Tight Junction Conductance Research Articles

Claudins and mineral metabolism.

The tight junction conductance made of the claudin-based paracellular channel is important in the regulation of calcium and magnesium reabsorption in the kidney. This review describes recent findings of the structure, the function, and the physiologic regulation of claudin-14, claudin-16, and claudin-19 channels that through protein interactions confer calcium and magnesium permeability to the tight junction. Mutations in two tight junction genes - claudin-16 and claudin-19 - cause the inherited renal disorder familial hypomagnesemia with hypercalciuria and nephrocalcinosis. A recent genome-wide association study has identified claudin-14 as a major risk gene of hypercalciuric nephrolithiasis. The crystal structure of claudin-19 has recently been resolved allowing the reconstruction of a claudin assembly model from cis-dimers made of claudin-16 and claudin-19 interaction. MicroRNAs have been identified as novel regulators of the claudin-14 gene. The microRNA-claudin-14 operon is directly regulated by the Ca sensing receptor gene in response to hypercalcemia. The paracellular pathway in the kidney is particularly important for mineral metabolism. Three claudin proteins - claudin-14, claudin-16, and claudin-19 - contribute to the structure and function of this paracellular pathway. Genetic mutations and gene expression changes in these claudins may lead to alteration of the paracellular permeability to calcium and magnesium, ultimately affecting renal mineral metabolism.

A proposed route to independent measurements of tight junction conductance at discrete cell junctions

Direct recording of tight junction permeability is of pivotal importance to many biologic fields. Previous approaches bear an intrinsic disadvantage due to the difficulty of separating tight junction conductance from nearby membrane conductance. Here, we propose the design of Double whole-cell Voltage Clamp - Ion Conductance Microscopy (DVC-ICM) based on previously demonstrated potentiometric scanning of local conductive pathways. As proposed, DVC-ICM utilizes two coordinated whole-cell patch-clamps to neutralize the apical membrane current during potentiometric scanning, which in models described here will profoundly enhance the specificity of tight junction recording. Several potential pitfalls are considered, evaluated and addressed with alternative countermeasures.

Dynamic properties of the tight junction barrier

A principal role of tight junctions is to seal the apical intercellular space and limit paracellular flux of ions and molecules. Despite the fact that tight junctions form heavily cross-linked structures, functional studies have fostered the hypothesis that the tight junction barrier is dynamic and defined by opening and closing events. However, it has been impossible to directly measure tight junction barrier function with sufficient resolution to detect such events. Nevertheless, recent electrophysiological and sieving studies have provided tremendous insight into the presence of at least two pathways of trans-tight junction flux: a high-capacity ion-selective "pore" pathway and a low-capacity "leak" pathway that allows the passage of macromolecules. Furthermore, it is now known that the tight junction molecular structure is highly dynamic and that dynamics are correlated with barrier function. Taken together, these data support a dynamic model of tight junction conductance and suggest that regulation of tight junction openings and closings may provide sensitive means of barrier regulation.

Identification of discrete single tight junction opening/closing events with ion channel‐like properties

Tight junctions establish the barrier to paracellular flux of ions and molecules. Recent quantitative studies of fluorescent‐tagged protein dynamics have shown that tight junction structure undergoes rapid and continuous molecular remodeling. While functional analyses have characterized charge‐ and size‐selectivity of the tight junction barrier, studies of submicron regions have been impossible due to the reliance of all available measures of barrier properties on population averages. To overcome this limitation, we studied submicron tight junction regions using high resolution patch clamp techniques. The data reveal stochastic, transient conductance increases (duration 1.4±0.2 ms, size 4.2±0.2 pS, frequency 13.4±0.4 s−1). Consistent with known selectivity of claudin‐2‐dependent pores, events persisted after replacement of Na+ with methylamine. Further, La3+ (a known inhibitor of claudin‐ 2 conductance) or stable claudin‐2 knockdown, but not transmembrane ion channel inhibitors, limited transient tight junction conductance increases. We conclude that the currents recorded correspond to trans‐tight junction channel openings and that tight junction channels have properties that resemble traditional ion channels. These single‐channel measurements will make biophysical definition of tight junction structure:function relationships possible with molecular resolution.

Comparative Aspects of Actions of a Short-Chain Phospholipid on Epithelial Na + Channels and Tight Junction Conductance

Ion transport in both the frog skin (a high-resistance epithelium) and the rabbit nasal airway epithelium (a low-resistance epithelium) are dominated by electrogenic Na + absorption via apical membrane amiloride-sensitive Na + channels, and short-circuit current (I SC) is essentially a measure of Na + absorption in both epithelia. In both epithelia, mucosal application of the short-chain phospholipid didecanoyl-L- α-phosphatidylcholine (DDPC) dose-dependently inhibited the amiloride-sensitive I SC and caused an initial decrease in epithelial conductance (G t) followed by an increase in G t to steady-state values above control level. The effects were reversible. It is concluded that DDPC (a) inhibits epithelial amiloride-sensitive Na + channels and (b) induces an increase in paracellular tight junction conductance. These effects may involve changes in non-specific lipid-protein interactions at the cell membrane level.

