Altered Ion Selectivity Research Articles

Functional impact of a congenital stationary night blindness type 2 mutation depends on subunit composition of Cav1.4 Ca2+ channels

Voltage-gated Cav1 and Cav2 Ca2+ channels are comprised of a pore-forming α1 subunit (Cav1.1-1.4, Cav2.1-2.3) and auxiliary β (β1-4) and α2δ (α2δ-1-4) subunits. The properties of these channels vary with distinct combinations of Cav subunits and alternative splicing of the encoding transcripts. Therefore, the impact of disease-causing mutations affecting these channels may depend on the identities of Cav subunits and splice variants. Here, we analyzed the effects of a congenital stationary night blindness type 2 (CSNB2)-causing mutation, I745T (IT), in Cav1.4 channels typical of those in human retina: Cav1.4 splice variants with or without exon 47 (Cav1.4+ex47 and Cav1.4Δex47, respectively), and the auxiliary subunits, β2X13 and α2δ-4. We find that IT caused both Cav1.4 splice variants to activate at significantly more negative voltages and with slower deactivation kinetics than the corresponding WT channels. These effects of the IT mutation, along with unexpected alterations in ion selectivity, were generally larger in channels lacking exon 47. The weaker ion selectivity caused by IT led to hyperpolarizing shifts in the reversal potential and large outward currents that were evident in channels containing the auxiliary subunits β2X13 and α2δ-4 but not in those with β2A and α2δ-1. We conclude that the IT mutation stabilizes channel opening and alters ion selectivity of Cav1.4 in a manner that is strengthened by exclusion of exon 47 and inclusion of β2X13 and α2δ-4. Our results reveal complex actions of IT in modifying the properties of Cav1.4 channels, which may influence the pathological consequences of this mutation in retinal photoreceptors.

Altered metal ion selectivity in signalling with heterocyclic tripodal receptor appended rhodamine-B derivatives

The rhodamine derivatives (2–7) were shown to exhibit metal ion complexation induced photophysical ‘turn-on’ signalling responses, where the selectivity in signalling were driven by stereo-electronic environment of the receptor units that structurally differ as derivatization of different heterocyclic units. The chromogenic and fluorogenic spectral enhancements of two order higher in logarithmic scale were observed in these tripodal receptor incorporated amino-ethyl-rhodamine probes, where substitution of a 2-[2-[(thiophen-2-yl-methyl)-(2-pyridinylmethyl)-amino]-ethyl]- (as in 3) has shown selectivity for Hg(II) ion, a 2-[2-[(furan-2-yl-methyl)-(2-pyridinyl methyl)-amino]-ethyl]- (as in 5) for Al(III) ion and a 2-[2-[(phen-2-ylmethyl)-(2-pyridinylmethyl)-amino]-ethyl]- (as in 7) for Fe(III) ion respectively. The higher order of association constants (log Ka > 9.0) of complexation, high sensitivity of detection(LOD < 2 nM) and higher rate of interaction between probe and metal ion (k ~ 0.001 s−1) have showed potential of these probes in selective detection of corresponding metal ions. In general, the investigations on complexation induced photophysical signalling in these probes in terms of selectivity and sensitivity of detection, association constants of complex formation, reversibility in signalling, execution at different pH range, kinetics of spiro-ring opening and theoretical structure elucidation have therefore put insights on their coordination mediated stereo-electronic environment to establish a rationality in structure-function correlation.

Zinc ameliorates intestinal barrier dysfunctions in shigellosis by reinstating claudin-2 and -4 on the membranes.

