Comprehensive Cysteine-scanning Mutagenesis Reveals Claudin-2 Pore-lining Residues with Different Intrapore Locations

The first extracellular loop (ECL1) of claudins forms paracellular pores in the tight junction that determine ion permselectivity. We aimed to map the pore-lining residues of claudin-2 by comprehensive cysteine-scanning mutagenesis of ECL1. We screened 45 cysteine mutations within the ECL1 by expression in polyclonal Madin-Darby canine kidney II Tet-Off cells and found nine mutants that displayed a significant decrease of conductance after treatment with the thiol-reactive reagent 2-(trimethylammonium)ethyl methanethiosulfonate, indicating the location of candidate pore-lining residues. Next, we stably expressed these candidates in monoclonal Madin-Darby canine kidney I Tet-Off cells and exposed them to thiol-reactive reagents. The maximum degree of inhibition of conductance, size selectivity of degree of inhibition, and size dependence of the kinetics of reaction were used to deduce the location of residues within the pore. Our data support the following sequence of pore-lining residues located from the narrowest to the widest part of the pore: Ser⁶⁸, Ser⁴⁷, Thr⁶²/Ile⁶⁶, Thr⁵⁶, Thr³²/Gly⁴⁵, and Met⁵². The paracellular pore appears to primarily be lined by polar side chains, as expected for a predominantly aqueous environment. Furthermore, our results strongly suggest the existence of a continuous sequence of residues in the ECL1 centered around Asp⁶⁵–Ser⁶⁸ that form a major part of the lining of the pore.     

Calcium Inhibits Paracellular Sodium Conductance through Claudin-2 by Competitive Binding

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.     

The Second Sodium Site in the Dopamine Transporter Controls Cation Permeation and Is Regulated by Chloride

The dopamine transporter (DAT) belongs to the family of neurotransmitter:sodium symporters and controls dopamine (DA) homeostasis by mediating Na⁺- and Cl⁻-dependent reuptake of DA. Here we used two-electrode voltage clamp measurements in Xenopus oocytes together with targeted mutagenesis to investigate the mechanistic relationship between DAT ion binding sites and transporter conductances. In Li⁺, DAT displayed a cocaine-sensitive cation leak current ∼10-fold larger than the substrate-induced current in Na⁺. Mutation of Na⁺ coordinating residues in the first (Na1) and second (Na2) binding sites suggested that the Li⁺ leak depends on Li⁺ interaction with Na2 rather than Na1. DA caused a marked inhibition of the Li⁺ leak, consistent with the ability of the substrate to interact with the Li⁺-occupied state of the transporter. The leak current in Li⁺ was also potently inhibited by low millimolar concentrations of Na⁺, which according to our mutational data conceivably depended on high affinity binding to Na1. The Li⁺ leak was further regulated by Cl⁻ that most likely increases Li⁺ permeation by allosterically lowering Na2 affinity. Interestingly, mutational lowering of Na2 affinity by substituting Asp-420 with asparagine dramatically increased cation permeability in Na⁺ to a level higher than seen in Li⁺. In addition to reveal a functional link between the bound Cl⁻ and the cation bound in the Na2 site, the data support a key role of Na2 in determining cation permeability of the transporter and thereby possibly in regulating the opening probability of the inner gate.     

Claudin-21 Has a Paracellular Channel Role at Tight Junctions

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.     

Claudin-8 Expression in Renal Epithelial Cells Augments the Paracellular Barrier by Replacing Endogenous Claudin-2

