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J Biol Chem. Nov 19, 2010; 285(47): 37060–37069.
Published online Aug 31, 2010. doi:  10.1074/jbc.M110.146621
PMCID: PMC2978634

Calcium Inhibits Paracellular Sodium Conductance through Claudin-2 by Competitive Binding*An external file that holds a picture, illustration, etc.
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Abstract

Claudins form paracellular pores at the tight junction in epithelial cells. Profound depletion of extracellular calcium is well known to cause loosening of the tight junction with loss of transepithelial resistance. However, moderate variations in calcium concentrations within the physiological range can also regulate transepithelial permeability. To investigate the underlying molecular mechanisms, we studied the effects of calcium on the permeability of claudin-2, expressed in an inducible MDCK I cell line. We found that in the physiological range, calcium acts as a reversible inhibitor of the total conductance and Na+ permeability of claudin-2, without causing changes in tight junction structure. The effect of calcium is enhanced at low Na+ concentrations, consistent with a competitive effect. Furthermore, mutation of an intrapore negatively charged binding site, Asp-65, to asparagine partially abrogated the inhibitory effect of calcium. This suggests that calcium competes with Na+ for binding to Asp-65. Other polyvalent cations had similar effects, including La3+, which caused severe and irreversible inhibition of conductance. Brownian dynamics simulations demonstrated that such inhibition can be explained if Asp-65 has a relatively high charge density, thus favoring binding of Ca2+ over that of Na+, reducing Ca2+ permeation by inhibiting its dissociation from this site, and decreasing Na+ conductance through repulsive electrostatic interaction with Ca2+. These findings may explain why hypercalcemia inhibits Na+ reabsorption in the proximal tubule of the kidney.

Keywords: Calcium, Epithelium, Ion Channels, Junctions, Sodium Transport, Claudin, Tight Junction

Introduction

The tight junction is the most apical component of the junctional complex between epithelial cells (1). It acts as a barrier that regulates the permeability of the paracellular transepithelial pathway (2,4). Profound depletion of extracellular calcium to the micromolar range is well known to cause loosening of the tight junction with loss of transepithelial resistance, disorganization of the junctional strands, and internalization of tight junction proteins (5,9). There have been reports that more modest changes in extracellular calcium within the physiological range also affect transepithelial resistance (7, 10, 11). However, the underlying molecular mechanism has not been established.

Claudins are four transmembrane domain proteins located at the tight junction between epithelial cells (12, 13). Their extracellular domains protrude into the paracellular space and form pores that regulate the paracellular permeability to small ions. The first extracellular loop appears to form the lining of the paracellular pore and determines charge selectivity (14, 15). The pore diameter, uniform among claudins, is ~8 Å as estimated from permeability to polyethylene glycols (16).

We have used claudin-2 overexpression in a high resistance strain of MDCK3 renal epithelial cells (MDCK I) as a model for investigating claudin pore permeability. In these cell lines, claudin-2 increases transepithelial conductance dramatically (17, 18) (10-fold in our hands), behaving as a cation-selective pore with permeability for Na+ relative to Cl (PNa/PCl) of 7.5 (19). We previously identified a negatively charged residue in the first extracellular loop, aspartate 65, as an intrapore electrostatic cation binding site that is largely responsible for conferring Na+ selectivity (19).

We now show that variations in extracellular calcium concentration within the physiological range regulate claudin-2 conductance and Na+ permeability. This regulation is rapid, reversible, and not associated with morphological derangements of the tight junction, indicating that it is distinct from the changes found with profound calcium depletion. Our experimental data and modeling suggest a mechanism in which calcium (and other polyvalent cations) binds with relatively high affinity to negatively charged site(s) within the pore, thereby reducing Na+ occupancy within the pore. These findings may explain how calcium regulates Na+ reabsorption in the proximal tubule of the kidney.

EXPERIMENTAL PROCEDURES

Tissue Culture and Electrophysiological Studies

The generation and maintenance of MDCK I TetOff cells stably expressing the wild type and the D65N mutant of claudin-2 were as reported previously (19). Cells were plated at confluent density on Snapwell filters (Corning) and cultured for 7–8 days in the presence (Dox+) or absence (Dox−) of 20 ng/ml doxycycline. The filters were then mounted in Ussing chambers, stirred with gas lifts in 100% O2 at 37 °C, current-clamped using Ag/AgCl electrodes bridged by 3 m KCl/3% agar pipettes, and interfaced via head-stage amplifiers to a microcomputer-controlled voltage/current clamp (DM-MC6 and VCC-MC6, respectively; Physiologic Instruments, San Diego). The standard Ringer solution used at base line contained (in mm): NaCl (140), CaCl2 (2), MgCl2 (1), glucose (10), mannitol (20), and Tris-HEPES (10), pH 7.4 (4-ml volume in each hemichamber). The conductance was determined at 1-s interval by measuring the voltage evoked by a 20-μA bipolar current pulse.

