The chloride channel ClC-4 co-localizes with cystic fibrosis transmembrane conductance regulator and may mediate chloride flux across the apical membrane of intestinal epithelia.

Cystic fibrosis (CF) causing mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) lead to mislocalization of CFTR protein from the brush border membrane of epithelial tissues and/or its dysfunction as a chloride channel. In initial reports, it was proposed that certain channels from the ClC family of chloride channels may provide compensatory or alternative pathways for epithelial chloride secretion in tissues from cystic fibrosis patients. In the present work, we provide the first evidence that ClC-4 protein is functionally expressed on the surface of the intestinal epithelium and hence, is appropriately localized to act as a therapeutic target in this CF-affected tissue. We show using confocal and electron microscopy that ClC-4 co-localizes with CFTR in the brush border membrane of the epithelium lining intestinal crypts in mouse and human tissues. In Caco-2 cells, a cell line thought to model human enterocytes, ClC-4 protein is expressed on the cell surface and also partially co-localizes with EEA1 and transferrin, marker molecules of early and recycling endosomes, respectively. Hence, like CFTR, ClC-4 may cycle between the plasma membrane and endosomal compartment. Furthermore, we show that ClC-4 functions as a chloride channel on the surface of these epithelial cells as antisense ClC-4 cDNA expression reduced the amplitude of endogenous chloride currents by 50%. These studies provide the first evidence that ClC-4 is endogenously expressed and may be functional in the brush border membrane of enterocytes and hence should be considered as a candidate channel to provide an alternative pathway for chloride secretion in the gastrointestinal tract of CF patients.     

GlialCAM, a protein defective in a leukodystrophy, serves as a ClC-2 Cl(-) channel auxiliary subunit

Ion fluxes mediated by glial cells are required for several physiological processes such as fluid homeostasis or the maintenance of low extracellular potassium during high neuronal activity. In mice, the disruption of the Cl(-) channel ClC-2 causes fluid accumulation leading to myelin vacuolation. A similar vacuolation phenotype is detected in humans affected with megalencephalic leukoencephalopathy with subcortical cysts (MLC), a leukodystrophy which is caused by mutations in MLC1 or GLIALCAM. We here identify GlialCAM as a ClC-2 binding partner. GlialCAM and ClC-2 colocalize in Bergmann glia, in astrocyte-astrocyte junctions at astrocytic endfeet around blood vessels, and in myelinated fiber tracts. GlialCAM targets ClC-2 to cell junctions, increases ClC-2 mediated currents, and changes its functional properties. Disease-causing GLIALCAM mutations abolish the targeting of the channel to cell junctions. This work describes the first auxiliary subunit of ClC-2 and suggests that ClC-2 may play a role in the pathology of MLC disease.     

Determinants of slow gating in ClC-0, the voltage-gated chloride channel of Torpedo marmorata

Membranehyperpolarization normally activates the slow gate of theTorpedo voltage-gated chloride channel(ClC-0). To elucidate the structural basis of this process, carboxyterminus truncation mutants and chimeras were constructed, expressed inXenopus oocytes, and evaluated using atwo-microelectrode voltage clamp. Introduction of stop codons atseveral positions between transmembrane domains 12 and 13 (D12 and D13)showed no expression, whereas a truncation just after D13 yieldedwild-type currents. A chimera (022) entailing the substitution of thecarboxy-terminal cytoplasmic tail after Lys-520 with the correspondingregion of ClC-2 lacked slow gating, whereas a more conservativeconstruct (chimera 002), in which D13 was replaced with its ClC-2analog, retained its capacity to slow gate. These findings suggest thatimportant structures reside within the interdomain stretch (IDS)between D12 and D13. Unlike ClC-2, in which transplantation of"ball" structures could restore gating to constitutively openmutants, transplantation of the ClC-0 IDS to the amino terminus ofchimera 022 did not restore gating. Surprisingly, replacement of theIDS by the analogous regions of either ClC-1 or ClC-2 showed slowvoltage-activated gating, although the gating was altered. Our findingslead us to conclude that both the functional expression and the slowvoltage gating of ClC-0 rely on structures at the carboxy terminus of the channel.