Claudins are transmembrane proteins of the tight junction that determine and regulate paracellular ion permeability. We previously reported that claudin-8 reduces paracellular cation permeability when expressed in low-resistance Madin-Darby canine kidney (MDCK) II cells. Here, we address how the interaction of heterologously expressed claudin-8 with endogenous claudin isoforms impacts epithelial barrier properties. In MDCK II cells, barrier improvement by claudin-8 is accompanied by a reduction of endogenous claudin-2 protein at the tight junction. Here, we show that this is not because of relocalization of claudin-2 into the cytosolic pool but primarily due to a decrease in gene expression. Claudin-8 also affects the trafficking of claudin-2, which was displaced specifically from the junctions at which claudin-8 was inserted. To test whether replacement of cation-permeable claudin-2 mediates the effect of claudin-8 on the electrophysiological phenotype of the host cell line, we expressed claudin-8 in high-resistance MDCK I cells, which lack endogenous claudin-2. Unlike in MDCK II cells, induction of claudin-8 in MDCK I cells (which did not affect levels of endogenous claudins) did not alter paracellular ion permeability. Furthermore, when endogenous claudin-2 in MDCK II cells was downregulated by epidermal growth factor to create a cell model with low transepithelial resistance and low levels of claudin-2, the permeability effects of claudin-8 were also abolished. Our findings demonstrate that claudin overexpression studies measure the combined effect of alterations in both endogenous and exogenous claudins, thus explaining the dependence of the phenotype on the host cell line.     

Structure-Function Studies of Claudin Extracellular Domains by Cysteine-scanning Mutagenesis

Claudins form size- and charge-selective pores in the tight junction that control the paracellular flux of inorganic ions and small molecules. However, the structural basis for ion selectivity of paracellular pores is poorly understood. Here we applied cysteine scanning to map the paracellular pathway of ion permeation across claudin-2-transfected Madin-Darby canine kidney type I cells. Four potential pore-lining amino acid residues in the first extracellular loop were mutated to cysteine and screened for their accessibility to thiol-reactive reagents. All mutants were functional except D65C, which formed dimers by intermolecular disulfide bonding, leading to a loss of charge and size selectivity. This suggests that claudin-2 pores are multimeric and that Asp⁶⁵ lies close to a protein-protein interface. Methanethiosulfonate reagents of different size and charge and the organic mercury derivate, p-(chloromercuri)benzenesulfonic acid, significantly decreased paracellular ion permeation across I66C-transfected cells by a mechanism that suggests steric blocking of the pore. The conductance of wild-type claudin-2 and the other cysteine mutants was only weakly affected. The rate of reaction with I66C decreased dramatically with increasing size of the reagent, suggesting that Ile⁶⁶ is buried deep within a narrow segment of the pore with its side group facing into the lumen. Furthermore, labeling with N-biotinoylaminoethyl methanethiosulfonate showed that I66C was weakly reactive, whereas Y35C was strongly reactive, suggesting that Tyr³⁵ is located at the protein surface outside of the pore.Sheets of polarized epithelia constitute barriers that separate fluid compartments of different chemical composition and mediate exchange of solutes and ions via transcellular and paracellular pathways. A large body of evidence suggests that transport via the paracellular pathway occurs through pores in the tight junctions that are formed by tetraspan membrane proteins, known as claudins (1–3).Our current understanding of paracellular pores is that they are size- and charge-selective water-filled channels that, in contrast to channels for transmembrane transport, are oriented parallel instead of perpendicular to the lipid layer of the cell membrane. Size exclusion experiments suggest a pore diameter of 6.4–8 Å (4, 5). Furthermore, site-directed mutagenesis and overexpression of claudins in epithelial cells identified the first extracellular domain as playing an important role in the charge selectivity of paracellular transport (6–8). The first extracellular domain of claudins contains various basic and acidic amino acids, some of which are conserved in different claudin isoforms, and these could be involved in the mechanism of ion permeation. Several studies have demonstrated homo- or heterotypic interaction of claudins, suggesting that paracellular pores are formed by oligomers of claudins (9–11). Taken together, significant progress has been made in uncovering the nature of the paracellular pathway and mechanisms of selectivity of paracellular ion permeation. However, it is unknown how the extracellular domains of claudins fold to form paracellular pores and which amino acid residues line the pathway of ion diffusion.Epithelia in vivo and epithelial cell lines express characteristic sets of different claudin isoforms that determine paracellular permeability and permselectivity. Claudin-2 is expressed in epithelia with a high capacity for passive paracellular cation transport, such as the epithelium lining the proximal renal tubules (12). The transfection of claudin-2 into high resistance Madin-Darby canine kidney (MDCK)² type I cells converts the tight junction from a “tight” into a “leaky” paracellular barrier by selectively increasing Na⁺ permeability (13, 14), suggesting a physiologic role of claudin-2 in creating paracellular Na⁺ channels. Because of the high signal/noise ratio of the claudin-2-induced permeability, this isoform provides an excellent model to study paracellular transport. We have recently generated a stable expression system of claudin-2 in MDCK I cells under the control of a TetOff promoter. This inducible system allows us to specifically determine the macroscopic conductance and permeability of claudin-2 pores by subtracting background measurements of uninduced cells. Using this expression system, we could recently demonstrate that the cation selectivity of claudin-2 cells is mediated by electrostatic interaction of partially dehydrated permeating cations with aspartate 65 (5). However, further investigations are necessary to study the position and function of this and other residues of the first extracellular domain and to elucidate their role in the transport mechanism of paracellular pores.The substituted cysteine accessibility method (SCAM), developed by the Karlin group, has proved to be a powerful tool in the mapping of the structures of membrane ion channels and transport proteins (15, 16). In SCAM, thiol-reactive reagents are used to covalently modify endogenous cysteines, or cysteines introduced into a protein by site-directed mutagenesis. SCAM can be used to study channel-lining amino acid side chains, the secondary structures of membrane-spanning segments, and the localization of selectivity filters, channel gates, and inhibitor binding sites.Here, we used SCAM to analyze the paracellular pathway of ion permeation across claudin-2 transfected MDCK I cells. Our data show that thiol-reactive reagents strongly block ion transport in at least one of the cysteine mutants that we have generated and, thus, provide a tool to map residues that line the paracellular pore.     

