Slc26a7 Chloride Channel Activity and Localization in Mouse Reissner’s Membrane Epithelium

Several members of the SLC26 gene family have highly-restricted expression patterns in the auditory and vestibular periphery and mutations in mice of at least two of these (SLC26A4 and SLC26A5) lead to deficits in hearing and/or balance. A previous report pointed to SLC26A7 as a candidate gene important for cochlear function. In the present study, inner ears were assayed by immunostaining for Slc26a7 in neonatal and adult mice. Slc26a7 was detected in the basolateral membrane of Reissner’s membrane epithelial cells but not neighboring cells, with an onset of expression at P5; gene knockout resulted in the absence of protein expression in Reissner’s membrane. Whole-cell patch clamp recordings revealed anion currents and conductances that were elevated for NO₃ ⁻ over Cl⁻ and inhibited by I⁻ and NPPB. Elevated NO₃ ⁻ currents were absent in Slc26a7 knockout mice. There were, however, no major changes to hearing (auditory brainstem response) of knockout mice during early adult life under constitutive and noise exposure conditions. The lack of Slc26a7 protein expression found in the wild-type vestibular labyrinth was consistent with the observation of normal balance. We conclude that SLC26A7 participates in Cl⁻ transport in Reissner’s membrane epithelial cells, but that either other anion pathways, such as ClC-2, possibly substitute satisfactorily under the conditions tested or that Cl⁻ conductance in these cells is not critical to cochlear function. The involvement of SLC26A7 in cellular pH regulation in other epithelial cells leaves open the possibility that SLC26A7 is needed in Reissner’s membrane cells during local perturbations of pH.     

Putative ClC-2 Chloride Channel Mediates Inward Rectification in <Emphasis Type="Italic">Drosophila </Emphasis>Retinal Photoreceptors

We report that Drosophila retinal photoreceptors express inwardly rectifying chloride channels that seem to be orthologous to mammalian ClC-2 inward rectifier channels. We measured inwardly rectifying Cl⁻ currents in photoreceptor plasma membranes: Hyperpolarization under whole-cell tight-seal voltage clamp induced inward Cl⁻ currents; and hyperpolarization of voltage-clamped inside-out patches excised from plasma membrane induced Cl⁻ currents that have a unitary channel conductance of ∼3.7 pS. The channel was inhibited by 1 mM Zn²⁺ and by 1 mM 9-anthracene, but was insensitive to DIDS. Its anion permeability sequence is Cl⁻ = SCN⁻> Br⁻>> I⁻, characteristic of ClC-2 channels. Exogenous polyunsaturated fatty acid, linolenic acid, enhanced or activated the inward rectifier Cl⁻ currents in both whole-cell and excised patch-clamp recordings. Using RT-PCR, we found expression in Drosophila retina of a ClC-2 gene orthologous to mammalian ClC-2 channels. Antibodies to rat ClC-2 channels labeled Drosophila photoreceptor plasma membranes and synaptic regions. Our results provide evidence that the inward rectification in Drosophila retinal photoreceptors is mediated by ClC-2-like channels in the non-transducing (extra-rhabdomeral) plasma membrane, and that this inward rectification can be modulated by polyunsaturated fatty acid. G. Ugarte and R. Delgado contributed equally to this work.     

Anion- and proton-dependent gating of ClC-4 anion/proton transporter under uncoupling conditions

ClC-4 is a secondary active transporter that exchanges Cl⁻ ions and H⁺ with a 2:1 stoichiometry. In external SCN⁻, ClC-4 becomes uncoupled and transports anions with high unitary transport rate. Upon voltage steps, the number of active transporters varies in a time-dependent manner, resembling voltage-dependent gating of ion channels. We here investigated modification of the voltage dependence of uncoupled ClC-4 by protons and anions to quantify association of substrates with the transporter. External acidification shifts voltage dependence of ClC-4 transport to more positive potentials and leads to reduced transport currents. Internal pH changes had less pronounced effects. Uncoupled ClC-4 transport is facilitated by elevated external [SCN⁻] but impaired by internal Cl⁻ and I⁻. Block by internal anions indicates the existence of an internal anion-binding site with high affinity that is not present in ClC channels. The voltage dependence of ClC-4 coupled transport is modulated by external protons and internal Cl⁻ in a manner similar to what is observed under uncoupling conditions. Our data illustrate functional differences but also similarities between ClC channels and transporters.     