     

Association between Hsp90 and the ClC-2 chloride channel upregulates channel function

The voltage-dependent ClC-2 chloride channel has been implicated in a variety of physiological functions, including fluid transport across specific epithelia. ClC-2 is activated by hyperpolarization, weakly acidic external pH, intracellular Cl^–, and cell swelling. To add more insight into the mechanisms involved in ClC-2 regulation, we searched for associated proteins that may influence ClC-2 activity. With the use of immunoprecipitation of ClC-2 from human embryonic kidney-293 cells stably expressing the channel, followed by electrophoretic separation of coimmunoprecipitated proteins and mass spectrometry identification, Hsp70 and Hsp90 were unmasked as possible ClC-2 interacting partners. Association of Hsp90 with ClC-2 was confirmed in mouse brain. Inhibition of Hsp90 by two specific inhibitors, geldanamycin or radicicol, did not affect total amounts of ClC-2 but did reduce plasma membrane channel abundance. Functional experiments using the whole cell configuration of the patch-clamp technique showed that inhibition of Hsp90 reduced ClC-2 current amplitude and impaired the intracellular Cl^– concentration [Cl^–]-dependent rightward shift of the fractional conductance. Geldanamycin and radicicol increased both the slow and fast activation time constants in a chloride-dependent manner. Heat shock treatment had the opposite effect. These results indicate that association of Hsp90 with ClC-2 results in greater channel activity due to increased cell surface channel expression, facilitation of channel opening, and enhanced channel sensitivity to intracellular [Cl^–]. This association may have important pathophysiological consequences, enabling increased ClC-2 activity in response to cellular stresses such as elevated temperature, ischemia, or oxidative reagents. heat shock; geldanamycin; cellular stress; channel trafficking; transepithelial chloride transport     

ClC-3 chloride channel prevents apoptosis induced by thapsigargin in PC12 cells

Cell volume can be altered by two different ways, swelling and shrinkage. Cell swelling is regulated by volume-regulated Cl⁻ channel (VRC). It is not well understood whether shrinkage is regulated by VRC. We previously found that antisense oligonucleotide specific to ClC-3 (ClC-3 antisense) prevented cell proliferation, which was related to cell swell volume regulation. In the present study, we further studied the role of ClC-3 Cl⁻ channel in cell apoptosis which was related to cell shrinkage volume regulation by using antisense oligonucleotide specific to ClC-3 (ClC-3 antisense) and ClC-3 cDNA transfection techniques. We found that thapsigargin (TG), a specific inhibitor of the endoplasmic reticulum calcium ATPase, evoked apoptotic morphological changes (including cytoplasmic blebbing, condensation of nuclear chromatin, and the formation of apoptotic bodies), DNA laddering, and caspase-3 activation in PC12 cells (Pheochromocytoma-derived cell line). TG increased the cell apoptotic population with a decrease in cell viability. These effects were consistent with the decrease in endogenous ClC-3 protein expression, which was also induced by TG. Overexpression of ClC-3 significantly inhibited TG effect on PC12 cell apoptosis, whereas the ClC-3 antisense produced opposite effects and facilitated apoptosis induced by TG. Our data strongly suggest that ClC-3 channel in PC12 cells mediates TG-induced apoptotic process through inhibitory mechanism. Thus, it appears that ClC-3 Cl⁻ channel mediates both cell proliferation and apoptosis through accelerative and inhibitory fashions, respectively. These authors contributed equally to this work.     