Claudin extracellular domains determine paracellular charge selectivity and resistance but not tight junction fibril architecture

Tight junctions (TJs) regulateparacellular permeability across epithelia and vary widely in theirtransepithelial electrical resistance (TER) and charge selectivity. Theclaudin family of transmembrane proteins influences these properties.We previously reported that claudin-4 increased TER ~300% whenexpressed in low-resistance Madin-Darby canine kidney (MDCK) II cellsand decreased the paracellular permeability for Na⁺ morethan Cl (Van Itallie C, Rahner C, and Anderson JM.J Clin Invest 107: 1319-1327, 2001). Incomparison, we report here that expression of claudin-2 increases TERby only ~20% and does not change the ionic selectivity of MDCK IIcells from their cation-selective background. To test whether theextracellular domains of claudins-4 and -2 determine their uniqueparacellular properties, we determined the effects of interchangingthese domains between claudins-4 and -2. Inducible expression ofwild-type claudins and extracellular domain chimeras increased both thenumber and depth of fibrils, but the characteristic fibril morphologiesof claudin-4 or -2 were not altered by switching extracellular domains.Like claudin-4, chimeras expressing the first or both extracellulardomains of claudin-4 on claudin-2 increased TER severalfold andprofoundly decreased the permeability of Na⁺ relative toCl. In contrast, chimeras expressing the first or bothextracellular domains of claudin-2 on claudin-4 increased the TER byonly ~60 and ~40%, respectively, and only modestly altered chargeselectivity. These results support a model in which the claudins createparacellular channels and the first extracellular domain is sufficientto determine both paracellular charge selectivity and TER.

     

A Claudin-9–Based Ion Permeability Barrier Is Essential for Hearing

Hereditary hearing loss is one of the most common birth defects, yet the majority of genes required for audition is thought to remain unidentified. Ethylnitrosourea (ENU)–mutagenesis has been a valuable approach for generating new animal models of deafness and discovering previously unrecognized gene functions. Here we report on the characterization of a new ENU–induced mouse mutant (nmf329) that exhibits recessively inherited deafness. We found a widespread loss of sensory hair cells in the hearing organs of nmf329 mice after the second week of life. Positional cloning revealed that the nmf329 strain carries a missense mutation in the claudin-9 gene, which encodes a tight junction protein with unknown biological function. In an epithelial cell line, heterologous expression of wild-type claudin-9 reduced the paracellular permeability to Na⁺ and K⁺, and the nmf329 mutation eliminated this ion barrier function without affecting the plasma membrane localization of claudin-9. In the nmf329 mouse line, the perilymphatic K⁺ concentration was found to be elevated, suggesting that the cochlear tight junctions were dysfunctional. Furthermore, the hair-cell loss in the claudin-9–defective cochlea was rescued in vitro when the explanted hearing organs were cultured in a low-K⁺ milieu and in vivo when the endocochlear K⁺-driving force was diminished by deletion of the pou3f4 gene. Overall, our data indicate that claudin-9 is required for the preservation of sensory cells in the hearing organ because claudin-9–defective tight junctions fail to shield the basolateral side of hair cells from the K⁺-rich endolymph. In the tight-junction complexes of hair cells, claudin-9 is localized specifically to a subdomain that is underneath more apical tight-junction strands formed by other claudins. Thus, the analysis of claudin-9 mutant mice suggests that even the deeper (subapical) tight-junction strands have biologically important ion barrier function.     