Whether zinc (Zn2+) regulates barrier functions by modulating tight-junction (TJ) proteins when pathogens such as Shigella alter epithelial permeability is still unresolved. We investigated the potential benefits of Zn2+ in restoring impaired barrier function in vivo in Shigella-infected mouse tissue and in vitro in T84 cell monolayers. Basolateral Shigella infection triggered a time-dependent decrease in transepithelial resistance followed by an increase in paracellular permeability of FITC-labeled dextran and altered ion selectivity. This led to ion and water loss into the intestinal lumen. Immunofluorescence studies revealed redistribution of claudin-2 and -4 to an intracellular location and accumulation of these proteins in the cytoplasm following infection. Zn2+ ameliorated this perturbed barrier by redistribution of claudin-2 and -4 back to the plasma membrane and by modulating the phosphorylation state of TJ proteins t hough extracellular signal-regulated kinase (ERK)1/2 dependency. Zn2+ prevents elevation of IL-6 and IL-8. Mice challenged with Shigella showed that oral Zn2+supplementation diminished diverse pathophysiological symptoms of shigellosis. Claudin-2 and -4 were susceptible to Shigella infection, resulting in altered barrier function and increased levels of IL-6 and IL-8. Zn2+ supplementation ameliorated this barrier dysfunction, and the inflammatory response involving ERK-mediated change of phosphorylation status for claudin-2 and -4. Thus, Zn2+ may have potential therapeutic value in inflammatory diarrhea and shigellosis. NEW & NOTEWORTHY Our study addresses whether Zn2+ could be an alternative strategy to reduce Shigella-induced inflammatory response and epithelial barrier dysfunction. We have defined a mechanism in terms of intracellular signaling pathways and tight-junction protein expression by Zn2+. Claudin-2 and -4 are susceptible to Shigella infection, whereas in the presence of Zn2+ they are resistant to infection-related barrier dysfunction involving ERK-mediated change of phosphorylation status of claudins.

A De Novo Mutation in the Sodium-Activated Potassium Channel KCNT2 Alters Ion Selectivity and Causes Epileptic Encephalopathy

Early infantile epileptic encephalopathies (EOEE) are a debilitating spectrum of disorders associated with cognitive impairments. We present a clinical report of a KCNT2 mutation in an EOEE patient. The de novo heterozygous variant Phe240Leu SLICK was identified by exome sequencing and confirmed by Sanger sequencing. Phe240Leu rSlick and hSLICK channels were electrophysiologically, heterologously characterized to reveal three significant alterations to channel function. First, [Cl-]i sensitivity was reversed in Phe240Leu channels. Second, predominantly K+-selective WT channels were made to favor Na+ over K+ by Phe240Leu. Third, and consequent to altered ion selectivity, Phe240Leu channels had larger inward conductance. Further, rSlick channels induced membrane hyperexcitability when expressed in primary neurons, resembling the cellular seizure phenotype. Taken together, our results confirm that Phe240Leu is a "change-of-function" KCNT2 mutation, demonstrating unusual altered selectivity in KNa channels. These findings establish pathogenicity of the Phe240Leu KCNT2 mutation in the reported EOEE patient.

SMIT1 Modifies KCNQ Channel Function and Pharmacology by Physical Interaction with the Pore

Voltage-gated potassium channels of the KCNQ (Kv7) subfamily are essential for control of cellular excitability and repolarization in a wide range of cell types. Recently, we and others found that some KCNQ channels functionally and physically interact with sodium-dependent solute transporters, including myo-inositol transporters SMIT1 and SMIT2, potentially facilitating various modes of channel-transporter signal integration. In contrast to indirect effects such as channel regulation by SMIT-transported, myo-inositol-derived phosphatidylinositol 4,5-bisphosphate (PIP2), the mechanisms and functional consequences of the physical interaction of channels with transporters have been little studied. Here, using co-immunoprecipitation with different channel domains, we found that SMIT1 binds to the KCNQ2 pore module. We next tested the effects of SMIT1 co-expression, in the absence of extracellular myo-inositol or other SMIT1 substrates, on fundamental functional attributes of KCNQ2, KCNQ2/3, KCNQ1, and KCNQ1-KCNE1 channels. Without exception, SMIT1 altered KCNQ ion selectivity, sensitivity to extracellular K+, and pharmacology, consistent with an impact on conformation of the KCNQ pore. SMIT1 also altered the gating kinetics and/or voltage dependence of KCNQ2, KCNQ2/3, and KCNQ1-KCNE1. In contrast, SMIT1 had no effect on Kv1.1 (KCNA1) gating, ion selectivity, or pharmacology. We conclude that, independent of its transport activity and indirect regulatory mechanisms involving inositol-derived increases in PIP2, SMIT1, and likely other related sodium-dependent solute transporters, regulates KCNQ channel ion selectivity, gating, and pharmacology by direct physical interaction with the pore module.