Common Gating of Both CLC Transporter Subunits Underlies Voltage-dependent Activation of the 2Cl?/1H+ Exchanger ClC-7/Ostm1

CLC anion transporters form dimers that function either as Cl⁻ channels or as electrogenic Cl⁻/H⁺ exchangers. CLC channels display two different types of “gates,” “protopore” gates that open and close the two pores of a CLC dimer independently of each other and common gates that act on both pores simultaneously. ClC-7/Ostm1 is a lysosomal 2Cl⁻/1H⁺ exchanger that is slowly activated by depolarization. This gating process is drastically accelerated by many CLCN7 mutations underlying human osteopetrosis. Making use of some of these mutants, we now investigate whether slow voltage activation of plasma membrane-targeted ClC-7/Ostm1 involves protopore or common gates. Voltage activation of wild-type ClC-7 subunits was accelerated by co-expressing an excess of ClC-7 subunits carrying an accelerating mutation together with a point mutation rendering these subunits transport-deficient. Conversely, voltage activation of a fast ClC-7 mutant could be slowed by co-expressing an excess of a transport-deficient mutant. These effects did not depend on whether the accelerating mutation localized to the transmembrane part or to cytoplasmic cystathionine-β-synthase (CBS) domains of ClC-7. Combining accelerating mutations in the same subunit did not speed up gating further. No currents were observed when ClC-7 was truncated after the last intramembrane helix. Currents and slow gating were restored when the C terminus was co-expressed by itself or fused to the C terminus of the β-subunit Ostm1. We conclude that common gating underlies the slow voltage activation of ClC-7. It depends on the CBS domain-containing C terminus that does not require covalent binding to the membrane domain of ClC-7.     

Regulation of ClC-2 chloride channels in T84 cells by TGF-alpha

The almost ubiquitously expressed ClC-2 chloride channel is activated by hyperpolarization and osmotic cell swelling. Osmotic swelling also activates a different class of outwardly rectifying chloride channels, and several reports point to a link between protein tyrosine phosphorylation and activation of these channels. This study examines the possibility that transforming growth factor-alpha (TGF-alpha) modulates ClC-2 activity in human colonic epithelial (T84) cells. TGF-alpha (0.17 nM) irreversibly inhibited ClC-2 current in nystatin-perforated whole cell patch-clamp experiments, whereas a superimposed reversible activation of the current was observed at 8.3 nM TGF-alpha. Both effects required activation of the intrinsic epidermal growth factor receptor (EGFR) tyrosine kinase activity, of phosphoinositide 3-kinase, and of protein kinase C. With microspectrofluorimetry of the pH-sensitive fluorescent dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein, TGF-alpha was shown to reversibly alkalinize T84 cells at 8.3 nM but not at 0.17 nM, suggesting that 8.3 nM TGF-alpha-induced alkalinization activates ClC-2 current. This study indicates that ClC-2 channels are targets for EGFR signaling in epithelial cells.     

Determinants of 4 alpha-phorbol sensitivity in transmembrane domains 3 and 4 of the cation channel TRPV4

TRPV4, a Ca(2+)-permeable member of the vanilloid subgroup of the transient receptor potential (TRP) channels, is activated by cell swelling and moderate heat (>27 degrees C) as well as by diverse chemical compounds including synthetic 4 alpha-phorbol esters, the plant extract bisandrographolide A, and endogenous epoxyeicosatrienoic acids (EETs; 5,6-EET and 8,9-EET). Previous work identified a tyrosine residue located in the first half of putative transmembrane segment 3 (TM3) as a crucial determinant for the activation of TRPV4 by its most specific agonist 4 alpha-phorbol 12,13-didecanoate (4 alpha-PDD), suggesting that 4 alpha-PDD interacts with the channel through its transmembrane segments. To obtain insight in the 4 alpha-PDD-binding site and in the mechanism of ligand-dependent TRPV4 activation, we investigated the consequences of specific point mutations in TM4 on the sensitivity of the channel to different chemical and physical stimuli. Mutations of two hydrophobic residues in the central part of TM4 (Leu(584) and Trp(586)) caused a severe reduction of the sensitivity of the channel to 4 alpha-PDD, bisandrographolide A, and heat, whereas responses to cell swelling, arachidonic acid, and 5,6-EET remained unaffected. In contrast, mutations of two residues in the C-terminal part of TM4 (Tyr(591) and Arg(594)) affected channel activation of TRPV4 by all stimuli, suggesting an involvement in channel gating rather than in interaction with agonists. Based on a comparison of the responses of WT and mutant TRPV4 to 4 alpha-PDD and different 4 alpha-phorbol esters, we conclude that the length of the fatty acid moiety determines the ligand binding affinity and propose a model for the interaction between 4 alpha-phorbol esters and the TM3/4 region of TRPV4.     