Functional Swapping between Transmembrane Proteins TMEM16A and TMEM16F

The transmembrane proteins TMEM16A and -16F each carry eight transmembrane regions with cytoplasmic N and C termini. TMEM16A carries out Ca²⁺-dependent Cl⁻ ion transport, and TMEM16F is responsible for Ca²⁺-dependent phospholipid scrambling. Here we established assay systems for the Ca²⁺-dependent Cl⁻ channel activity using 293T cells and for the phospholipid scramblase activity using TMEM16F^−/− immortalized fetal thymocytes. Chemical cross-linking analysis showed that TMEM16A and -16F form homodimers in both 293T cells and immortalized fetal thymocytes. Successive deletion from the N or C terminus of both proteins and the swapping of regions between TMEM16A and -16F indicated that their cytoplasmic N-terminal (147 amino acids for TMEM16A and 95 for 16F) and C-terminal (88 amino acids for TMEM16A and 68 for 16F) regions were essential for their localization at plasma membranes and protein stability, respectively, and could be exchanged. Analyses of TMEM16A and -16F mutants with point mutations in the pore region (located between the fifth and sixth transmembrane regions) indicated that the pore region is essential for both the Cl⁻ channel activity of TMEM16A and the phospholipid scramblase activity of TMEM16F. Some chemicals such as epigallocatechin-3-gallate and digallic acid inhibited the Cl⁻ channel activity of TMEM16A and the scramblase activity of TMEM16F with an opposite preference. These results indicate that TMEM16A and -16F use a similar mechanism for sorting to plasma membrane and protein stabilization, but their functional domains significantly differ.     

Claudins create charge-selective channels in the paracellular pathway between epithelial cells

Epithelia separate tissuespaces by regulating the passage of ions, solutes, and water throughboth the transcellular and paracellular pathways. Paracellularpermeability is defined by intercellular tight junctions, which varywidely among tissues with respect to solute flux, electricalresistance, and ionic charge selectivity. To test the hypothesis thatmembers of the claudin family of tight junction proteins create chargeselectivity, we assessed the effect of reversing the charge of selectedextracellular amino acids in two claudins using site-directedmutagenesis. Claudins were expressed in cultured Madin-Darby caninekidney cell monolayers under an inducible promoter, and clones werecompared with and without induction for transmonolayer electricalresistance and dilution potentials. Expression and localization ofclaudins were determined by immunoblotting, immunofluorescencemicroscopy, and freeze-fracture electron microscopy. We observed thatsubstituting a negative for a positive charge at position 65 in thefirst extracellular domain of claudin-4 increased paracellularNa⁺ permeability. Conversely, substituting positive fornegative charges at three positions in the first extracellular domainof claudin-15, singly and in combination, reversed paracellular charge selectivity from a preference for Na⁺ to Cl.These results support a model where claudins create charge-selective channels in the paracellular space.