Na+-independent,electrogenic Cl- uptake across the posterior gills of the Chinese crab (Eriocheir sinensis): Voltage-clamp and microelectrode studies

Summary Single gill lamellae from posterior gills of Chinese crabs (Eriocheir sinensis) were isolated, separated into halves and mounted in a modified Ussing chamber. Area-related short-circuit current (I_sc) and conductance (G_tot) of this preparation were measured. Epithelial cells were impaled with microelectrodes through the basolateral membrane and cellular potentials (V_i under open- and V_sc under short-circuit conditions) as well as the voltage divider ratios (F_i, F_o) were determined.With NaCl salines on both sides an outside positive PD_te (22±2 mV) and an I_sc (-64±13 A·cm^-2) with a polarity corresponding to an uptake of negative charges (inward negative) were obtained. Trough-like potential profiles were recorded across the preparation under open- as well as short-circuit conditions (V_o=-101±5 mV, external bath as reference; V_i=-78±2 mV, internal bath as reference; V_sc=-80±2 mV, extracellular space as reference). The voltage divider ratios of the external (apical membrane plus cuticle) and internal (basolateral membrane) barrier were F_o=0.92±0.01 and F_i=0.08±0.01, respectively. To investigate a Cl^--related contribution to the above parameters, Na⁺-free solutions in the external bath (basolateral NaCl-saline) were used. Inward negative I_sc under these conditions almost completely depended on external Cl^-. Elimination of Cl^- in the external bath reversed I_sc, and G_tot decreased substantially. Concomitantly, V_sc depolarised and F_o increased. Cl^--dependent current and conductance showed saturation kinetics with increasing external [Cl^-]. Addition of 20 mmol·1^-1 thiocyanate to the external bath had similar, although less pronounced, effects as Cl^- substitution. Equally, external SITS (1 mmol·1^-1) inhibited the current and, concomitantly, G_tot decreased substantially. Addition of 1 mmol·1^-1 acetazolamide to, and omission of NaHCO₃ from, the basolateral bath resulted in a decrease of I_sc while G_tot remained unchanged. The Cl^--channel blocker DPC inhibited I_sc almost completely when added to the basolateral saline, whereas G_tot decreased moderately; however, V_sc depolarised without significant change of F_i. Ouabain had no influence on I_sc and G_tot. Increasing the basolateral [K⁺] resulted in a decrease in I_sc, while G_tot was not affected. At the same time V_sc largely depolarised and F_i decreased. Addition of the K⁺-channel blocker Ba⁺⁺ (5 mmol·1^-1) to the basolateral solution resulted in a two-step alteration of the transepithelial (I_sc, Gtot) and cellular (V_sc, F_i) parameters. The results are discussed with regard to (i) the mechanisms responsible for active transbranchial Cl^- uptake, and (ii) the technical improvement of being able to perform transport studies with crab gill preparations in an Ussing chamber.Abbreviations DMSO dimethylsulfoxide - DPC diphenylamine-2-carboxylate - F _{o, i} voltage divider ratio for external (o) and internal (i) barrier, respectively - G _Cl conductance related to the external [Cl^-] - G _tot total tissue conductance - I _Cl short-circuit current related to the external [Cl^-] - I _sc short-circuit current - PD _te transepithelial potential difference - R _ME resistance of the microelectrode - SITS 4-acetamido-4-isothiocyanato-stilbene-2,2-disulfonic acid - V _{o, i} open-circuit voltage across the external (o) and internal (i) barrier, respectively - V _sc intracellular potential under short-circuit conditions     

Mechanism of Cl- sensitivity in internal ion receptors of the leech; an inward current gated off by Cl- in the nephridial nerve cells