Intracellular β-nicotinamide adenine dinucleotide inhibits the skeletal muscle ClC-1 chloride channel

ClC-1 is the dominant sarcolemmal chloride channel and plays an important role in regulating membrane excitability that is underscored by ClC-1 mutations in congenital myotonia. Here we show that the coenzyme β-nicotinamide adenine dinucleotide (NAD), an important metabolic regulator, robustly inhibits ClC-1 when included in the pipette solution in whole cell patch clamp experiments and when transiently applied to inside-out patches. The oxidized (NAD(+)) form of the coenzyme was more efficacious than the reduced (NADH) form, and inhibition by both was greatly enhanced by acidification. Molecular modeling, based on the structural coordinates of the homologous ClC-5 and CmClC proteins and in silico docking, suggest that NAD(+) binds with the adenine base deep in a cleft formed by ClC-1 intracellular cystathionine β-synthase domains, and the nicotinamide base interacts with the membrane-embedded channel domain. Consistent with predictions from the models, mutation of residues in cystathionine β-synthase and channel domains either attenuated (G200R, T636A, H847A) or abrogated (L848A) the effect of NAD(+). In addition, the myotonic mutations G200R and Y261C abolished potentiation of NAD(+) inhibition at low pH. Our results identify a new biological role for NAD and suggest that the main physiological relevance may be the exquisite sensitivity to intracellular pH that NAD(+) inhibition imparts to ClC-1 gating. These findings are consistent with the reduction of sarcolemmal chloride conductance that occurs upon acidification of skeletal muscle and suggest a previously unexplored mechanism in the pathophysiology of myotonia.     

Role of charged residues in coupling ligand binding and channel activation in the extracellular domain of the glycine receptor

The glycine receptor is a member of the ligand-gated ion channel receptor superfamily that mediates fast synaptic transmission in the brainstem and spinal cord. Following ligand binding, the receptor undergoes a conformational change that is conveyed to the transmembrane regions of the receptor resulting in the opening of the channel pore. Using the acetylcholine-binding protein structure as a template, we modeled the extracellular domain of the glycine receptor alpha1-subunit and identified the location of charged residues within loops 2 and 7 (the conserved Cys-loop). These loops have been postulated to interact with the M2-M3 linker region between the transmembrane domains 2 and 3 as part of the receptor activation mechanism. Charged residues were substituted with cysteine, resulting in a shift in the concentration-response curves to the right in each case. Covalent modification with 2-(trimethylammonium) ethyl methanethiosulfonate was demonstrated only for K143C, which was more accessible in the open state than the closed state, and resulted in a shift in the EC50 toward wild-type values. Charge reversal mutations (E53K, D57K, and D148K) also impaired channel activation, as inferred from increases in EC50 values and the conversion of taurine from an agonist to an antagonist in E53K and D57K. Thus, each of the residues Glu-53, Asp-57, Lys-143, and Asp-148 are implicated in channel gating. However, the double reverse charge mutations E53K:K276E, D57K:K276E, and D148K:K276E did not restore glycine receptor function. These results indicate that loops 2 and 7 in the extracellular domain play an important role in the mechanism of activation of the glycine receptor although not by a direct electrostatic mechanism.     

