alkalinization might induce the release of Mg2+ from intracellular stores.

Sodium-magnesium antiport in Retzius neurones of the leech Hirudo medicinalis.

1. Intracellular free magnesium ([Mg2+]i) and sodium ([Na+]i) concentrations were measured in Retzius neurones of the leech Hirudo medicinalis using ion-sensitive microelectrodes. 2. The mean steady-state values for [Mg2+]i and [Na+]i were 0.46 mM (pMg, 3.34 +/- 0.23; range, 0.1-1.2 mM; n = 32) and 8.95 mM (pNa, 2.05 +/- 0.15; range, 5.1-15.5 mM, n = 21), respectively, at a mean membrane potential (Em) of -35.6 +/- 6.1 mV (n = 32). Thus, [Mg2+]i is far below the value calculated for a passive distribution (16.9 mM) but close to the equilibrium value calculated for a hypothetical 1 Na(+)-1 Mg2+ antiport (0.41 mM). 3. Simultaneous measurements of [Mg2+]i, [Na+]i and Em in Retzius neurones showed that an increase in the extracellular Mg2+ concentration ([Mg2+]o) resulted in an increase in [Mg2+]i, a parallel decrease in [Na+]i and a membrane depolarization, while a decrease in [Mg2+]o had opposite effects. These results are compatible with calculations based on a 1 Na(+)-1 Mg2+ antiport. 4. Na+ efflux at high [Mg2+]o still occurred when the Na(+)-K+ pump was inhibited by the application of ouabain or in K(+)-free solutions. This efflux was blocked by amiloride. 5. In the absence of extracellular Na+ ([Na+]o), no Mg2+ influx occurred. Mg2+ influx at high [Mg2+]o was even lower than in the presence of [Na+]o. Mg2+ efflux was blocked in the absence of [Na+]o. 6. The rate of Mg2+ extrusion was reduced by lowering [Na+]o, even if the Na+ gradient across the membrane remained almost unchanged. 7. Mg2+ efflux was blocked by amiloride (half-maximal effect at 0.25 mM amiloride; Hill coefficient, 1.3) but not by 5-(N-ethyl-N-isopropyl)-amiloride (EIPA). 8. No changes in intracellular Ca2+ and pH (pHi) could be detected when [Mg2+]o was varied between 1 and 30 mM. 9. Changing pHi by up to 0.4 pH units had no effect on [Mg2+]i. 10. The results suggest the presence of an electrogenic 1 Na(+)-1 Mg2+ antiport in leech Retzius neurones. This antiport can be reversed and is inhibited by low extracellular and/or intracellular Na+ and by amiloride.

Non-uniformity of sarcomere lengths can explain the 'catch-like' effect of arthropod muscle.

The 'catch-like' effect, a hysteresis phenomenon in arthropod skeletal muscle contraction thought to be related to the catch of molluscan smooth muscle, was investigated in the closer muscle of the crab Eriphia spinifrons. Several parameters were varied to determine their influence on the catch-like effect. These parameters were (1) the frequency of repetitive stimulation of the slow excitatory neuron, (2) additional stimulation of the inhibitory neuron, (3) the amount of stretch applied to the muscle and (4) the stiffness of the mechano-electrical transducer. The results show that the catch-like effect is not related to the catch of molluscan smooth muscle but rather to the 'residual force enhancement' or 'creep' phenomenon described for vertebrate muscle. A hypothesis for residual force enhancement implies that the increase in force is caused by non-uniformity of sarcomere lengths along the muscle fibre. Based on this hypothesis and the actual force-length relationship of the crab muscle studied, calculations were carried out to determine, if the observed catch-like effect can be explained by such a model. The calculations corroborate the experimental evidence. The catch-like effect of arthropod muscles can thus be explained by the same mechanism responsible for residual force enhancement and creep in vertebrate muscle. A physiological relevance of the catch-like effect in arthropod muscle is inferred.

Intracellular free Mg2+ concentration in skeletal muscle fibres of frog and crayfish.

The free intracellular Mg2+ concentration ([Mg2+]i) was investigated in frog sartorius and crayfish phasic and tonic skeletal muscle fibres, using a new Mg2(+)-sensitive microelectrode based on the ionophore ETH 5214 [Hu et al. (1989) Anal Chem 61:574-576]. In Ringer solution containing 0.5 mmol/l MgCl2, the mean [Mg2+]i of the frog muscle fibres was 1.3 mmol/l. In phasic crayfish muscle fibres, [Mg2+]i was about twice as high (mean 3.5 mmol/l) as in tonic fibres (mean 1.5 mmol/l), measured in van Harreveld solution containing 1.2 mmol/l MgCl2. Long-lasting (3-12 h) incubation of frog skeletal muscle fibres in Na(+)-free solution produced a reversible increase of [Mg2+]i by a factor of about 1.7. A tenfold rise of extracellular Mg2+ led to an increase in [Mg2+]i in the presence as well as in the absence of Na+. In these experiments, mean [Mg2+]i values of 3.2 mmol/l were never exceeded. Thus, [Mg2+]i remained at least 60 times lower than predicted from a passive distribution across the cell membrane. The results suggest the existence of a Na(+)-dependent and a Na(+)-independent Mg2+ extrusion mechanism, which is regulated by actual Mg2+ concentrations.