Pattern recognition channels in anti-viral host defense in humans and an insect vector

Abstract A novel mechanism of innate immune signaling is proposed. The central hypothesis is that viral-associated molecular patterns directly activate intracellular splice variants of voltage-gated sodium channels to increase transcription of anti-viral genes. Past work revealed that splice variants of human voltage-gated sodium channels are expressed intracellularly on endosomes and contain exon deletions that alter channel kinetics. Here it is shown that intracellular dsRNA activates the human macrophage SCN5A variant to initiate a signaling cascade that leads to increased transcription of type I interferons and SP100-related genes. This pathway is regulated by sequential downstream activation of a calcium-dependent adenylate cyclase, adcy8, and the cyclic AMP-dependent transcription factor ATF-2. Knockdown of channel expression in human macrophages reduces the host response to HSV-1. Additional work reveals expression of an evolutionarily related sodium channel gene, AAEL006019, in Aedes aegypti, the yellow fever mosquito. This intracellular channel contains an alternative exon deletion that reduces peak current but does not alter ion selectivity. In contrast to human macrophage SCN5A, the AAEL006019 channel is activated by mimics of virally-derived ssRNA, but not dsRNA. Channel activation increases transcription of mediators of insect innate immunity, RelA and Ago2. These data suggest that channel variants initiate anti-viral responses independently of other pattern recognition receptor pathways. It is postulated that pattern recognition by channel variants are an evolutionarily conserved mechanism of innate immune signaling that reflect a complex interplay between human host, vector, and virus.

SCN4A Pore Mutation Pathogenetically Contributes to Autosomal Dominant Essential Tremor and May Increase Susceptibility to Epilepsy

Essential tremor (ET) is a very wide spread movement disorder with unknown genetic causes. A positive family history is a highly risk factor for ET, affecting millions of people alone in the United States. To identify the genetic cause of ET, we performed whole-exom sequencing analyses in a large Spanish family with ET, where two patients also develop epilepsy. A disease-segregating mutation (p.Gly1537Ser) in the SCN4A gene was identified and analyzed functional with the patch clamp technique. We found more rapid kinetics at near-threshold potentials, altered ion selectivity and facilitated conductance of potassium and ammonium ions, which could contribute to tremor and increase susceptibility to epilepsy, respectively. We associated for the first time the genetic variability of SCN4A with the development of essential tremor, adding it to the growing list of neurological channelopathies.

Metal-binding and redox properties of substituted linear and cyclic ATCUN motifs

The amino-terminal copper and nickel binding (ATCUN) motif is a short peptide sequence found in human serum albumin and other proteins. Synthetic ATCUN–metal complexes have been used to oxidatively cleave proteins and DNA, cross-link proteins, and damage cancer cells. The ATCUN motif consists of a tripeptide that coordinates Cu(II) and Ni(II) ions in a square planar geometry, anchored by chelation sites at the N-terminal amine, histidine imidazole and two backbone amides. Many studies have shown that the histidine is required for tight binding and square planar geometry. Previously, we showed that macrocyclization of the ATCUN motif can lead to high-affinity binding with altered metal ion selectivity and enhanced Cu(II)/Cu(III) redox cycling (Inorg. Chem. 2013, 52, 2729–2735). In this work, we synthesize and characterize several linear and cyclic ATCUN variants to explore how substitutions at the histidine alter the metal-binding and catalytic properties. UV–visible spectroscopy, EPR spectroscopy and mass spectrometry indicate that cyclization can promote the formation of ATCUN-like complexes even in the absence of imidazole. We also report several novel ATCUN-like complexes and quantify their redox properties. These findings further demonstrate the effects of conformational constraints on short, metal-binding peptides, and also provide novel redox-active metallopeptides suitable for testing as catalysts for stereoselective or regioselective oxidation reactions.