ClC-2 chloride channels contribute to HTC cell volume homeostasis

Membrane Cl(-) channels play an important role in cell volume homeostasis and regulation of volume-sensitive cell transport and metabolism. Heterologous expression of ClC-2 channel cDNA leads to the appearance of swelling-activated Cl(-) currents, consistent with a role in cell volume regulation. Since channel properties in heterologous models are potentially modified by cellular background, we evaluated whether endogenous ClC-2 proteins are functionally important in cell volume regulation. As shown by whole cell patch clamp techniques in rat HTC hepatoma cells, cell volume increases stimulated inwardly rectifying Cl(-) currents when non-ClC-2 currents were blocked by DIDS (100 microM). A cDNA closely homologous with rat brain ClC-2 was isolated from HTC cells; identical sequence was demonstrated for ClC-2 cDNAs in primary rat hepatocytes and cholangiocytes. ClC-2 mRNA and membrane protein expression was demonstrated by in situ hybridization, immunocytochemistry, and Western blot. Intracellular delivery of antibodies to an essential regulatory domain of ClC-2 decreased ClC-2-dependent currents expressed in HEK-293 cells. In HTC cells, the same antibodies prevented activation of endogenous Cl(-) currents by cell volume increases or exposure to the purinergic receptor agonist ATP and delayed HTC cell volume recovery from swelling. These studies provide further evidence that mammalian ClC-2 channel proteins are functional and suggest that in HTC cells they contribute to physiological changes in membrane Cl(-) permeability and cell volume homeostasis.     

The muscle chloride channel ClC-1 has a double-barreled appearance that is differentially affected in dominant and recessive myotonia

Single-channel recordings of the currents mediated by the muscle Cl- channel, ClC-1, expressed in Xenopus oocytes, provide the first direct evidence that this channel has two equidistant open conductance levels like the Torpedo ClC-0 prototype. As for the case of ClC-0, the probabilities and dwell times of the closed and conducting states are consistent with the presence of two independently gated pathways with approximately 1.2 pS conductance enabled in parallel via a common gate. However, the voltage dependence of the common gate is different and the kinetics are much faster than for ClC-0. Estimates of single-channel parameters from the analysis of macroscopic current fluctuations agree with those from single-channel recordings. Fluctuation analysis was used to characterize changes in the apparent double-gate behavior of the ClC-1 mutations I290M and I556N causing, respectively, a dominant and a recessive form of myotonia. We find that both mutations reduce about equally the open probability of single protopores and that mutation I290M yields a stronger reduction of the common gate open probability than mutation I556N. Our results suggest that the mammalian ClC-homologues have the same structure and mechanism proposed for the Torpedo channel ClC-0. Differential effects on the two gates that appear to modulate the activation of ClC-1 channels may be important determinants for the different patterns of inheritance of dominant and recessive ClC-1 mutations.     

A Serine Residue in ClC-3 Links Phosphorylation–Dephosphorylation to Chloride Channel Regulation by Cell Volume

In many mammalian cells, ClC-3 volume-regulated chloride channels maintain a variety of normal cellular functions during osmotic perturbation. The molecular mechanisms of channel regulation by cell volume, however, are unknown. Since a number of recent studies point to the involvement of protein phosphorylation/dephosphorylation in the control of volume-regulated ionic transport systems, we studied the relationship between channel phosphorylation and volume regulation of ClC-3 channels using site-directed mutagenesis and patch-clamp techniques. In native cardiac cells and when overexpressed in NIH/3T3 cells, ClC-3 channels were opened by cell swelling or inhibition of endogenous PKC, but closed by PKC activation, phosphatase inhibition, or elevation of intracellular Ca²⁺. Site-specific mutational studies indicate that a serine residue (serine51) within a consensus PKC-phosphorylation site in the intracellular amino terminus of the ClC-3 channel protein represents an important volume sensor of the channel. These results provide direct molecular and pharmacological evidence indicating that channel phosphorylation/dephosphorylation plays a crucial role in the regulation of volume sensitivity of recombinant ClC-3 channels and their native counterpart, I_Cl.vol.     