     

Claudin-3 acts as a sealing component of the tight junction for ions of either charge and uncharged solutes

The paracellular barrier of epithelia and endothelia is established by several tight junction proteins including claudin-3. Although claudin-3 is present in many epithelia including skin, lung, kidney, and intestine and in endothelia, its function is unresolved as yet. We therefore characterized claudin-3 by stable transfection of MDCK II kidney tubule cells with human claudin-3 cDNA. Two clone systems were analyzed, exhibiting high or low claudin-2 expression, respectively. Expression of other claudins was unchanged. Ultrastructurally, tight junction strands were changed toward uninterrupted and rounded meshwork loops. Functionally, the paracellular resistance of claudin-3-transfected monolayers was strongly elevated, causing an increase in transepithelial resistance compared to vector controls. Permeabilities for mono- and divalent cations and for anions were decreased. In the high-claudin-2 system, claudin-3 reduced claudin-2-induced cation selectivity, while in the low-claudin-2 system no charge preference was observed, the latter thus reflecting the "intrinsic" action of claudin-3. Furthermore, the passage of the paracellular tracers fluorescein (332 Da) and FD-4 (4 kDa) was decreased, whereas the permeability to water was not affected. We demonstrate that claudin-3 alters the tight junction meshwork and seals the paracellular pathway against the passage of small ions of either charge and uncharged solutes. Thus, in a kidney model epithelium, claudin-3 acts as a general barrier-forming protein.     

Molecular Basis for Cation Selectivity in Claudin-2–based Paracellular Pores: Identification of an Electrostatic Interaction Site

Paracellular ion transport in epithelia is mediated by pores formed by members of the claudin family. The degree of selectivity and the molecular mechanism of ion permeation through claudin pores are poorly understood. By expressing a high-conductance claudin isoform, claudin-2, in high-resistance Madin-Darby canine kidney cells under the control of an inducible promoter, we were able to quantitate claudin pore permeability. Claudin-2 pores were found to be narrow, fluid filled, and cation selective. Charge selectivity was mediated by the electrostatic interaction of partially dehydrated permeating cations with a negatively charged site within the pore that is formed by the side chain carboxyl group of aspartate-65. Thus, paracellular pores use intrapore electrostatic binding sites to achieve a high conductance with a high degree of charge selectivity.     

The Cystic Fibrosis Transmembrane Conductance Regulator Impedes Proteolytic Stimulation of the Epithelial Na+ Channel

Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR) that prevent its proper folding and trafficking to the apical membrane of epithelial cells. Absence of cAMP-mediated Cl⁻ secretion in CF airways causes poorly hydrated airway surfaces in CF patients, and this condition is exacerbated by excessive Na⁺ absorption. The mechanistic link between missing CFTR and increased Na⁺ absorption in airway epithelia has remained elusive, although substantial evidence implicates hyperactivity of the epithelial Na⁺ channel (ENaC). ENaC is known to be activated by selective endoproteolysis of the extracellular domains of its α- and γ-subunits, and it was recently reported that ENaC and CFTR physically associate in mammalian cells. We confirmed this interaction in oocytes by co-immunoprecipitation and found that ENaC associated with wild-type CFTR was protected from proteolytic cleavage and stimulation of open probability. In contrast, ΔF508 CFTR, the most common mutant protein in CF patients, failed to protect ENaC from proteolytic cleavage and stimulation. In normal airway epithelial cells, ENaC was contained in the anti-CFTR immunoprecipitate. In CF airway epithelial cultures, the proportion of full-length to total α-ENaC protein signal was consistently reduced compared with normal cultures. Our results identify limiting proteolytic cleavage of ENaC as a mechanism by which CFTR down-regulates Na⁺ absorption.     

Permeability of Wild-Type and Mutant Cystic Fibrosis Transmembrane Conductance Regulator Chloride Channels to Polyatomic Anions

Permeability of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel to polyatomic anions of known dimensions was studied in stably transfected Chinese hamster ovary cells by using the patch clamp technique. Biionic reversal potentials measured with external polyatomic anions gave the permeability ratio (P_X/P_Cl) sequence NO₃ ⁻ > Cl⁻ > HCO₃ ⁻ > formate > acetate. The same selectivity sequence but somewhat higher permeability ratios were obtained when anions were tested from the cytoplasmic side. Pyruvate, propanoate, methane sulfonate, ethane sulfonate, and gluconate were not measurably permeant (P_X/P_Cl < 0.06) from either side of the membrane. The relationship between permeability ratios from the outside and ionic diameters suggests a minimum functional pore diameter of ∼5.3 Å. Permeability ratios also followed a lyotropic sequence, suggesting that permeability is dependent on ionic hydration energies. Site-directed mutagenesis of two adjacent threonines in TM6 to smaller, less polar alanines led to a significant (24%) increase in single channel conductance and elevated permeability to several large anions, suggesting that these residues do not strongly bind permeating anions, but may contribute to the narrowest part of the pore.     