Histone-induced damage of a mammalian epithelium: the conductive effect

Human semen has been reported to be cytotoxic to rat descending colon by a mechanism involving polyamines (cationic molecules) and collagenase. In this study, we report that histones, cationic proteins found in human semen, can contribute to semen's cytotoxicity. Histones H1, H4, and H5, when added to the mucosal side of rabbit urinary bladder epithelium, were found to alter the transepithelial conductance (Gt) in a voltage-sensitive manner. When the cell interior was negative, the conductance rapidly increased and plateaued. When the cell interior was positive, the induced conductance decreased to control values. Histone increased the Gt by increasing the apical membrane conductance rather than the tight junction conductance. The magnitude of the Gt increase was dose dependent, and the histone-induced conductance was nonselective for Na+, K+, and Cl-. The induced conductance could be reversed by either increasing mucosal Ca2+ concentration or by removal of histone from the mucosal solution. Prolonged exposure of the epithelium to histone was toxic as determined by the irreversible loss of transepithelial resistance. These results indicate that histone increases membrane ionic permeability, is cytotoxic, and thus may contribute to human semen's toxic effect on colonic epithelium.

Short-term effects of uninephrectomy on electrical properties of the cortical collecting duct from rabbit remnant kidneys.

Microelectrode techniques were used to assess the electrical properties of the collecting duct cell in the isolated perfused cortical collecting duct from remnant kidneys 3, 6, and 24 h after uninephrectomy (UNX); results were compared with those from sham-operated kidneys. Plasma aldosterone levels did not change during the time course after UNX. The lumen-negative transepithelial voltage was elevated significantly 3 h after UNX, and was increased further 24 h after UNX. The basolateral membrane voltage (VB) was elevated 6 h after UNX, and then was increased further at 24 h. Although the tight junction conductance and the fractional apical membrane resistance (fRA) were not altered at any time points after UNX, the apical membrane conductance as well as the transepithelial (GT) and basolateral membrane conductances increased 6 and 24 h after UNX. The changes in apical membrane voltage, GT, and fRA upon addition of luminal amiloride increased just 3 h after UNX, and then remained elevated at 6 and 24 h. The changes in apical membrane voltage and GT upon addition of luminal Ba2+, the changes in VB upon addition of bath ouabain, and the changes in VB, GT, and fRA upon raising bath K+ were not influenced 3 h after UNX, but increased at 6 and 24 h. At these latter periods after UNX, the transference number of Cl- of the basolateral membrane decreased significantly, whereas the transference number of K+ of the basolateral membrane increased significantly. Simultaneously, addition of Ba2+ to the bath caused the VB to hyperpolarize in parallel with decreases in GT and fRA. We conclude: (a) the initial effect of UNX (3 h) in the collecting duct cell is an increase in apical membrane Na+ conductance; (b) the delayed effects of UNX (6 and 24 h) are increases in apical membrane K+ conductance as well as basolateral membrane Na(+)-K+ pump activity and K+ conductance; (c) the hyperpolarization of VB at 6 and 24 h after UNX may result in the decrease of the ratio of the relative Cl- conductance to the relative K+ conductance of the basolateral membrane and also may increase passive K+ entry into the cell across the basolateral membrane; (d) these time-dependent electrical changes occur independently of plasma aldosterone levels.

Effects of uninephrectomy on electrical properties of the cortical collecting duct from rabbit remnant kidneys.

Microelectrode techniques were used to determine the Na+ and K+ transport properties of the collecting duct cell in the isolated cortical collecting duct (CCD) from rabbits 14 d after uninephrectomy (UNX); results were compared with those from sham-operated rabbits (control). UNX had no effects on plasma aldosterone levels. The CCDs from UNX rabbits exhibited structural hypertrophy. The lumen negative transepithelial voltage and the basolateral membrane voltage (VB) were elevated in the UNX group. Although the transepithelial conductance (GT) and the fractional apical membrane resistance (fRA) were not different between the two groups, the conductances of the apical and the basolateral membranes were increased, and the tight junction conductance was decreased in the UNX group. The amiloride-sensitive changes in apical membrane voltage (VA), fRA, and GT were greater in the UNX group. The changes in VA upon raising the perfusate K+ concentration and the changes in VA and GT upon addition of Ba2+ to the perfusate were elevated in the UNX group. Upon raising K+ in the bath, a large depolarization of VB was observed in the UNX group. Lowering the bath Cl- resulted in a small depolarization of VB in the UNX group. Addition of Ba2+ to the bath in the UNX group caused the VB to hyperpolarize in parallel with decreases in GT and fRA whereas in the control group it had no effect on VB. Addition of ouabain to the bath resulted in a large depolarization of VB in the UNX group. We conclude that (a) UNX stimulates conductances of Na+ and K+ in the apical membrane, active Na(+)-K+ pump activity, and K+ conductance in the basolateral membrane, independently of plasma aldosterone; (b) The basolateral membrane in the tubules of UNX rabbits is more selective to K+; and (c) the hyperpolarization of VB upon UNX may increase passive K+ entry into the cell across the basolateral membrane.