Ste20-like proline/alanine-rich kinase (SPAK) deficiency exacerbates experimental colitis in mouse (CAM1P.239)

Abstract Inflammatory bowel diseases (IBDs) are characterized by epithelial barrier disruption and alterations in immune regulation. Ste20-like proline/alanine-rich kinase (SPAK) plays a role in intestinal inflammation, but the underlying mechanisms need to be defined. Here, SPAK knockout (KO) C57BL/6 mice exhibited significant increase in translocation of intestinal bacteria into mucosa, accompanying with marked elevation in paracellular permeability to FITC-dextran and altered apical side tight junction sodium ion selectivity, compared to wild type (WT) mice. In murine models of colitis induced by dextran sulfate sodium (DSS) and trinitrobenzene sulfuric acid (TNBS), KO mice were more sensitive than WT mice, as demonstrated by colonoscopy features, histological characteristics and MPO activities. Consistent with these findings, KO mice showed decreased IL-10 levels and increased proinflammatory cytokine secretion. Co-immunoprecipitation and immunohistochemical assays demonstrated that SPAK is associated with PKCtheta and CD46, regulators of regulatory T cell differentiation and activation. Flow cytometry analysis found that KO mice had significant lower amount of T regulatory 1 (Tr1) cells in comparison with WT mice. In conclusions, SPAK deficiency compromises intestinal innate immune homeostasis, implying its important role as a potential intervention target against pathological responses in IBD.

Knockout of Ste20-Like Proline/Alanine-Rich Kinase (SPAK) Attenuates Intestinal Inflammation in Mice

Inflammatory bowel diseases are characterized by epithelial barrier disruption and alterations in immune regulation. Ste20-like proline/alanine-rich kinase (SPAK) plays a role in intestinal inflammation, but the underlying mechanisms need to be defined. Herein, SPAK knockout (KO) C57BL/6 mice exhibited significant increases in intestinal transepithelial resistance, a marked decrease in paracellular permeability to fluorescence isothiocyanate-dextran, and altered apical side tight junction sodium ion selectivity, compared with wild-type mice. Furthermore, the expression of junction protein, claudin-2, decreased. In contrast, expressions of occludin, E-cadherin, β-catenin, and claudin-5 increased significantly, whereas no obvious change of claudin-1, claudin-4, zonula occludens protein 1, and zonula occludens protein 2 expressions was observed. In murine models of colitis induced by dextran sulfate sodium and trinitrobenzene sulfuric acid, KO mice were more tolerant than wild-type mice, as demonstrated by colonoscopy features, histological characteristics, and myeloperoxidase activities. Consistent with these findings, KO mice showed increased IL-10 levels and decreased proinflammatory cytokine secretion, ameliorated bacterial translocation on treatment with dextran sulfate sodium, and regulation of with no lysine (WNK) kinase activity. Together, these features may reduce epithelial permeability. In conclusion, SPAK deficiency increases intestinal innate immune homeostasis, which is important for control or attenuation of pathological responses in inflammatory boweldiseases.

β-Barrel Mobility Underlies Closure of the Voltage-Dependent Anion Channel

The voltage-dependent anion channel (VDAC) is themajor protein in the outer mitochondrial membrane, where it mediates transport of ATP and ADP. Changes in its permeability, induced by voltage or apoptosis-related proteins, have been implicated in apoptotic pathways. The three-dimensional structure of VDAC has recently been determined asa 19-stranded β-barrel with an in-lying N-terminal helix. However, its gating mechanism is still unclear. Using solid-state NMR spectroscopy, molecular dynamics simulations, and electrophysiology, we show that deletion of the rigid N-terminal helix sharply increases overall motion in VDAC's β-barrel, resulting in elliptic, semicollapsed barrel shapes. These states quantitatively reproduce conductance and selectivity of the closed VDAC conformation. Mutation of the N-terminal helix leads to a phenotype intermediate to the open and closed states. These data suggest that the N-terminal helix controls entry into elliptic β-barrel states which underlie VDAC closure. Our results also indicate that β-barrel channels are intrinsically flexible.

