GlialCAM,a CLC-2 Cl- Channel Subunit,Activates the Slow Gate of CLC Chloride Channels

GlialCAM, a glial cell adhesion molecule mutated in megalencephalic leukoencephalopathy with subcortical cysts, targets the CLC-2 Cl^- channel to cell contacts in glia and activates CLC-2 currents in vitro and in vivo. We found that GlialCAM clusters all CLC channels at cell contacts in vitro and thus studied GlialCAM interaction with CLC channels to investigate the mechanism of functional activation. GlialCAM slowed deactivation kinetics of CLC-Ka/barttin channels and increased CLC-0 currents opening the common gate and slowing its deactivation. No functional effect was seen for common gate deficient CLC-0 mutants. Similarly, GlialCAM targets the common gate deficient CLC-2 mutant E211V/H816A to cell contacts, without altering its function. Thus, GlialCAM is able to interact with all CLC channels tested, targeting them to cell junctions and activating them by stabilizing the open configuration of the common gate. These results are important to better understand the physiological role of GlialCAM/CLC-2 interaction.     

Modulation of the slow/common gating of CLC channels by intracellular cadmium

Members of the CLC family of Cl⁻ channels and transporters are homodimeric integral membrane proteins. Two gating mechanisms control the opening and closing of Cl⁻ channels in this family: fast gating, which regulates opening and closing of the individual pores in each subunit, and slow (or common) gating, which simultaneously controls gating of both subunits. Here, we found that intracellularly applied Cd²⁺ reduces the current of CLC-0 because of its inhibition on the slow gating. We identified CLC-0 residues C229 and H231, located at the intracellular end of the transmembrane domain near the dimer interface, as the Cd²⁺-coordinating residues. The inhibition of the current of CLC-0 by Cd²⁺ was greatly enhanced by mutation of I225W and V490W at the dimer interface. Biochemical experiments revealed that formation of a disulfide bond within this Cd²⁺-binding site is also affected by mutation of I225W and V490W, indicating that these two mutations alter the structure of the Cd²⁺-binding site. Kinetic studies showed that Cd²⁺ inhibition appears to be state dependent, suggesting that structural rearrangements may occur in the CLC dimer interface during Cd²⁺ modulation. Mutations of I290 and I556 of CLC-1, which correspond to I225 and V490 of CLC-0, respectively, have been shown previously to cause malfunction of CLC-1 Cl⁻ channel by altering the common gating. Our experimental results suggest that mutations of the corresponding residues in CLC-0 change the subunit interaction and alter the slow gating of CLC-0. The effect of these mutations on modulations of slow gating of CLC channels by intracellular Cd²⁺ likely depends on their alteration of subunit interactions.     

Proton Sensing of CLC-0 Mutant E166D

CLC Cl- channels are homodimers in which each subunit has a proper pore and a (fast) gate. An additional slow gate acts on both pores. A conserved glutamate (E166 in CLC-0) is a major determinant of gating in CLC-0 and is crucially involved in Cl-/H+ antiport of CLC-ec1, a CLC of known structure. We constructed tandem dimers with one wild-type (WT) and one mutant subunit (E166A or E166D) to show that these mutations of E166 specifically alter the fast gate of the pore to which they belong without effect on the fast gate of the neighboring pore. In addition both mutations activate the common slow gate. E166A pores have a large, voltage-independent open probability of the fast gate (popen), whereas popen of E166D pores is dramatically reduced. Similar to WT, popen of E166D was increased by lowering pHint. At negative voltages, E166D presents a persistent inward current that is blocked by p-chlorophenoxy-acetic acid (CPA) and increased at low pHext. The pHext dependence of the persistent current is analogous to a similar steady inward current in WT CLC-0. Surprisingly, however, the underlying unitary conductance of the persistent current in E166D is about an order of magnitude smaller than that of the transient deactivating inward Cl- current. Collectively, our data support the possibility that the mutated CLC-0 channel E166D can assume two distinct open states. Voltage-independent protonation of D166 from the outside favors a low conductance state, whereas protonation from the inside favors the high conductance state.     

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.     