Summary The nephridial nerve cells of the leech, Hirudo medicinalis, 34 sensory cells, each associated with one nephridium, are sensitive to changes in extracellular Cl^- concentration, an important factor in ion homeostasis. Using single-electrode current- and voltage clamp and ion substitution techniques, the specificity and mechanism of Cl^- sensitivity of the nephridial nerve cell was studied in isolated preparations. Increase of the normally low external Cl^- concentration leads to immediate and sustained hyperpolarization, decrease of the frequency of bursts and decrease of membrane conductance. The response is halogen specific: Cl^- can be replaced by Br^–, but not by organic mono- or divalent anions or inorganic divalent anions.At physiological Cl^- concentrations (36mM extra-cellular Cl^-), the nephridial nerve cell has a high resting conductance for Cl^- and the membrane potential is governed by Cl^-. In high extracellular Cl^- concentrations (110–130 mM), membrane conductance is low, most likely due to the gating off of Cl^- channels. Under these conditions, membrane potential is dominated by the K⁺ distribution and the nephridial nerve cell hyperpolarizes towards E_K.Abbreviations NNC nephridial nerve cell - V _m membrane potential - E _Cl(k) equilibrium potential for Cl (K) - IV-curve current-voltage relationship     

Ca2+-Activated Cl? Channels of the ClCa Family Express in the Cilia of a Subset of Rat Olfactory Sensory Neurons

The Ca²⁺-activated Cl⁻ channel is considered a key constituent of odor transduction. Odorant binding to a specific receptor in the cilia of olfactory sensory neurons (OSNs) triggers a cAMP cascade that mediates the opening of a cationic cyclic nucleotide-gated channel (CNG), allowing Ca²⁺ influx. Ca²⁺ ions activate Cl⁻ channels, generating a significant Cl⁻ efflux, with a large contribution to the receptor potential. The Anoctamin 2 channel (ANO2) is a major constituent of the Cl⁻ conductance, but its knock-out has no impairment of behavior and only slightly reduces field potential odorant responses of the olfactory epithelium. Likely, an additional Ca²⁺-activated Cl⁻ channel of unknown molecular identity is also involved. In addition to ANO2, we detected two members of the ClCa family of Ca²⁺-activated Cl⁻ channels in the rat olfactory epithelium, ClCa4l and ClCa2. These channels, also expressed in the central nervous system, may correspond to odorant transduction channels. Whole Sprague Dawley olfactory epithelium nested RT-PCR and single OSNs established that the mRNAs of both channels are expressed in OSNs. Real time RT-PCR and full length sequencing of amplified ClCa expressed in rat olfactory epithelium indicated that ClCa4l is the most abundant. Immunoblotting with an antibody recognizing both channels revealed immunoreactivity in the ciliary membrane. Immunochemistry of olfactory epithelium and OSNs confirmed their ciliary presence in a subset of olfactory sensory neurons. The evidence suggests that ClCa4l and ClCa2 might play a role in odorant transduction in rat olfactory cilia.     

A dynamic model of saliva secretion

We construct a mathematical model of the parotid acinar cell with the aim of investigating how the distribution of K⁺ and Cl⁻ channels affects saliva production. Secretion of fluid is initiated by Ca²⁺ signals acting on Ca²⁺ dependent K⁺ and Cl⁻ channels. The opening of these channels facilitates the movement of Cl⁻ ions into the lumen which water follows by osmosis. We use recent results into both the release of Ca²⁺ from internal stores via the inositol (1,4,5)-trisphosphate receptor (IP₃R) and IP₃ dynamics to create a physiologically realistic Ca²⁺ model which is able to recreate important experimentally observed behaviours seen in parotid acinar cells. We formulate an equivalent electrical circuit diagram for the movement of ions responsible for water flow which enables us to calculate and include distinct apical and basal membrane potentials to the model. We show that maximum saliva production occurs when a small amount of K⁺ conductance is located at the apical membrane, with the majority in the basal membrane. The maximum fluid output is found to coincide with a minimum in the apical membrane potential. The traditional model whereby all Cl⁻ channels are located in the apical membrane is shown to be the most efficient Cl⁻ channel distribution.     