Halide Permeation in Wild-Type and Mutant Cystic Fibrosis Transmembrane Conductance Regulator Chloride Channels

Permeation of cystic fibrosis transmembrane conductance regulator (CFTR) Cl⁻ channels by halide ions was studied in stably transfected Chinese hamster ovary cells by using the patch clamp technique. In cell-attached patches with a high Cl⁻ pipette solution, the CFTR channel displayed outwardly rectifying currents and had a conductance near the membrane potential of 6.0 pS at 22°C or 8.7 pS at 37°C. The current–voltage relationship became linear when patches were excised into symmetrical, N-tris(hydroxymethyl)methyl-2-aminomethane sulfonate (TES)-buffered solutions. Under these conditions, conductance increased from 7.0 pS at 22°C to 10.9 pS at 37°C. The conductance at 22°C was ∼1.0 pS higher when TES and HEPES were omitted from the solution, suggesting weak, voltage-independent block by pH buffers. The relationship between conductance and Cl⁻ activity was hyperbolic and well fitted by a Michaelis-Menten–type function having a K _m of ∼38 mM and maximum conductance of 10 pS at 22°C. Dilution potentials measured with NaCl gradients indicated high anion selectivity (P_Na/P_Cl = 0.003–0.028). Biionic reversal potentials measured immediately after exposure of the cytoplasmic side to various test anions indicated P_I (1.8) > P_Br (1.3) > P_Cl (1.0) > P_F (0.17), consistent with a “weak field strength” selectivity site. The same sequence was obtained for external halides, although inward F⁻ flow was not observed. Iodide currents were protocol dependent and became blocked after 1–2 min. This coincided with a large shift in the (extrapolated) reversal potential to values indicating a greatly reduced I⁻/Cl⁻ permeability ratio (P_I/P_Cl < 0.4). The switch to low I⁻ permeability was enhanced at potentials that favored Cl⁻ entry into the pore and was not observed in the R347D mutant, which is thought to lack an anion binding site involved in multi-ion pore behavior. Interactions between Cl⁻ and I⁻ ions may influence I⁻ permeation and be responsible for the wide range of P_I/P_Cl ratios that have been reported for the CFTR channel. The low P_I/P_Cl ratio usually reported for CFTR only occurred after entry into an altered permeability state and thus may not be comparable with permeability ratios for other anions, which are obtained in the absence of iodide. We propose that CFTR displays a “weak field strength” anion selectivity sequence.     

The ClC-3 Cl?/H+ Antiporter Becomes Uncoupled at Low Extracellular pH

Adenovirus expressing ClC-3 (Ad-ClC-3) induces Cl⁻/H⁺ antiport current (I_ClC-3) in HEK293 cells. The outward rectification and time dependence of I_ClC-3 closely resemble an endogenous HEK293 cell acid-activated Cl⁻ current (ICl_acid) seen at extracellular pH ≤ 5.5. ICl_acid was present in smooth muscle cells from wild-type but not ClC-3 null mice. We therefore sought to determine whether these currents were related. ICl_acid was larger in cells expressing Ad-ClC-3. Protons shifted the reversal potential (E_rev) of I_ClC-3 between pH 8.2 and 6.2, but not pH 6.2 and 5.2, suggesting that Cl⁻ and H⁺ transport become uncoupled at low pH. At pH 4.0 E_rev was completely Cl⁻ dependent (55.8 ± 2.3 mV/decade). Several findings linked ClC-3 with native ICl_acid; 1) RNA interference directed at ClC-3 message reduced native ICl_acid; 2) removal of the extracellular “fast gate” (E224A) produced large currents that were pH-insensitive; and 3) wild-type I_ClC-3 and ICl_acid were both inhibited by (2-sulfonatoethyl)methanethiosulfonate (MTSES; 10–500 μm)-induced alkanethiolation at exposed cysteine residues. However, a ClC-3 mutant lacking four extracellular cysteine residues (C103_P130del) was completely resistant to MTSES. C103_P130del currents were still acid-activated, but could be distinguished from wild-type I_ClC-3 and from native ICl_acid by a much slower response to low pH. Thus, ClC-3 currents are activated by protons and ClC-3 protein may account for native ICl_acid. Low pH uncouples Cl⁻/H⁺ transport so that at pH 4.0 ClC-3 behaves as an anion-selective channel. These findings have important implications for the biology of Cl⁻/H⁺ antiporters and perhaps for pH regulation in highly acidic intracellular compartments.     