Gating competence of constitutively open CLC-0 mutants revealed by the interaction with a small organic Inhibitor

Opening of CLC chloride channels is coupled to the translocation of the permeant anion. From the recent structure determination of bacterial CLC proteins in the closed and open configuration, a glutamate residue was hypothesized to form part of the Cl--sensitive gate. The negatively charged side-chain of the glutamate was suggested to occlude the permeation pathway in the closed state, while opening of a single protopore of the double-pore channel would reflect mainly a movement of this side-chain toward the extracellular pore vestibule, with little rearrangement of the rest of the channel. Here we show that mutating this critical residue (Glu166) in the prototype Torpedo CLC-0 to alanine, serine, or lysine leads to constitutively open channels, whereas a mutation to aspartate strongly slowed down opening. Furthermore, we investigated the interaction of the small organic channel blocker p-chlorophenoxy-acetic acid (CPA) with the mutants E166A and E166S. Both mutants were strongly inhibited by CPA at negative voltages with a >200-fold larger affinity than for wild-type CLC-0 (apparent KD at -140 mV approximately 4 micro M). A three-state linear model with an open state, a low-affinity and a high-affinity CPA-bound state can quantitatively describe steady-state and kinetic properties of the CPA block. The parameters of the model and additional mutagenesis suggest that the high-affinity CPA-bound state is similar to the closed configuration of the protopore gate of wild-type CLC-0. In the E166A mutant the glutamate side chain that occludes the permeation pathway is absent. Thus, if gating consists only in movement of this side-chain the mutant E166A should not be able to assume a closed conformation. It may thus be that fast gating in CLC-0 is more complex than anticipated from the bacterial structures.     

Large movement in the C terminus of CLC-0 chloride channel during slow gating

Chloride channels and transporters of the CLC gene family are expressed in virtually all cell types and are crucial in the regulation of membrane potential, chloride homeostasis and intravesicular pH. There are two gating processes that open CLC channels-fast and slow. The fast gating process in CLC channels has recently been linked to a small movement of a glutamate side chain. However, the molecular mechanism underlying the slow gating process is still elusive. Using spectroscopic microscopy, we observed a large backbone movement in the C terminus of the CLC-0 chloride channel that was functionally linked to slow gating. We further showed that the C-terminal movement had a time course similar to slow gating. In addition, a mutation known to lock the slow gate open prevented movement of the C terminus. When combined with recent structural information on the CLC C terminus, our findings provide a structural model for understanding the conformational changes linked to slow gating in CLC transport proteins.     

Molecular cloning of CLC chloride channels in Oreochromis mossambicus and their functional complementation of yeast CLC gene mutant

We have cloned two members of the CLC chloride channel family (OmCLC-3 and OmCLC-5) from gill cDNA libraries of the euryhaline tilapia Oreochromis mosammbicus. At the amino acid level, OmCLC-3 is 90.5% identical to rat CLC-3 and OmCLC-5 is 79.2% identical to rat CLC-5. Ribonuclease protection assay revealed that OmCLC-5 was mainly expressed in the gill, kidney, and intestine in both freshwater- (FW) and seawater- (SW) adapted tilapia. Although the mRNA of OmCLC-3 was broadly expressed in tissues of FW- and SW-adapted tilapia, the most intense signals were observed in the gill, kidney, intestine, and brain. Injection of OmCLC-3 and OmCLC-5 cRNAs into Xenopus oocytes did not elicit chloride currents, but these clones did functionally complement the gef1 phenotype of YPH250(gef), a yeast strain in which a single CLC channel (GEF1) has been disrupted by homologous recombination. These results clearly indicated that CLC channels closely related to the mammalian CLC-3, -4, and -5 subfamily exist also in tilapia and that OmCLC-3 and OmCLC-5 function as intracellular chloride channels.     