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.     

The bestrophin- and TMEM16A-associated Ca2+-activated Cl– channels in vascular smooth muscles

The presence of Ca²⁺-activated Cl^– currents (I_Cl(Ca)) in vascular smooth muscle cells (VSMCs) is well established. I_Cl(Ca) are supposedly important for arterial contraction by linking changes in [Ca²⁺]_i and membrane depolarization. Bestrophins and some members of the TMEM16 protein family were recently associated with I_Cl(Ca). Two distinct I_Cl(Ca) are characterized in VSMCs; the cGMP-dependent I_Cl(Ca) dependent upon bestrophin expression and the ‘classical’ Ca²⁺-activated Cl^– current, which is bestrophin-independent. Interestingly, TMEM16A is essential for both the cGMP-dependent and the classical I_Cl(Ca). Furthermore, TMEM16A has a role in arterial contraction while bestrophins do not. TMEM16A’s role in the contractile response cannot be explained however only by a simple suppression of the depolarization by Cl^– channels. It is suggested that TMEM16A expression modulates voltage-gated Ca²⁺ influx in a voltage-independent manner and recent studies also demonstrate a complex role of TMEM16A in modulating other membrane proteins.     

Extracellular Zinc Ion Inhibits ClC-0 Chloride Channels by Facilitating Slow Gating

Extracellular Zn²⁺ was found to reversibly inhibit the ClC-0 Cl⁻ channel. The apparent on and off rates of the inhibition were highly temperature sensitive, suggesting an effect of Zn²⁺ on the slow gating (or inactivation) of ClC-0. In the absence of Zn²⁺, the rate of the slow-gating relaxation increased with temperature, with a Q₁₀ of ∼37. Extracellular Zn²⁺ facilitated the slow-gating process at all temperatures, but the Q₁₀ did not change. Further analysis of the rate constants of the slow-gating process indicates that the effect of Zn²⁺ is mostly on the forward rate (the rate of inactivation) rather than the backward rate (the rate of recovery from inactivation) of the slow gating. When ClC-0 is bound with Zn²⁺, the equilibrium constant of the slow-gating process is increased by ∼30-fold, reflecting a 30-fold higher Zn²⁺ affinity in the inactivated channel than in the open-state channel. As examined through a wide range of membrane potentials, Zn²⁺ inhibits the opening of the slow gate with equal potency at all voltages, suggesting that a two-state model is inadequate to describe the slow-gating transition. Following a model originally proposed by Pusch and co-workers (Pusch, M., U. Ludewig, and T.J. Jentsch. 1997. J. Gen. Physiol. 109:105–116), the effect of Zn²⁺ on the activation curve of the slow gate can be well described by adding two constraints: (a) the dissociation constant for Zn²⁺ binding to the open channel is 30 μM, and (b) the difference in entropy between the open state and the transition state of the slow-gating process is increased by 27 J/ mol/°K for the Zn²⁺-bound channel. These results together indicate that extracellular Zn²⁺ inhibits ClC-0 by facilitating the slow-gating process.     

Characterization of whole-cell k<Superscript>+</Superscript> currents across the plasma membrane of pollen grain and tube protoplasts of <Emphasis Type="Italic">Lilium longiflorum</Emphasis>

Outward and inward currents, mainly carried by K⁺, were detected in protoplasts of pollen grains (PG) and pollen tubes (PT) of Lilium longiflorum Thunb. by using the whole-cell configuration of the patch-clamp technique. The outward K⁺ current (I_{K+ out}) was similar in both protoplast types, while the inward K⁺ current (I_{K+ in}) was higher in pollen tube protoplasts. In PT but not in PG protoplasts, inward K⁺ currents were already detectable at negative membrane voltages usually monitored in lily pollen. I_{K+ in} consisted of a slow and a fast current component, as revealed by fitting a sum of two exponential functions to the time-dependent current. The contribution of the fast component to the total inward current was higher in PT than in PG protoplasts, which was even more evident at acidic pH of the external medium. Therefore, based on the measured characteristics, the I_{K+ in} of PT protoplasts may contribute to the endogenous K⁺ currents surrounding a growing pollen tube. Abbreviations: BS, bath solution; BTP, bis-Tris-propane; MES, 2-N-morpholinoethane sulfonic acid; V_act, activation voltage; V_M, membrane voltage; E_rev, reversal potential; I_{K+ in}, inward K⁺ current; I_{K+ out}, outward K⁺ current; PG, pollen grain; PT, pollen tube; PM, pipette medium     