Solutions with different Na+ and Ca2+ concentrations were made up with the appropriate concentrations of NaCl and CaCl2, and the osmolality was balanced by adjusting the concentration of mannitol. Solution exchanges were performed by one of two methods: (a) each hemichamber was continuously perfused with 10 volumes of the new solution for ~1 min or (b) serial exchanges of fixed volumes (up to 2 ml) of high or low Ca2+ solution were performed by rapid aspiration and pipetting. For determination of the Ca2+ concentration-conductance relationship at fixed Na+ concentrations (Fig. 4A), filters were bathed in 2 mm Ca2+ plus the indicated Na+ concentration (50 or 100 mm), and then solution was exchanged by serial pipetting (method b) with solution containing the same Na+ concentration and either 35.3 mm Ca2+ or 0 mm Ca2+. For studies of mole fraction dependence (Fig. 4B), the standard Ringer solution (140 mm Na+, 2 mm Ca2+) was exchanged by serial pipetting with solution containing either 130 mm Na+, 12 mm Ca2+, or 142 mm Na+ and 0 mm Ca2+.

FIGURE 4.
Interaction between Na+ and Ca2+ in the claudin-2 pore. A, effect of Ca2+ on conductance at different Na+ concentrations. Conductances were normalized to the conductance in 0.26 mm Ca2+. Curves were fitted by nonlinear regression to the Michaelis-Menten ...

For measurements of Na+ permeability (Fig. 2), the 2:1 NaCl dilution potential was measured by exchanging the basolateral solution with Ringer solution containing 70 mm NaCl (with mannitol added to balance the osmolality). This was corrected for the difference in liquid junction potentials between the basolateral and apical pipettes using the method described previously (see Ref. 19, supplemental material therein). The ion permeability ratio, β = PCl/PNa, was then calculated from the Goldman-Hodgkin-Katz voltage equation,

equation image

where V is the apical voltage with respect to the basolateral side and α is the activity ratio of NaCl in the apical compartment compared with the basolateral compartment. The absolute permeability to Na+ was then estimated by the method of Kimizuka and Koketsu (20),

equation image

where GM is the transepithelial conductance and a is the Na+ activity.

FIGURE 2.
Effect of extracellular calcium concentration on claudin-2 Na+ permeability (A) and charge selectivity (B). Note that for zero Ca2+, 5 mm EGTA was also added.

Immunofluorescence Staining

Monolayers grown on filters and exposed to the indicated Ca2+ concentrations were fixed by overnight incubation in methanol at −20 °C and rinsed in 100% acetone. They were then blocked by incubation in phosphate-buffered saline (PBS) containing 1% bovine serum albumin, 5% goat serum, and 0.3% Triton X-100 for 1 h. The filters were incubated in primary antibodies (mouse anti-occludin, 1:100, and rabbit anti-claudin-2, 1:100, both from Invitrogen) for 1 h, washed in PBS, incubated in secondary antibodies (Alexa Fluor 488-conjugated anti-rabbit IgG and Alexa Fluor 555-conjugated anti-mouse IgG, 1:1000, both from Invitrogen), washed again, and mounted in ProLong anti-fade agent (Invitrogen). Slides were visualized with a Leica TCS SP2 multi-photon confocal microscope.

Brownian Dynamics Modeling

Recently we developed a Brownian dynamics (BD) model to investigate the ion permeation characteristics and charge selectivity of different monovalent alkali solutions in claudin-2 (full details can be found in Ref. 19). Briefly, the claudin-2 channel was represented in a simplistic manner as a cylindrical pore with a diameter of 6.5 Å, which was connected to two cone-like vestibules on either end. The critical charged residues at aspartate 65 were modeled as six spheres located hexagonally in the middle of the pore, each sphere characterized by radius RD, charge qD, and the distance between the channel centerline and the sphere center, RC (see supplemental Fig. S1). The protein channel and water were treated as static continua, but ions were treated explicitly and underwent high friction regime Brownian motion in which the movement of ion k was guided by the following effective potential,