Mechanisms of block of muscle type CLC chloride channels (Review)

CLC proteins are a nine-member gene family of Cl- channels that have diverse roles in the plasma membrane and in intracellular organelles. The recent structure determination of bacterial CLC homologues by Dutzler et al. was a breakthrough for the structure-function analysis of CLC channels. This review describes the mechanisms of inhibition of muscle type CLC channels by two classes of small organic substances: 9-anthracene carboxylic acid (9AC) and p-chlorophenoxy propionic acid (CPP). Both substances block muscle type CLC channels (CLC-0 and CLC-1) from the intracellular side. For CPP, one could show that it inhibits the individual protopores of the double-barrelled channel. A major difference between the two types of blockers is the extremely slow binding- and unbinding-kinetics of 9AC (time scale of min), compared to that of CPP block (time scale of s), while the general mechanism of block seems to be quite similar. In the case of the chiral CPP only the S(-) enantiomer is effective. Both substances exhibit a strongly voltage-dependent block with strong inhibition at negative voltages and relief of block at depolarizing potentials at which the channels tend to open maximally. A quantitative kinetic model was developed for the CPP block of CLC-0 in which the closed state has a much larger affinity for CPP than the open state and opening of drug-bound channels is greatly slowed compared to drug-free channels. First experiments with mutated CLC-0 channels and with derivatives of CPP strongly support the pore localization of the CPP binding site. This work provides the basis for the use of these small organic substances as tools to investigate the pharmacological properties of mammalian CLC channels guided by the crystallographic structure of bacterial CLC homologues. They might also turn out to be useful to obtain information about the intricate coupling of gating and permeation that characterizes CLC channels.     

Mechanisms of block of muscle type CLC chloride channels (Review)

CLC proteins are a nine-member gene family of Cl - channels that have diverse roles in the plasma membrane and in intracellular organelles. The recent structure determination of bacterial CLC homologues by Dutzler et al. was a break-through for the structure-function analysis of CLC channels. This review describes the mechanisms of inhibition of muscle type CLC channels by two classes of small organic substances: 9-anthracene carboxylic acid (9AC) and p-chlorophenoxy propionic acid (CPP). Both substances block muscle type CLC channels (CLC-0 and CLC-1) from the intracellular side. For CPP, one could show that it inhibits the individual protopores of the double-barrelled channel. A major difference between the two types of blockers is the extremely slow binding- and unbinding-kinetics of 9AC (time scale of min), compared to that of CPP block (time scale of s), while the general mechanism of block seems to be quite similar. In the case of the chiral CPP only the S(-) enantiomer is effective. Both substances exhibit a strongly voltage-dependent block with strong inhibition at negative voltages and relief of block at depolarizing potentials at which the channels tend to open maximally. A quantitative kinetic model was developed for the CPP block of CLC-0 in which the closed state has a much larger affinity for CPP than the open state and opening of drug-bound channels is greatly slowed compared to drug-free channels. First experiments with mutated CLC-0 channels and with derivatives of CPP strongly support the pore localization of the CPP binding site. This work provides the basis for the use of these small organic substances as tools to investigate the pharmacological properties of mammalian CLC channels guided by the crystallographic structure of bacterial CLC homologues. They might also turn out to be useful to obtain information about the intricate coupling of gating and permeation that characterizes CLC channels.     

Localization of mouse CLC-6 and CLC-7 mRNA and their functional complementation of yeast CLC gene mutant

CLC-6 and CLC-7 belong to the family of voltage-dependent chloride channels. To learn more about the in vivo roles of CLC-6 and CLC-7, we performed in situ hybridization of these CLC channels in various mouse organs. Mouse CLC-6 (mCLC-6) was expressed in the peripheral region of seminiferous tubules in the testis, tracheal epithelium, epithelium of bronchioles, alveolar cells in the lung, acinar cells in the pancreas, and intestinal epithelium, but we could not detect signals from pancreatic islets. Mouse CLC-7 (mCLC-7) was expressed in neurons in the medulla oblongata, Purkinje cells in the cerebellum, proximal tubules in the kidney, and hepatocytes in the liver. The distribution of mCLC-6 and mCLC-7 were similar in the lung, pancreas, and testis. mCLC-6 functionally complemented the gef1 phenotype of a yeast strain in which a single CLC channel (GEF1) had been disrupted by homologous recombination. In contrast, mCLC-7 did not complement this gef1 phenotype. This study identified the cell types that express mCLC-6 and mCLC-7 in the mouse tissues, and the complementation assay suggested that mCLC-6 functions as an intracellular chloride channel.     