Characterization of sarcoplasmic reticulum Ca2+ ATPase nucleotide binding domain mutants using NMR spectroscopy

T-type Ca²⁺ channels have been implicated in tremorogenesis and motor coordination. The α1 subunit of the Ca_V3.1 T-type Ca²⁺ channel is highly expressed in motor pathways in the brain, but knockout of the Ca_V3.1 gene (α1G^-/-) per se causes no motor defects in mice. Thus, the role of Ca_V3.1 channels in motor control remains obscure in vivo. Here, we investigated the effect of the Ca_V3.1 knockout in the null genetic background of α1 GABA_A receptor (α1^−/−) by generating the double mutants (α1^−/−/α1G^-/-). α1^−/−/α1G^-/- mice showed severer motor abnormalities than α1^−/− mice as measured by potentiated tremor activities at 20 Hz and impaired motor learning. Propranolol, an anti-ET drug that is known to reduce the pathologic tremor in α1^−/− mice, was not effective for suppressing the potentiated tremor in α1^−/−/α1G^-/- mice. In addition, α1^−/−/α1G^-/- mice showed an age-dependent loss of cerebellar Purkinje neurons. These results suggest that α1^−/−/α1G^-/- mice are a novel mouse model for a distinct subtype of ET in human and that Ca_V3.1 T-type Ca²⁺ channels play a role in motor coordination under pathological conditions.     

Suppression of L-type Ca current by fluid pressure in rat ventricular myocytes: Possible role of Cl–OH exchange

The application of fluid pressure (FP) in ventricular myocytes using pressurized fluid flow inhibits L-type Ca²⁺ current (I_Ca), with approximately 80% of this effect coming through the enhancement of Ca²⁺ releases from the sarcoplasmic reticulum. In the present study, we explored the remaining mechanisms for the inhibition of I_Ca by FP. Since FP significantly increases H⁺ concentration and H⁺ is known to inhibit I_Ca, we examined whether pH regulation plays a role in the inhibitory effect by FP on I_Ca. A flow of pressurized (∼16.3 dyne/cm²) fluid, identical to that bathing the myocytes, was applied onto single rat ventricular myocytes for which the I_Ca was monitored using whole-cell patch-clamp under HEPES-buffered conditions. Extracellular application of the alkalizing agent, NH₄Cl (20 mM), enhanced I_Ca by ∼34% in the control conditions while increasing I_Ca significantly less (by ∼21%) in FP-pretreated myocytes, suggesting an inhibition of the effect of NH₄Cl on I_Ca possibly by FP-induced acidosis. Application of DIDS (4,4′-diisothiocyanatostilbene-2,2′-disulphonic acid, 500 μM), which blocks exchange but not Cl⁻–OH⁻ exchange, did not alter the inhibitory effect of FP on I_Ca. Replacement of external Cl⁻ with aspartate attenuated the inhibitory effect of FP on I_Ca. In highly Ca²⁺-buffered cells, where Ca²⁺-dependent inhibition of I_Ca was minimized, the external Cl⁻ removal eliminated the inhibitory effect of FP on I_Ca. These results suggest that the decrease of I_Ca in the presence of FP is at least partly caused by intracellular acidosis via activation of Cl⁻–OH⁻ exchange in rat ventricular myocytes.     