SARAF Inactivates the Store Operated Calcium Entry Machinery to Prevent Excess Calcium Refilling

Store operated calcium entry (SOCE) is a principal cellular process by which cells regulate basal calcium, refill intracellular Ca(2+) stores, and execute a wide range of specialized activities. STIM and Orai proteins have been identified as the essential components enabling the reconstitution of Ca(2+) release-activated Ca(2+) (CRAC) channels that mediate SOCE. Here, we report the molecular identification of SARAF as a negative regulator of SOCE. Usingheterologous expression, RNAi-mediated silencing and site directed mutagenesis combined with electrophysiological, biochemical and imaging techniques we show that SARAF is an endoplasmic reticulum membrane resident protein that associates with STIM to facilitate slow Ca(2+)-dependent inactivation of SOCE. SARAF plays a key role in shaping cytosolic Ca(2+) signals and determining the content of the major intracellular Ca(2+) stores, a role that is likely to be important in protecting cells from Ca(2+) overfilling.

The Pore of the Voltage-Gated Proton Channel

In classical tetrameric voltage-gated ion channels four voltage-sensing domains (VSDs), one from each subunit, control one ion permeation pathway formed by four pore domains. The human Hv1 proton channel has a different architecture, containinga VSD, but lacking a pore domain. Since its location is not known, we searched for the Hv permeation pathway. We find that mutation of the S4 segment's third arginine R211 (R3) compromises proton selectivity, enabling conduction of a metal cation and even of the large organic cation guanidinium, reminiscent of Shaker's omega pore. In the open state, R3 appears to interact with an aspartate (D112) that is situated in the middle of S1 and is unique to Hv channels. The double mutation of both residues further compromises cation selectivity. We propose that membrane depolarization reversibly positions R3 next to D112 inthe transmembrane VSD to form the ion selectivity filter in the channel's open conformation.

Molecular Coupling in the Human ether-a-go-go-related gene-1 (hERG1) K+ Channel Inactivation Pathway

Emerging evidence suggests that K(+) channel inactivation involves coupling between residues in adjacent regions of the channel. Human ether-a-go-go-related gene-1 (hERG1) K(+) channels undergo a fast inactivation gating process that is crucial for maintaining electrical stability in the heart. The molecular mechanisms that drive inactivation in hERG1 channels are unknown. Using alanine scanning mutagenesis, we show that a pore helix residue (Thr-618) that points toward the S5 segment is critical for normal inactivation gating. Amino acid substitutions at position 618 modulate the free energy of inactivation gating, causing enhanced or reduced inactivation. Mutation of an S5 residue that is predicted to be adjacent to Thr-618 (W568L) abolishes inactivation and alters ion selectivity. The introduction of the Thr-618-equivalent residue in Kv1.5 enhances inactivation. Molecular dynamic simulations of the Kv1.2 tetramer reveal van der Waals coupling between hERG1 618- and 568-equivalent residues and a significant increase in interaction energies when threonine is introduced at the 618-equivalent position. We propose that coupling between the S5 segment and pore helix may participate in the inactivation process in hERG1 channels.

The Third Transmembrane Segment of Orai1 Protein Modulates Ca2+ Release-activated Ca2+ (CRAC) Channel Gating and Permeation Properties

Orai1, the pore subunit of Ca(2+) release-activated Ca(2+) channels, has four transmembrane segments (TMs). The first segment, TMI, lines the pore and plays an important role in channel activation and ion permeation. TMIII, on the other hand, does not line the pore but still regulates channel gating and permeation properties. To understand the role of TMIII, we have mutated and characterized several residues in this domain. Mutation of Trp-176 to Cys (W176C) and Gly-183 to Ala (G183A) had dramatic effects. Unlike wild-type channels, which exhibit little outward current and are activated by STIM1, W176C mutant channels exhibited a large outward current at positive potentials and were constitutively active in the absence of STIM1. G183A mutant channels also exhibited substantial outward currents but were active only in the presence of 2-aminoethoxydiphenyl borate (2-APB), irrespective of STIM1. With W176C mutant channels inward, monovalent currents were blocked by Ca(2+) with a high affinity similar to the wild type, but the Ca(2+)-dependent blocking of outward currents differed in the two cases. Although a 50% block of the WT outward current required 250 μm Ca(2+), more than 6 mm was necessary to have the same effect on W176C mutant channels. In the presence of extracellular Ca(2+), W176C and G183A outward currents developed slowly in a voltage-dependent manner, whereas they developed almost instantaneously in the absence of Ca(2+). These changes in permeation and gating properties mimic the changes induced by mutations of Glu-190 in TMIII and Asp-110/Asp-112 in the TMI/TMII loop. On the basis of these data, we propose that TMIII maintains negatively charged residues at or near the selectivity filter in a conformation that facilitates Ca(2+) inward currents and prevents outward currents of monovalent cations. In addition, to controlling selectivity, TMIII may also stabilize channel gating in a closed state in the absence of STIM1 in a Trp-176-dependent manner.