equation image

where qk, qj, and qDi are the charges of ions k and j and one of the six Asp-65 residues, respectively. Dk0 and Dk(z) are the diffusion constants of ion k in the bulk and at position z in the channel direction, respectively. [var phi]kstat is the electrostatic potential due to effective protein charges (excluding Asp-65) and an externally applied membrane potential. These effective protein charges included all other charged residues (Glu-53, Asp-76, Arg-30, and Lys-48) in the first extracellular domain and were treated as point charges of 0.2e and −0.2e for positively and negatively charged residues, respectively (19). [var phi]kself is the dielectric contribution (see below) to the self-energy, or image potential, for an ion with a unit (proton) charge. Both [var phi]kstat and [var phi]kself were obtained by solving Poisson's equation on a three-dimensional grid using a standard finite difference method (21, 22). [var phi]coul(rkj) (or [var phi]coul(rkDi)) is a truncated Coulomb potential (21), and the term [var phi]kjdiel (or [var phi]kDidiel) (21, 22) arises from the fact that our simulation system is dielectrically inhomogeneous (19), here rkj is the distance between mobile ions k and j, and rkDi is the distance between mobile ion k and the charge at the center of the sphere representing the ith Asp-65 residue. The dielectric constant of the aqueous regions (both inside and outside the pore region) is taken to be ϵw = 80, and the dielectric constant in the protein/membrane regions is taken as ϵp = 20. The third and fourth terms together account for Coulombic interactions between pairs of ions in a dielectrically inhomogeneous medium. The fifth and sixth terms estimate the Coulombic interactions between a mobile ion and the charged residues Asp-65. The effects of the dielectric inhomogeneity of the channel environment on the ion-ion and ion-Asp-65 electrostatic interactions (the fourth and sixth terms in Equation 3) were implemented using an efficient empirical pair potential (22, 23),

equation image

In the present study, the channel length was taken as L = 32 Å, and the empirically determined value c = 2.0 was employed (22, 23). The last term in Equation 3 accounts for the variation of the diffusion constant characterizing ion k along the permeation path way (i.e. the channel z direction) (24).

Radii of 1.8, 0.95, and 0.99 Å were taken for Cl, Na+, and Ca2+, respectively (25). Bulk diffusion coefficients for Cl, Na+, and Ca2+ were assumed to be 2.0 × 10−5, 1.33 × 10−5, and 0.8 × 10−5 cm2/s, respectively (25). We assumed that La3+ had the same parameters as Ca2+ except for the charge it carried. Inside the claudin-2 channel, the diffusivities for different ions were assumed to be half of their bulk value (19). All simulation parameters followed our previous BD model (19) except that the effective charge carried on one Asp-65 residue was varied in order to investigate the effects of the strength of the Asp-65 binding site charge on calcium inhibition. Bulk solutions with different Na+ and Ca2+ concentrations were treated in the BD simulations by distributing fixed numbers of ions within the boundary buffer regions at each Monte Carlo cycle. The desired numbers of Na+, Ca2+, and Cl ions in the buffer regions were obtained by integrating the given boundary concentrations over the volumes of the boundary buffer regions. In the present study, the size of the boundary regions was adjusted appropriately to simulate different bath concentrations in an efficient way. Unless otherwise expressed explicitly, a transmembrane potential of −60 mV was applied to calculate the channel conductance and mobile ion density profiles.

RESULTS

Extracellular Ca2+ Inhibits Claudin-2 Conductance

To determine the effect of extracellular Ca2+ concentration on claudin-2 conductance, we used MDCK I TetOff claudin-2 cells cultured either in the absence of doxycycline to induce claudin-2 expression (Dox−) or in its presence to suppress claudin-2 (Dox+). Exchanging the extracellular solution from a standard Ringer solution containing 2 mm Ca2+ to a solution containing 5 mm Ca2+ caused a prompt increase in transepithelial resistance (TER), reflecting a drop in transepithelial conductance (Fig. 1). Conversely, reducing the extracellular Ca2+ to the submillimolar range caused a rapid decrease in TER (increase in conductance). Both of these effects were greater in Dox− cells than in Dox+ cells (5 mm Ca2+ caused a 9.2 ± 3.9% increase in TER in Dox+ cells versus 19.0 ± 3.3% in Dox− cells; 0.5 mm Ca2+ caused a 8.5 ± 3.6% decrease in TER in Dox+ cells versus 13.4 ± 0.6% in Dox− cells), indicating that these effects were mediated largely by regulation of claudin-2 conductance. In subsequent studies, we investigated the effect of Ca2+ on claudin-2 conductance in isolation by subtracting the conductance in Dox+ cells from that in Dox− cells.