The Late Endosomal ClC-6 Mediates Proton/Chloride Countertransport in Heterologous Plasma Membrane Expression

Members of the CLC protein family of Cl⁻ channels and transporters display the remarkable ability to function as either chloride channels or Cl⁻/H⁺ antiporters. Due to the intracellular localization of ClC-6 and ClC-7, it has not yet been possible to study the biophysical properties of these members of the late endosomal/lysosomal CLC branch in heterologous expression. Whereas recent data suggest that ClC-7 functions as an antiporter, transport characteristics of ClC-6 have remained entirely unknown. Here, we report that fusing the green fluorescent protein (GFP) to the N terminus of ClC-6 increased its cell surface expression, allowing us to functionally characterize ClC-6. Compatible with ClC-6 mediating Cl⁻/H⁺ exchange, Xenopus oocytes expressing GFP-tagged ClC-6 alkalinized upon depolarization. This alkalinization was dependent on the presence of extracellular anions and could occur against an electrochemical proton gradient. As observed in other CLC exchangers, ClC-6-mediated H⁺ transport was abolished by mutations in either the “gating” or “proton” glutamate. Overexpression of GFP-tagged ClC-6 in CHO cells elicited small, outwardly rectifying currents with a Cl⁻ > I⁻ conductance sequence. Mutating the gating glutamate of ClC-6 yielded an ohmic anion conductance that was increased by additionally mutating the “anion-coordinating” tyrosine. Additionally changing the chloride-coordinating serine 157 to proline increased the NO₃⁻ conductance of this mutant. Taken together, these data demonstrate for the first time that ClC-6 is a Cl⁻/H⁺ antiporter.     

Downregulation of Chloride Channel ClC-2 by Janus Kinase 3

Janus kinase-3 (JAK3) fosters proliferation and counteracts apoptosis of lymphocytes and tumor cells. The gain of function mutation ^A572VJAK3 has been discovered in acute megakaryoplastic leukemia. JAK3 is inactivated by replacement of lysine by alanine in the catalytic subunit (^K855AJAK3). Regulation of cell proliferation and apoptosis involves altered activity of Cl^? channels. The present study, thus, explored whether JAK3 modifies the function of the small conductance Cl^? channel ClC-2. To this end, ClC-2 was expressed in Xenopus oocytes with or without wild-type JAK3, ^A568VJAK3 or ^K851AJAK3, and the Cl^? channel activity determined by dual-electrode voltage clamp. Channel protein abundance in the cell membrane was determined utilizing chemiluminescence. As a result, expression of ClC-2 was followed by a marked increase of cell membrane conductance. The conductance was significantly decreased following coexpression of JAK3 or ^A568VJAK3, but not by coexpression of ^K851AJAK3. Exposure of the oocytes expressing ClC-2 together with ^A568VJAK3 to the JAK3 inhibitor WHI-P154 (4-[(3’-bromo-4’-hydroxyphenyl)amino]-6,7-dimethoxyquinazoline, 22 μM) increased the conductance. Coexpression of ^A568VJAK3 decreased the ClC-2 protein abundance in the cell membrane of ClC-2 expressing oocytes. The decline of conductance in ClC-2 and ^A568VJAK3 coexpressing oocytes following inhibition of channel protein insertion by brefeldin A (5 μM) was similar in oocytes expressing ClC-2 with ^A568VJAK3 and oocytes expressing ClC-2 alone, indicating that ^A568VJAK3 might slow channel protein insertion into rather than accelerating channel protein retrieval from the cell membrane. In conclusion, JAK3 downregulates ClC-2 activity and thus counteracts Cl^? exit—an effect possibly influencing cell proliferation and apoptosis.     

ClC-2 is required for rapid restoration of epithelial tight junctions in ischemic-injured murine jejunum

Background and aims

Involvement of the epithelial chloride channel ClC-2 has been implicated in barrier recovery following ischemic injury, possibly via a mechanism involving ClC-2 localization to the tight junction. The present study investigated mechanisms of intestinal barrier repair following ischemic injury in ClC-2^−/− mice.