Voltage-gated sodium channel (NaV) protein dissection creates a set of functional pore-only proteins

Many voltage-gated ion channel (VGIC) superfamily members contain six-transmembrane segments in which the first four form a voltage-sensing domain (VSD) and the last two form the pore domain (PD). Studies of potassium channels from the VGIC superfamily together with identification of voltage-sensor only proteins have suggested that the VSD and the PD can fold independently. Whether such transmembrane modularity is common to other VGIC superfamily members has remained untested. Here we show, using protein dissection, that the Silicibacter pomeroyi voltage-gated sodium channel (Na(V)Sp1) PD forms a stand-alone, ion selective pore (Na(V)Sp1p) that is tetrameric, α-helical, and that forms functional, sodium-selective channels when reconstituted into lipid bilayers. Mutation of the Na(V)Sp1p selectivity filter from LESWSM to LDDWSD, a change similar to that previously shown to alter ion selectivity of the bacterial sodium channel Na(V)Bh1 (NaChBac), creates a calcium-selective pore-only channel, Ca(V)Sp1p. We further show that production of PDs can be generalized by making pore-only proteins from two other extremophile Na(V)s: one from the hydrocarbon degrader Alcanivorax borkumensis (Na(V)Ab1p), and one from the arsenite oxidizer Alkalilimnicola ehrlichei (Na(V)Ae1p). Together, our data establish a family of active pore-only ion channels that should be excellent model systems for study of the factors that govern both sodium and calcium selectivity and permeability. Further, our findings suggest that similar dissection approaches may be applicable to a wide range of VGICs and, thus, serve as a means to simplify and accelerate biophysical, structural, and drug development efforts.

Structural Basis of an Inherited hERG1 Long QT Mutant

Mutations in human ether-a-go-go related K+ channel gene hERG1 are a common cause of Long QT syndrome (LQTS), an electrical disorder of the heart that predisposes individuals to sudden death. Of these, changes to the pore domain are associated with a greater risk of life-threatening arrhythmias. One such mutation N629D, located adjacent to the selectivity filter, causes loss of function and altered ion selectivity. We have shown that the selectivity filter sequence of non-selective cation channel NaK shows striking similarity to that of hERG1, and that mutagenic transformation of the NaK filter into hERG1's generates a structurally canonical K+ filter. Here, using this hERG1-like NaK as a structural model, we reveal the molecular mechanisms that underlie channel dysfunction in the N629D mutant. The NaK/hERG N629D filter crystal structure obtained at 2.8 A show that N629D induces a dramatic conformational rearrangements in the extracellular vestibule that extend to the filter, disrupting K+ binding sites to cause loss of ion selectivity. These changes involve a 180 deg rotation of the Phenylalanine 66 at the selectivity filter (equivalent to position Phe627 in hERG1) and an intrinsic sensitivity to the nature of the permeant ion. These findings provide insight into the hERG1 selectivity filter and offer a new biophysical tool for the study of the molecular mechanisms underlying LQTS pore mutants at atomic level.