FIGURE 1.
Effect of changes in extracellular calcium concentration on TER of MDCK I TetOff claudin-2 cells. A, cells grown in the presence of doxycycline (Dox+) to suppress claudin-2 expression. B, cells grown in the absence of doxycycline (Dox−) to induce ...

Effect of Moderate Changes in Ca2+ Is Distinct from That of Profound Ca2+ Depletion

Changes in claudin-2 conductance due to moderate changes in extracellular Ca2+ concentration (up to 5 mm and down to 0.25 mm) for at least 15 min were rapidly and fully reversible. By contrast, profound extracellular Ca2+ depletion (zero Ca2+ plus 5 mm EGTA) caused TER to drop to almost zero in both Dox+ and Dox− cells; this could not be reversed by restoring the Ca2+ concentration to 2 mm Ca2+ (Fig. 1) even after up to an hour of observation (not shown). This suggests that the effects of moderate changes in extracellular Ca2+ are distinct from those due to profound Ca2+ depletion.

Two other lines of evidence are consistent with this hypothesis. First, moderate reductions in extracellular Ca2+ (down to 0.25 mm) increased claudin-2 conductance by selectively increasing the permeability to Na+ (PNa), which was reflected in an increase in the ratio of permeabilities to Na+ relative to Cl (PNa/PCl); moderate elevations in extracellular Ca2+ had the opposite effect (Fig. 2). However, more extreme reductions in Ca2+, down to 0.1 or 0 mm, decreased PNa/PCl progressively to a minimum of 0.71, similar to the ratio of their free solution mobilities (μNaCl = 0.66). This suggests that moderate changes in Ca2+ caused a functional interference with Na+ permeation through the claudin-2 pore, whereas extreme reductions in Ca2+ opened up a free solution shunt pathway.

The second line of evidence for a biphasic effect of Ca2+ is shown in Fig. 3. When we performed immunofluorescence staining for claudin-2 or for occludin, another tight junction membrane protein that is constitutively expressed in all epithelial cells, we found a typical chicken wire pattern of staining in cells incubated in normal (2 mm) Ca2+ and also in cells exposed to moderate changes in Ca2+ (down to 0.25 mm or up to 5 mm). However, after even brief exposure to extreme reductions in Ca2+, the tight junction was morphologically grossly disrupted, with separation of the lateral membranes (see Fig. 3, 0 Ca2+, 5 mm EGTA). We observed occasional areas with loss of claudin-2 from the tight junction, although using digital image quantitation we were unable to detect a difference in the proportion of claudin-2 at the junction between monolayers exposed to any of the Ca2+ concentrations (see supplemental Fig. S2 and Table S1).

FIGURE 3.
Effect of extracellular calcium on tight junction morphology. Monolayers were exposed to the indicated concentrations of calcium ± EGTA for 15 min and then fixed and immunostained with antibodies to claudin-2 (green) and occludin (red). The upper ...

Ca2+ Competes with Na+ for Occupancy within the Claudin Pore

We showed previously that claudin-2 is permeable to Ca2+ and that permeating Ca2+ ions, like Na+, interact electrostatically with the negatively charged side chain of aspartate 65 located within the pore (19). We therefore hypothesized that within the range of moderate variations in Ca2+ concentration, Ca2+ inhibits Na+ conductance by competing with Na+ for occupancy at this site within the claudin-2 pore. To test this hypothesis, we looked for intrapore interactions between Na+ and Ca2+ ions in two ways. First, we determined the Ca2+ concentration-conductance relationship at two different Na+ concentrations. As shown in Fig. 4A, claudin-2 conductance fell progressively with increasing Ca2+ concentrations from 0.26 to 18 mm. The concentration of Ca2+ that caused half-maximal inhibition, Ki (best fit value, 95% CI), was lower in the presence of 50 mm Na+ (3.5 mm, 3.3–3.8 mm) than in 100 mm Na+ (5.5 mm, 5.2–5.7 mm) consistent with competitive binding. Second, we measured the conductance while varying the mole fraction of Ca2+, keeping the sum of Na+ and Ca2+ concentrations constant (Fig. 4B). If Na+ and Ca2+ ions permeate claudin-2 independently of each other, then conductance should be a linear function of the mole fraction of Ca2+. We found that the claudin-2 conductance was a nonlinear function of the mole fraction of Ca2+ but that it was monotonic (i.e. not anomalous mole fraction behavior). Such non-ideal mole fraction behavior can be explained by preferential binding selectivity of one ion species over another (26, 27). In this model, the mole fraction that produces the average of the limiting conductances (midpoint mole fraction (MMF)) can be used to determine the ion that is preferred by the channel. In claudin-2, we estimated that the MMF was 0.014, suggesting that Ca2+ binds preferentially over Na+ within the pore.