ATP depletion inhibits the endocytosis of ClC-2

The chloride channel, ClC-2 is expressed ubiquitously and participates in multiple physiological processes. In particular, ClC-2 has been implicated in the regulation of neuronal chloride ion homeostasis and mutations in ClC-2 are associated with idiopathic generalized epilepsy. Despite the physiological and pathophysiological significance of this channel, its regulation remains incompletely understood. The functional expression of ClC-2 at the cell surface has been shown to be enhanced by depletion of cellular ATP, implicating its possible role in cellular energy sensing. In the present study, biochemical assays of cell surface expression suggest that this gain of function reflects, in part, an increase in channel number due to the reduction in ClC-2 internalization by endocytosis. Cell surface expression of the disease-causing mutant: G715E, thought to lack wild-type nucleotide binding affinity, is similarly affected, suggesting that ATP-depletion modifies the function of proteins in the endocytic pathway rather than ClC-2 directly. Using a combination of immunofluorescence and biochemical studies, we confirmed that ClC-2 is internalized via dynamin-dependent endocytosis and that the change in surface expression evoked by ATP depletion is partially mimicked by inhibition of dynamin function using a dynamin dominant-negative mutant (DynK44A). Furthermore, trafficking via the early endosomal compartment occurs in part through rab5-associated vesicles and recycling of ClC-2 to the cell surface occurs through a rab11 dependent pathway. In summary, we have determined that the internalization of ClC-2 by endocytosis is inhibited by metabolic stress, highlighting the importance for understanding the molecular mechanisms mediating the endosomal trafficking of this channel.     

Biophysical properties of ClC-3 differentiate it from swelling-activated chloride channels in Chinese hamster ovary-K1 cells

ClC-3 is a highly conserved voltage-gated chloride channel, which together with ClC-4 and ClC-5 belongs to one subfamily of the larger group of ClC chloride channels. Whereas ClC-5 is localized intracellularly, ClC-3 has been reported to be a swelling-activated plasma membrane channel. However, recent studies have shown that native ClC-3 in hepatocytes is primarily intracellular. Therefore, we reexamined the properties of ClC-3 in a mammalian cell expression system and compared them with the properties of endogenous swelling-activated channels. Chinese hamster ovary (CHO)-K1 cells were transiently transfected with rat ClC-3. The resulting chloride currents were Cl(-) > I(-) selective, showed extreme outward rectification, and lacked inactivation at positive voltages. In addition, they were insensitive to the chloride channel blockers, 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB) and 4, 4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) and were not inhibited by phorbol esters or activated by osmotic swelling. These properties are identical to those of ClC-5 but differ from those previously attributed to ClC-3. In contrast, nontransfected CHO-K1 cells displayed an endogenous swelling-activated chloride current, which was weakly outward rectifying, inactivated at positive voltages, sensitive to NPPB and DIDS, and inhibited by phorbol esters. These properties are identical to those previously attributed to ClC-3. Therefore, we conclude that when expressed in CHO-K1 cells, ClC-3 is an extremely outward rectifying channel with similar properties to ClC-5 and is neither activated by cell swelling nor identical to the endogenous swelling-activated channel. These data suggest that ClC-3 cannot be responsible for the swelling-activated chloride channel under all circumstances.     