Computational Electrophysiology on Vdac-1 reveals Mechanism of Anion Flux

The voltage-gated anion channel VDAC-1 in mitochondrial outer membranes is the principal passageway between mitochondria and the cytoplasm, transferring ATP, the major source of chemical energy in cells. It is also implicated in rupture of the mitochondrial membrane during mitochondria-dependent cell apoptosis. We used our newly developed method, computational electrophysiology, capable of modelling detailed ion transfer processes under steady flux conditions in molecular dynamics simulations, to study VDAC-1 embedded in lipid bilayers. In particular, we investigated how changes in transmembrane voltage, alterations in membrane state, functionally important mutations, and the intrinsic dynamics of VDAC-1 impact on channel current and how it is related to the surrounding bilayer. We found that the beta-barrel structure of VDAC-1 is unexpectedly dynamic, owing to the complete lack of a hydrophobic core, and undergoes large elliptic deformations. Strikingly, the N-terminal helix proves to be the most rigid section of the protein and confers stability to the global structure. Its removal results in greatly enhanced fluctuations and can give rise to a partial collapse of the barrel, especially if coupled to induced shock waves in the membrane. In electrophysiology simulations, the partially collapsed states exhibit subconductance behavior and altered ion selectivity.

Epithelial Myosin Light Chain Kinase Activation Induces Mucosal Interleukin-13 Expression to Alter Tight Junction Ion Selectivity

Intestinal barrier function is reduced in inflammatory bowel disease (IBD). Tumor necrosis factor (TNF) and interleukin (IL)-13, which are up-regulated in IBD, induce barrier defects that are associated with myosin light chain kinase (MLCK) activation and increased claudin-2 expression, respectively, in cultured intestinal epithelial monolayers. Here we report that these independent signaling pathways have distinct effects on tight junction barrier properties and interact in vivo. MLCK activation alters size selectivity to enhance paracellular flux of uncharged macromolecules without affecting charge selectivity and can be rapidly reversed by MLCK inhibition. In contrast, IL-13-dependent claudin-2 expression increases paracellular cation flux in vitro and in vivo without altering tight junction size selectivity but is unaffected by MLCK inhibition in vitro. In vivo, MLCK activation increases paracellular flux of uncharged macromolecules and also triggers IL-13 expression, claudin-2 synthesis, and increased paracellular cation flux. We conclude that reversible, MLCK-dependent permeability increases cause mucosal immune activation that, in turn, feeds back on the tight junction to establish long-lasting barrier defects. Interactions between these otherwise distinct tight junction regulatory pathways may contribute to IBD pathogenesis.

Stim-Dependent and Independent Effects of 2-APB on Orai3 Crac Channels

The compound 2-aminoethyldiphenyl borate (2-APB) has received widespread attention for its ability to modulate store-operated CRAC channels. 2-APB elicits complex effects in native and ectopic CRAC channels arising from the over-expression of Orai1 (the pore subunit), causing a several-fold enhancement of ICRAC at low concentrations (20 μM). However, recent studies indicate that 2-APB produces strikingly different effects in the Orai3 variant. Here, high 2-APB concentrations activate (rather than inhibit) Orai3 channels. Moreover, the 2-APB activated Orai3 currents differ from store-operated Orai3 (and Orai1) currents in manifesting altered ion selectivity. The multiplicity of 2-APB effects in the different Orai isoforms has confounded efforts to understand its mode of action. Here, we find that 2-APB (50 μM) induces Orai3 current in two kinetically distinct phases: an initial increase in current with no change in ion selectivity is followed by secondary activation of Orai3 channels with altered ion selectivity. Lower concentrations of 2-APB (< 10 μM) potentiated Orai3 currents with no change in ion selectivity, resembling effects seen in Orai1. In contrast to the activation of Orai3 channels by high concentrations of 2-APB, the potentiation by low concentrations of 2-APB was entirely dependent on STIM1. High concentrations of 2-APB also eliminated fast Ca2+-dependent inactivation of Orai3 currents. Collectively, our results indicate that as seen with Orai1 and native CRAC channels, 2-APB causes dual effects on Orai3 channels: low concentrations potentiate Orai3 currents with no change in ion selectivity, whereas high concentrations activate Orai3 currents while also altering ion selectivity and removing fast inactivation. Our results suggest that the complex effects of 2-APB on Orai1 and Orai3 channels share common mechanisms.