We showed previously that an acidic residue in the first extracellular domain of claudin-2, aspartate 65, is an intrapore binding site for Na+ and a major determinant of its conductance and cation selectivity (19). We therefore tested the hypothesis that aspartate 65 is part of the binding site that mediates Ca2+ inhibition of Na+ conductance. As shown in Fig. 5, mutating aspartate 65 to a polar, uncharged residue (D65N) reduced the Ca2+ inhibition of conductance by approximately half, suggesting that aspartate 65 is an important Ca2+ binding site.

FIGURE 5.
Role of Asp-65 in calcium inhibition. A, effect of changing bath Ca2+ from 2 to 5 mm (left) or 0.5 mm (right) on conductance in wild-type claudin-2 (WT) or the D65N mutant is shown. *, p < 0.05; **, p < 0.005 compared with WT. B, Western ...

Inhibition of Claudin-2 by Other Polyvalent Cations

On the basis of our findings, we postulated that other polyvalent cations small enough to enter the pore might also inhibit Na+ conductance. As shown in Fig. 6, several inorganic cations were able to inhibit claudin-2 conductance (La3+ [dbl greater-than sign] Ba2+ = Ca2+ > Mg2+). Interestingly, La3+ inhibition was poorly reversible, suggesting that its high charge density leads to particularly tight binding to negatively charged sites within the pore. The organic polycations 2,4,6-triaminopyridine and protamine, which have been used as paracellular blockers (28, 29), were also found to inhibit claudin-2 conductance moderately.

FIGURE 6.
Inhibition of claudin-2 conductance by polyvalent cations. A and B, MDCK I TetOff claudin-2 cells under Dox− conditions were bathed in Ringer solution containing Ca2+ and Mg2+ each at 0.25 mm. At the times indicated by the gray bars, the bath ...

Insights from Brownian Dynamics Modeling into the Inhibition Mechanism Associated with Multivalent Ions

We recently performed extensive Brownian dynamics simulations of ion permeation through the claudin-2 pore (19). The behavior of claudin-2 was found to be well described by a very simple model in which the pore was assumed to be a 6.5 Å diameter cylinder with conical vestibules (supplemental Fig. S1). The negatively charged side chain of Asp-65 was positioned at the center, facing into the lumen. A hexagonally distributed array of such Asp-65 spheres was constructed, each characterized by a partial charge, −0.1e. We then tested whether this model could explain the mechanism of Ca2+ inhibition of claudin-2 conductance.

Using the parameters in our original model, increasing the extracellular Ca2+ concentration from 0 up to 75 mm did not significantly reduce the conductance (not shown). Known binding sites with high affinity for Ca2+ and selectivity over Na+ generally tend to have high charge density and hence high electrostatic field strength (30, 31). We therefore tested whether increasing the effective charge carried by Asp-65 (from −0.1e in our original model) would reproduce Ca2+ inhibition. We were indeed able to observe Ca2+ inhibition of Na+ conductance when we increased the charge carried by Asp-65 to −0.3e (but not at −0.2e) (Fig. 7A). The effect on total conductance was saturable with respect to Ca2+ concentration, with maximum inhibition of about 50% of the conductance and a Ki for Ca2+ of about 6.5 mm. Decreasing the Na+ concentration caused a leftward shift in the Ca2+ concentration-conductance curve and reduction in Ki (Fig. 7B). Furthermore, there was a good concordance in the magnitude of the changes in conductance induced by moderate changes in extracellular Ca2+ in the presence of different Na+ concentrations between what was predicted in our simulations and what we observed in our experiments (Table 1).