Molecular Determinants of the Interaction between Clostridium perfringens Enterotoxin Fragments and Claudin-3

Clostridium perfringens enterotoxin (CPE) binds to the extracellular loop 2 of a subset of claudins, e.g. claudin-3. Here, the molecular mechanism of the CPE-claudin interaction was analyzed. Using peptide arrays, recombinant CPE-(116–319) bound to loop 2 peptides of mouse claudin-3, -6, -7, -9, and -14 but not of 1, 2, 4, 5, 8, 10–13, 15, 16, 18–20, and 22. Substitution peptide mapping identified the central motif ¹⁴⁸NPL¹⁵⁰VP, supposed to represent a turn region in the loop 2, as essential for the interaction between CPE and murine claudin-3 peptides. CPE-binding assays with claudin-3 mutant-transfected HEK293 cells or lysates thereof demonstrated the involvement of Asn¹⁴⁸ and Leu¹⁵⁰ of full-length claudin-3 in the binding. CPE-(116–319) and CPE-(194–319) bound to HEK293 cells expressing claudin-3, whereas CPE-(116–319) bound to claudin-5-expressing HEK293 cells, also. This binding was inhibited by substitutions T151A and Q156E in claudin-5. In contrast, removal of the aromatic side chains in the loop 2 of claudin-3 and -5, involved in trans-interaction between claudins, increased the amount of CPE-(116–319) bound. These findings and molecular modeling indicate different molecular mechanisms of claudin-claudin trans-interaction and claudin-CPE interaction. Confocal microscopy showed that CPE-(116–319) and CPE-(194–319) bind to claudin-3 at the plasma membrane, outside cell-cell contacts. Together, these findings demonstrate that CPE binds to the hydrophobic turn and flanking polar residues in the loop 2 of claudin-3 outside tight junctions. The data can be used for the specific design of CPE-based modulators of tight junctions, to improve drug delivery, and as chemotherapeutics for tumors overexpressing claudins.The clinical use of many promising drug candidates is impeded by unacceptable pharmacokinetics (1). The ability of a drug to pass through tissue barriers is a major determinant for its delivery. In epithelia and endothelia, the paracellular route is blocked by tight junctions (TJ).⁴ Different approaches have been used to enhance transcellular drug delivery. These include the use of influx transporters, blocking of efflux transporters, or receptor-mediated endocytosis (2). Alternative approaches aim to enhance paracellular permeation of drugs by loosening the TJ (3, 4). This strategy has the advantage that it could improve the delivery of structurally unrelated drugs, and the drug itself does not have to be modified. Although different TJ modulators have been described, most of these are based on surfactants or chelators (3). These often have low tissue specificity and cause severe side effects, e.g. exfoliation of cells, which irreversibly compromise the barrier functions (5, 6). Fewer side effects may be obtained by more specific modulation of a molecular key component of the TJ (7).TJ consist of transmembrane proteins, mainly the tetraspan proteins of the claudin family, as well as occludin and tricellulin (8). Other molecules associated with TJ include membrane-bound scaffolding and signaling proteins (9). However, claudins (Cld) are the major functional constituent of TJ (10). Claudins tighten the paracellular space, selectively for tissue, size, and charge. The tissue-specific combination of the claudin subtypes present in heteropolymers is assumed to determine the permeability properties of TJ (11). It was therefore proposed that tissue-specific drug delivery via the paracellular route would be possible by modulation of the barrier-function of claudins in a subtype-specific manner (7).A subset of claudins, e.g. Cld3 and -4 but not -1 and -2, have been shown to be receptors for Clostridium perfringens enterotoxin (CPE) with high association constants of about 10⁸ m⁻¹ (12). CPE causes one of the most common food-borne diseases (13). It consists of two functional domains, an N-terminal region that mediates the cytotoxic effect and the C-terminal region (CPE-(184–319)), which binds to extracellular loop 2 (ECL2) of Cld3 but not of Cld1 nor to the ECL1 of Cld3 (12). Treatment of epithelial monolayers with non-cytotoxic CPE-(184–319) increases paracellular permeability (14). CPE-(184–319) enhanced drug absorption in rat jejunum 400-fold relative to sodium caprate, which is in clinical use (15). Thus, CPE is a promising tool to specifically modulate claudins, the key constituents of TJ, and thereby to enhance paracellular drug delivery. In addition, some studies have suggested the use of CPE for the chemotherapy of tumors overexpressing claudins (16–18).Cld1 and -5 are potential targets for transepidermal and brain drug delivery, respectively (19, 20). However, it has been reported that these claudins do not interact with CPE (12). Modification of CPE could enhance and/or shift its claudin-subtype specificity. Therefore, the design of CPE-based TJ modulators could permit efficient claudin subtype-specific modulation, which would also be tissue-specific modulation of TJ. To achieve this, an understanding of the molecular mechanism of the CPE-claudin interaction is a necessary prerequisite. In this study, we identify the residues within the ECL2 of Cld3 that are involved in interaction with CPE.     