Methods

Wild type, ClC-2 heterozygous and ClC-2^−/− murine jejunal mucosa was subjected to complete ischemia, after which recovery of barrier function was monitored by measuring in vivo blood-to-lumen clearance of ³H-mannitol. Tissues were examined by light and electron microscopy. The role of ClC-2 in re-assembly of the tight junction during barrier recovery was studied by immunoblotting, immunolocalization and immunoprecipitation.

Results

Following ischemic injury, ClC-2^−/− mice had impaired barrier recovery compared to wild type mice, defined by increases in epithelial paracellular permeability independent of epithelial restitution. The recovering ClC-2^−/− mucosa also had evidence of ultrastructural paracellular defects. The tight junction proteins occludin and claudin-1 shifted significantly to the detergent soluble membrane fraction during post-ischemic recovery in ClC-2^−/− mice whereas wild type mice had a greater proportion of junctional proteins in the detergent insoluble fraction. Occludin was co-immunoprecipitated with ClC-2 in uninjured wild type mucosa, and the association between occludin and ClC-2 was re-established during ischemic recovery. Based on immunofluorescence studies, re-localization of occludin from diffuse sub-apical areas to apical tight junctions was impaired in ClC-2^−/− mice.

Conclusions

These data demonstrate a pivotal role of ClC-2 in recovery of the intestinal epithelium barrier by anchoring assembly of tight junctions following ischemic injury.     

IRBIT: A regulator of ion channels and ion transporters

IRBIT (also called AHCYL1) was originally identified as a binding protein of the intracellular Ca^2 + channel inositol 1,4,5-trisphosphate (IP₃) receptor and functions as an inhibitory regulator of this receptor. Unexpectedly, many functions have subsequently been identified for IRBIT including the activation of multiple ion channels and ion transporters, such as the Na⁺/HCO₃⁻ co-transporter NBCe1-B, the Na⁺/H⁺ exchanger NHE3, the Cl⁻ channel cystic fibrosis transmembrane conductance regulator (CFTR), and the Cl⁻/HCO₃⁻ exchanger Slc26a6. The characteristic serine-rich region in IRBIT plays a critical role in the functions of this protein. In this review, we describe the evolution, domain structure, expression pattern, and physiological roles of IRBIT and discuss the potential molecular mechanisms underlying the coordinated regulation of these diverse ion channels/transporters through IRBIT. This article is part of a Special Issue entitled: Calcium signaling in health and disease. Guest Editors: Geert Bultynck, Jacques Haiech, Claus W. Heizmann, Joachim Krebs, and Marc Moreau.     

Chloride and potassium channels in U937 human monocytes

Summary Ionic channels in a human monocyte cell line (U937) were studied with the inside-out patch-clamp technique. A Ca²⁺-activated K⁺ channel and three Cl^–-selective channels were observed. The Ca²⁺-activated K⁺ channel had an inward-rectifying current-voltage relationship with slope conductance of 28 pS, and was not dependent on membrane potential. Among the three Cl^– channels, and outward-rectifying 28-pS channel was most frequently observed. The permeability ratio (Cl^–/Na⁺) was 4–5 and CH₃SO ₄ ^– was also permeant. The channel became less active with increasing polarizations in either direction, and was inactive beyond ±120 mV. The channel, observed as bursts, occasionally had rapid events within the bursts, suggesting the presence of another mode of kinetics. Diisothiocyanatostilbene-disulfonic acid (DIDS) blocked the channel reversibly in a dose-dependent manner. The second 328-pS Cl^– channel had a linear currentvoltage relationship and permeability ratio (Cl^–/Na⁺) of 5–6. This channel became less active with increasing polarizations and inactive beyond ±50 mV. DIDS blocked the channel irreversibly. The channel had multiple subconductance states. The third 15-pS Cl^– channel was least frequently observed and least voltage sensitive among the Cl^– channels. Intracellular Ca²⁺ or pH affected none of the three Cl^– channels. All three Cl^– channels had a latent period before being observed, suggesting inhibitory factor(s) presentin situ. Activation of the cells with interferon-, interferon-A or 12-O-tetradecanoylphorbol-13-acetate (TPA) caused no change in the properties on any of the channels.