FIGURE 7.
Brownian dynamics modeling of Ca2+ inhibition in the claudin-2 pore, assuming an effective charge qD = −0.3e on each Asp-65 residue. A, effect of extracellular Ca2+ concentration (with total Na+ concentration maintained at 150 mm) on the partial ...
TABLE 1
Comparison between Ca2+ inhibition effects determined experimentally and by BD modeling

To investigate the mechanism by which the binding site charge strength affects Ca2+ inhibition, we calculated in silico the predicted single channel conductances in symmetric bath concentrations of 0.15 m CaCl2 or NaCl for a range of charges on the Asp-65 residues. As the effective charge carried by Asp-65 was progressively increased from −0.1 to −0.3e, the Ca2+ conductance first increased to a maximum (at −0.2e) and then dropped significantly (at −0.3e) (Table 2). By contrast, the Na+ conductance increased monotonically and appeared to saturate as the effective charge was increased up to a value of −5e (cf. Ref. 19, supplemental material therein). Further augmentation of the charge on Asp-65 (i.e. > −5e) produced a decrease in Na+ conductance. The reduction in conductance of Ca2+ as the charge carried by Asp-65 was increased (or in conductance of Na+ at a very high and nonphysiologically relevant electrostatic field (19)) can be explained as arising from excessively strong electrostatic attractions between the cation and claudin-2 so that the rate of its dissociation from the channel is greatly reduced (25). Inspection of the ion density profiles along the claudin-2 pore during our simulations in mixtures of NaCl and CaCl2 at different ratios revealed that increasing concentrations of Ca2+ reduced occupancy by Na+, predominantly at the site of Asp-65 but also along the entire length of the pore (Fig. 7C and Table 1). Thus Ca2+ inhibits Na+ conductance by competing for binding to Asp-65, presumably through repulsive electrostatic forces. Moreover, because the Ca2+ conductance was almost 12 times less than that of Na+ when the charge on Asp-65 was chosen to be −0.3e (Table 2), the increase in Ca2+ conductance with increasing Ca2+ concentration was insufficient to offset the loss of Na+ conductance, and consequently the overall channel conductance was inhibited.

TABLE 2
Comparison of binding site charge strength on the conductance

We then investigated the inhibition mechanism associated with La3+ ions. BD simulations were performed in symmetric bulk solutions containing 150 mm NaCl and 5 mm LaCl3. Assuming qD (the effective charge carried by Asp-65) to be −0.3, −0.35, and −0.4e, we found that the total channel conductance was inhibited by 20 ± 10, 52 ± 15, and 85 ± 14%, respectively (Table 3). In the BD simulations with qD = −0.4e, any La3+ ion entering the center portion of the channel pore became bound near Asp-65 throughout the entire simulation (~17 μs), and no La3+ ions were observed to transport through the channel.

TABLE 3
Comparison of binding site charge strength on La3+ inhibition

DISCUSSION

We have found that Ca2+ acts as an inhibitory ion, reducing the overall conductance of claudin-2 when present at high concentration in Ringer solution and increasing overall conductance when its concentration is moderately reduced. The major conducting ion for claudin-2 in Ringer solution is Na+, and indeed we showed that these effects of Ca2+ were due to changes in Na+ permeability. Importantly, the effects of moderate changes in Ca2+ concentration were distinct from those due to profound Ca2+ depletion, which is known to cause loosening of the tight junction with the appearance of a nonselective free solution shunt, disorganization of the junctional strands, and internalization of tight junction proteins and to require many hours to reverse (5,9). We found, by contrast, that a moderate reduction in Ca2+ concentration increased Na+ selectivity, did not alter the distribution of the tight junction proteins, and was rapidly and fully reversible (t½ ~ 3–6 s).

Diamond and colleagues (10, 11) first described this phenomenon in the gall bladder epithelium. They found that decreasing the extracellular Ca2+ concentration from 5 to 0.25 mm increased transepithelial Na+ permeability and hence charge selectivity and showed that this was qualitatively different from the effects of exposure to the Ca2+ chelator, EDTA, which abolished selectivity altogether. They postulated that there exists a transepithelial permeability pathway in the gall bladder that is lined by fixed negative charges and that Ca2+ acts by binding and masking these charges. In a 1980 paper by Martinez-Palomo et al. (7) that investigated the effects of low Ca2+ in MDCK cells, it was found that decreasing the Ca2+ concentration from 10 mm to “a fraction of a millimole” caused a graded decrease in TER of ~25–30%, whereas a further reduction to nominally zero Ca2+ concentration, or the addition of 2.4 mm EGTA, caused a sharp drop in TER (see Fig. 1 in Ref. 7), again consistent with the idea that the effects of Ca2+ are biphasic. More recently, Tang and Goodenough (32) observed a similar inhibition of conductance by Ca2+ in MDCK II cells but not in MDCK I or T84 cells.