Cellular variability in the development of tight junctions after activation of protein kinase C.

Phorbol 12-myristate 13-acetate (PMA) decreases the tight junction conductance (TJC) during the reorganization of LLC-PK1A monolayers, but has the opposite effect in LLC-PK1B4, MDCK, and MDCK4 cells. Because no protein synthesis was required for the effects of PMA on the TJC of LLC-PK1A monolayers, we conclude that the regulation of the tight junction by protein kinase C (PKC) is a posttranslational event. In LLC-PK1A monolayers with existing tight junctions, PMA produced an initial increase in the TJC that reverted later to control values despite the continuous presence of PMA and cycloheximide. The inhibitory effect of PMA on the other cell lines was not revertible. A downregulation of total PKC activity and phorbol ester receptors was only observed during the reorganization of LLC-PK1A monolayers. PMA further increases this downregulation. This indicates that the peculiar response to PMA observed in LLC-PK1A monolayers is the result of two concurrent events: 1) the early activation of the enzyme just before the reorganization of the tight junctions begin, and 2) its late downregulation induced after prolonged exposure to phorbol esters. We conclude that PKC regulates the development of the occluding junctions, but through different mechanisms dependent on the characteristics of the cells.

Determination of the driving force for the sodium pump (E Na) and of active and passive conductances (G Na and G sh) in isolated toad skin: Influence of antidiuretic hormone

1. Values of the sodium potential (E Na), active conductance (G Na) and passive conductance (G sh) were measured in the isolated skin of the toad Pleurodema thaul placed in an Ussing chamber, and Isaacson's test was performed with 2,4,6-triminopyrimidine (TAP) and with amiloride. 2. The numerical estimates obtained in the presence of TAP were E Na122.85 ± 15.17 mV, G Na0.493 ± 0.09 mS/cm 2 and G sh1.145 ± 0.23 mS/cm 2. 3. After exposure to ADH these values were as follows: E Na85.76 ± 12.17 mV, G Na1.191 ± 0.20 mS/cm 2 and G sh0.935 ± 0.14 mS/cm 2. 4. Addition of 0.5 × 10 −2 TAP produced a 53.90 ± 5.10% decrease in transepithelial potential and a 37.90 ± 4.90% fall in short-circuit current. 5. Exposure to ADH increased the transepithelial potential difference 34.20 ± 13.20% and the short-circuit current to 78.00 ± 20.50% above the control values. 6. Comparison of the efficiency and mechanism of action of TAP and amiloride in the determination of electrical parameters shows that both agents induce a similar decrease in G sh, a finding which could indicate that TAP blocks toad skin apical membrane Na + channels without affecting tight junction conductance.

Mineralocorticoid regulation of apical cell membrane Na+ and K+ transport of the cortical collecting duct.

The effects of mineralocorticoid (DOCA) treatment of rabbits on the Na+ and K+ transport properties of the cortical collecting duct apical cell membrane were assessed using microelectrode techniques. Applying standard cable techniques and equivalent circuit analysis to the isolated perfused tubule, the apical cell membrane K+ and Na+ currents and conductances could be estimated from the selective effects of the K+ channel blocker Ba2+ and the Na+ channel blocker amiloride on the apical membrane; amiloride treatment was observed also to decrease the tight junction conductance by an average of 10%. After 1 day of DOCA treatment, the Na+ conductance and current (Na+ influx) of the apical cell membrane doubled and remained elevated with prolonged treatment for up to 2 wk. The apical cell membrane K+ conductance was not influenced after 1 day, although the K+ current (K+ secretion) increased significantly due to an increased driving force for K+ exit. After 4 days or more of DOCA treatment the K+ conductance doubled, resulting in a further modest stimulation in K+ secretion. After 2 wk of DOCA treatment the tight junction conductance decreased by near 30%, resulting in an additional hyperpolarization of the transepithelial voltage, thereby favoring K+ secretion. It is concluded that the acute effect (within 1 day) of mineralocorticoids on Na+ and K+ transport is an increase in the apical membrane Na+ conductance followed by delayed chronic alterations in the apical membrane K+ conductance and tight junction conductance, thereby resulting in a sustained increased capacity of the tubule to reabsorb Na+ and secrete K+.

Intracellular electric potential in the epithelial cells of rabbit gallbladder

Intracellular electric potential, ionic concentrations and membrane impedances have been measured in rabbit gall bladder epithelium. The contribution of tight junction conductance to the total transmural conductance is confirmed to be very large. The cell membrane potentials across the mucosal and the serosal barrier are both sensitive to changes in K + concentrations of the media. None of the main ions (Na +, K +, Cl −) is at electrochemical equilibrium.