Anoctamin-6 Controls Bone Mineralization by Activating the Calcium Transporter NCX1

Anoctamin-6 (Ano6, TMEM16F) belongs to a family of putative Ca²⁺-activated Cl⁻ channels and operates as membrane phospholipid scramblase. Deletion of Ano6 leads to reduced skeleton size, skeletal deformities, and mineralization defects in mice. However, it remains entirely unclear how a lack of Ano6 leads to a delay in bone mineralization by osteoblasts. The Na⁺/Ca²⁺ exchanger NCX1 was found to interact with Ano6 in a two-hybrid split-ubiquitin screen. Using human osteoblasts and osteoblasts from Ano6^−/− and WT mice, we demonstrate that NCX1 requires Ano6 to efficiently translocate Ca²⁺ out of osteoblasts into the calcifying bone matrix. Ca²⁺-activated anion currents are missing in primary osteoblasts isolated from Ano6 null mice. Our findings demonstrate the importance of NCX1 for bone mineralization and explain why deletion of an ion channel leads to the observed mineralization defect: Ano6 Cl⁻ currents are probably required to operate as a Cl⁻ bypass channel, thereby compensating net Na⁺ charge movement by NCX1.     

Tight junction proteins claudin-2 and -12 are critical for vitamin D-dependent Ca2+ absorption between enterocytes

Ca²⁺ is absorbed across intestinal epithelial monolayers via transcellular and paracellular pathways, and an active form of vitamin D₃, 1α,25-dihydroxyvitamin D₃ [1α,25(OH)₂D₃], is known to promote intestinal Ca²⁺ absorption. However, the molecules driving the paracellular Ca²⁺ absorption and its vitamin D dependency remain obscure. Because the tight junction proteins claudins are suggested to form paracellular channels for selective ions between neighboring cells, we hypothesized that specific intestinal claudins might facilitate paracellular Ca²⁺ transport and that expression of these claudins could be induced by 1α,25(OH)₂D₃. Herein, we show, by using RNA interference and overexpression strategies, that claudin-2 and claudin-12 contribute to Ca²⁺ absorption in intestinal epithelial cells. We also provide evidence showing that expression of claudins-2 and -12 is up-regulated in enterocytes in vitro and in vivo by 1α,25(OH)₂D₃ through the vitamin D receptor. These findings strongly suggest that claudin-2- and/or claudin-12-based tight junctions form paracellular Ca²⁺ channels in intestinal epithelia, and they highlight a novel mechanism behind vitamin D-dependent calcium homeostasis.