Our findings extend these prior observations by demonstrating that the effects of moderate changes in Ca2+ concentration on epithelial permeability are due to effects on claudin-2 and, by implication, on the paracellular pathway at the tight junction. Interestingly, both the mammalian gall bladder (33, 34) and low resistance strains of MDCK cells such as MDCK II (17) express claudin-2, which likely accounts for the majority of the Na+ conductance found by previous investigators to be inhibitable by Ca2+. However, in our cells, Ca2+ also had similar, albeit lesser, effects on Dox+ cells that did not express claudin-2 (Fig. 1). This suggests that the effects of moderate changes in Ca2+ are probably not specific to claudin-2 but may be shared to some extent by other claudins as well, including those claudins endogenously expressed in MDCK I cells.

The mechanism of Ca2+ inhibition is not due to pore block, because we know from radiotracer flux studies that Ca2+ itself can pass through the claudin-2 pore (19). However, our finding that the Ki for Ca2+ inhibition is dependent on the extracellular Na+ concentration suggests that Ca2+ and Na+ do interact in some way within the pore. Furthermore, we showed previously that Ca2+ permeability is strongly dependent on the negative charge at Asp-65, which is also an intrapore Na+ binding site (19). Thus, the simplest explanation is that Ca2+ competes with Na+ for binding to negatively charged site(s) within the pore. Indeed when we measured conductance at different mole fractions of Ca2+/Na+, we found a monotonic but nonlinear relationship, which is consistent with the preferential binding of Ca2+ over Na+ at a common site within the pore (26, 27).

Note that we did not find anomalous mole fraction dependence, which is defined as the appearance of a minimum or maximum in the conductance curve and implies the presence of a single file, multi-ion channel (35). Our findings are in contrast to those of Tang and Goodenough (32), who observed apparent anomalous mole fraction behavior of Ca2+ versus Na+ in MDCK II cells. However, this was based on an inflection point of their curve at ~20 mm Ca2+ and a decline in TER as extracellular Ca2+ was further increased to 150 mm, concentrations that could have pleiotropic toxic effects on cell function (32). Furthermore, previous estimates of the claudin-2 pore diameter by us (6.5 Å (19)) and by Van Itallie et al. (8 Å (16)) suggest that the pore is too large to be consistent with a single file permeation mechanism (the Pauling ionic diameters of Na+ and Ca2+ are 1.9 and 2.0 Å, respectively).

We found that mutation of Asp-65 to asparagine partially abrogated Ca2+ inhibition, confirming that the side-chain carboxylate group at Asp-65 is part of the binding site shared by Na+ and Ca2+. That Ca2+ inhibition is not totally abolished by the D65N mutation suggests the possibility that there are other negatively charged cation-binding residue(s) within the pore. Alternatively, there could be residual cation binding to asparagine due to strong polarization by the Ca2+ ions of its amide group.

Our BD simulations shed light on the probable structural determinants of Ca2+ inhibition of claudin-2. Two effects were found necessary for Ca2+ inhibition. First, along the permeation pathway there must be a common binding site (presumably at Asp-65) shared by Ca2+ and Na+, to which Ca2+ binds much more strongly than Na+. In this way, increases in extracellular Ca2+ can decrease Na+ ion occupancy within the pore (Fig. 7C and Table 1) and hence Na+ conductance. For this condition to be satisfied, our simulations show that the binding site must have a relatively high charge density (at least −0.3e/residue), a property that favors Ca2+ over Na+ binding.

Second, the permeability ratio for Ca2+ relative to Na+ must be small enough that any increase of Ca2+ conductance cannot compensate for the reduction of Na+ conductance. In general, the conductance of Ca2+ is lower than Na+ because its ion diffusion constant is smaller (by almost 50%) and the dielectric energy barrier encountered by Ca2+ in partitioning from the bulk fluid to the protein-enclosed pore is higher (4 times higher than for Na+ under the assumptions of our model). In addition, however, our model predicts that the binding site charge density is also a major determinant of Ca2+ conductance. Specifically, small increases in intrapore negative charge (from −0.1 to −0.2e) favor Ca2+ conductance, presumably by electrostatically attracting more Ca2+ ions into the pore, whereas further increases (to −0.3e and greater) dramatically inhibit Ca2+ conductance (Table 2), presumably by binding tightly to Ca2+ ions and preventing their dissociation from the site. Similar principles apply for Na+, but because its binding to the site is much weaker, the charge density at which its conductance peaks is much higher (−5e). Thus, a binding site with a moderately high charge density could explain the experimental findings.