An internalization signal in ClC-5, an endosomal Cl-channel mutated in dent's disease

The ClC-5 chloride channel resides mainly in vesicles of the endocytotic pathway and contributes to their acidification. Its disruption in mice entails a broad defect in renal endocytosis and causes secondary changes in calciotropic hormone levels. Inactivating mutations in Dent's disease lead to proteinuria and kidney stones. Possibly by recycling, a small fraction of ClC-5 also reaches the plasma membrane. Here we identify a carboxyl-terminal internalization motif in ClC-5. It resembles the PY motif, which is crucial for the endocytosis and degradation of epithelial Na(+) channels. Mutating this motif increases surface expression and currents about 2-fold. This is probably because of interactions with WW domains, because dominant negative mutants of the ubiquitin-protein ligase WWP2 increased surface expression and currents of ClC-5 only when its PY motif was intact. Stimulating endocytosis by expressing rab5 or its GTPase-deficient Q79L mutant decreased WT ClC-5 currents but did not affect channels with mutated motifs. Similarly, decreasing endocytosis by expressing the inactive S34N mutant of rab5 increased ClC-5 currents only if its PY-like motif was intact. Thus, the endocytosis of ClC-5, which itself is crucial for the endocytosis of other proteins, depends on the interaction of a carboxyl-terminal internalization signal with ubiquitin-protein ligases containing WW domains.     

ClC-3 is a candidate of the channel proteins mediating acid-activated chloride currents in nasopharyngeal carcinoma cells

Acid-activated chloride currents have been reported in several cell types and may play important roles in regulation of cell function. However, the molecular identities of the channels that mediate the currents are not defined. In this study, activation of the acid-induced chloride current and the possible candidates of the acid-activated chloride channel were investigated in human nasopharyngeal carcinoma cells (CNE-2Z). A chloride current was activated when extracellular pH was reduced to 6.6 from 7.4. However, a further decrease of extracellular pH to 5.8 inhibited the current. The current was weakly outward-rectified and was suppressed by hypertonicity-induced cell shrinkage and by the chloride channel blockers 5-nitro-2-3-phenylpropylamino benzoic acid (NPPB), tamoxifen, and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid disodium salt hydrate (DIDS). The permeability sequence of the channel to anions was I(-) > Br(-) > Cl(-) > gluconate(-). Among the ClC chloride channels, ClC-3 and ClC-7 were strongly expressed in CNE-2Z cells. Knockdown of ClC-3 expression with ClC-3 small interfering (si)RNA prevented the activation of the acid-induced current, but silence of ClC-7 expression with ClC-7 siRNA did not significantly affect the current. The results suggest that the chloride channel mediating the acid-induced chloride current was volume sensitive. ClC-3 is a candidate of the channel proteins that mediate or regulate the acid-activated chloride current in nasopharyngeal carcinoma cells.     

SPI-0211 activates T84 cell chloride transport and recombinant human ClC-2 chloride currents

The purpose of this study was to determine the mechanism of action of SPI-0211 (lubiprostone), a novel bicyclic fatty acid in development for the treatment of bowel dysfunction. Adult rabbit intestine was shown to contain mRNA for ClC-2 using RT-PCR, Northern blot analysis, and in situ hybridization. T84 cells grown to confluence on permeable supports were shown to express ClC-2 channel protein in the apical membrane. SPI-0211 increased electrogenic Cl- transport across the apical membrane of T84 cells, with an EC50 of approximately 18 nM measured by short-circuit current (Isc) after permeabilization of the basolateral membrane with nystatin. SPI-0211 effects on Cl- currents were also measured by whole cell patch clamp using the human embryonic kidney (HEK)-293 cell line stably transfected with either recombinant human ClC-2 or recombinant human cystic fibrosis transmembrane regulator (CFTR). In these studies, SPI-0211 activated ClC-2 Cl- currents in a concentration-dependent manner, with an EC50 of approximately 17 nM, and had no effect in nontransfected HEK-293 cells. In contrast, SPI-0211 had no effect on CFTR Cl- channel currents measured in CFTR-transfected HEK-293 cells. Activation of ClC-2 by SPI-0211 was independent of PKA. Together, these studies demonstrate that SPI-0211 is a potent activator of ClC-2 Cl- channels and suggest a physiologically relevant role for ClC-2 Cl- channels in intestinal Cl- transport after SPI-0211 administration.