Not surprisingly, the effects of Ca2+ on claudin-2 could be mimicked by other divalent inorganic cations such as Mg2+ and Ba2+. Interestingly, even large organic polycations such as protamine and 2,4,6-triaminopyridine were inhibitory, validating their use as tools to inhibit paracellular permeability (28, 29). One prediction ensuing from our model is that cations with extremely high charge density would bind so tightly to the intrapore site that inhibition of conductance would be profound and effectively irreversible. Our findings with La3+, a trivalent cation with a small ionic radius, are entirely consistent with this prediction.

All of our current simulations (of both Ca2+ and La3+ inhibition) indicate that Asp-65 must bear a higher charge density than suggested by our original model (which only accounted for Na+ and Cl permeation) (19). One possible explanation is that the presence of Ca2+ or La3+ near the binding site may significantly change the protein structure and thus the effective charges carried by Asp-65, which would then differ considerably from that generated by the binding of Na+ (36). Asp-65 is an aspartate residue that bears a charge of −1.0e. However, this charge is distributed over more than one atom (e.g. over the side-chain carboxylate group), and may be partly shielded by other protein atoms in the immediate vicinity of the Asp-65 residue, thus reducing the effective charge employed in a simplistic static sphere model of the critical Asp-65 residue. The degree of effective Asp-65 charge reduction may differ slightly for Na+, Ca2+, and La3+, because the deformation/polarization of the Asp-65 residue induced by the approaching ion will undoubtedly be different. Our BD simulations suggest that the effective charge carried by Asp-65 may increase with the charge of the binding ions. This hypothesis is in accordance with MD simulations of Na+ and Ca2+ binding to an acid-sensing ion channel (36), where 40% more oxygen atoms from acidic residues were observed to coordinate the bound Ca2+ than Na+. Such variations at the molecular level may not be appropriately captured by the present BD model, as this model assumes the same static protein structure for all permeant ions. However, the model can, in principle, be refined by calculating the single or even multi-ion potential mean forces for Ca2+ (or La3+) and Na+ once an atomic level structure of claudin-2 becomes available.

It is worth noting that our findings may well have physiological relevance. Claudin-2 is highly expressed in the proximal tubule of the kidney (37, 38), where it has recently been shown to play a major role in paracellular Na+ reabsorption (39). Acute hypercalcemia, which leads to a high filtered load of Ca2+ in the renal tubule, induces a profound natriuresis, at least in part by inhibiting Na+ reabsorption in the proximal tubule (40). We postulate that this effect may be due to the inhibition of Na+ diffusion through claudin-2 by luminal Ca2+. This is an important protective mechanism for keeping urinary Ca2+ concentrations from exceeding their solubility threshold and thus preventing kidney stone formation in the setting of hypercalcemia.

Finally, the identification of claudin-2 inhibitors may also have pharmacological utility in gastrointestinal diseases. Claudin-2 is expressed in the crypt epithelia (41), where it may mediate paracellular Na+ secretion coupled to transcellular Cl secretion. Intestinal claudin-2 is up-regulated in human and mouse models of inflammatory bowel disease, where it is thought to play a role both in the pathogenesis of the disease and in the development of leak flux diarrhea (42,44). Interestingly, bismuth salts have been used for more than 30 years in the treatment of diarrhea (45), and even lanthanum chloride has been found to inhibit intestinal secretions in response to Escherichia coli enterotoxin (46). We therefore speculate that orally administered polyvalent cation salts may act by inhibiting intestinal claudin-2 permeability and may have a potential clinical role in the treatment of inflammatory bowel and secretory diarrheal diseases.

Supplementary Material

Supplemental Data:

*This work was supported, in whole or in part, by National Institutes of Health Grants DK062283 (to A. S. L. Y.) and 1S10RR024754 (shared instrumentation grant to the University of Southern California Multi-Photon Microscopy Core).

An external file that holds a picture, illustration, etc.
Object name is sbox.jpgThe on-line version of this article (available at http://www.jbc.org) contains supplemental “Methods” and “Results,” Figs. S1 and S2, and Table S1.

3The abbreviations used are:

MDCK
Madin-Darby canine kidney
DOX
doxycycline
BD
Brownian dynamics
TER
transepithelial resistance.

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