Use of cholesterol-loaded cyclodextrin: An alternative for bad cooler stallions

During the cooling process, sperm may suffer irreversible damage that compromises the fertility rate. Incorporating cholesterol-loaded cyclodextrin (CLC) represents a strategy to increase sperm resistance at low temperatures; however, high levels of cholesterol in the cell membrane can interfere with sperm capacitation. The goals of this study were to determine the CLC concentration and cooling temperature that produce optimal kinetic parameters and viability of sperm from stallions identified as bad coolers (BCs) and good coolers (GCs), as well as the effect of adding CLC on the occurrence of the acrosome reaction (ACR) and on the fertility rate of cooled sperm. In experiment 1, each ejaculate was divided into four groups: Control and treated with 1 (CLC-1), 1.5 (CLC-1.5), or 2 mg (CLC-2) of CLC/120 × 10⁶ sperm and cooled for 48 hours at 5 °C. In experiment 2, each ejaculate was divided into four groups: Control and CLC-1.5 cooled at 15 °C or 5 °C for 24 hours. For experiment 3, GC and BC stallions were used, and the ejaculates were divided into control and CLC-1.5 cooled at 5 °C for 48 hours. According to experiment, the sperm kinetics (SK) and plasma membrane integrity (PMI) were analyzed before and after 24 and 48 hours of cooling. In experiment 4, the ejaculates were divided into four groups: Control and CLC-1.5 maintained at room temperature or cooled at 5 °C for 24 hours. Each group was incubated with ionophore calcium at 37 °C for 3 hours. The incidence of ACR was analyzed before and after 1, 2, and 3 hours of incubation. For experiment 5, two cycles of 10 mares for a GC stallion and two cycles of 25 for a BC stallion were used. The inseminations were performed with control and CLC-1.5 groups cooled at 5 °C for 24 hours. According to results, all groups treated with CLC exhibited higher PMI compared with controls, and CLC-1.5 and CLC-2 exhibited the best SK results. The cooling temperature of 5 °C was superior to 15 °C when the sperm was treated with CLC. The GC and BC stallions benefited from the CLC-1.5 treatment, but the BCs were more evident, which presented greatly increased PMI and SK. There was a delay in capacitation of at least 3 hours for the fresh sperm and at least 1 hour for cooled sperm supplemented with CLC-1.5. After adding CLC-1.5, the fertility of BC stallion significantly increased, but that of the GC was not altered. Thus, incorporating CLC is an effective technique to cool equine semen, although it is indicated mainly for BC stallions.     

Structural insights into chloride and proton-mediated gating of CLC chloride channels

CLC Cl(-) channels fulfill numerous physiological functions as demonstrated by their involvement in several human genetic diseases. They have an unusual homodimeric architecture in which each subunit forms an individual pore whose open probability is regulated by various physicochemical factors, including voltage, Cl(-) concentration, and pH. The voltage dependence of Torpedo channel CLC-0 is derived probably indirectly from the translocation of a Cl(-) ion through the pore during the opening step. Recent structure determinations of bacterial CLC homologues marked a breakthrough for the structure-function analysis of CLC channels. The structures revealed a complex fold with 18 alpha-helices and two Cl(-) ions per subunit bound in the center of the protein. The side chain of a highly conserved glutamate residue that resides in the putative permeation pathway appears to be a major component of the channel gate. First studies have begun to exploit the bacterial structures as guides for a rational structure-function analysis. These studies confirm that the overall structure seems to be conserved from bacteria to humans. A full understanding of the mechanisms of gating of eukaryotic CLC channels is, however, still lacking.     

Cytoplasmic ATP inhibition of CLC-1 is enhanced by low pH

The CLC-1 Cl(-) channel is abundantly expressed on the plasma membrane of muscle cells, and the membrane potential of muscle cells is largely controlled by the activity of this Cl(-) channel. Previous studies showed that low intracellular pH increases the overall open probability of recombinant CLC-1 channels in various expression systems. Low intracellular pH, however, is known to inhibit the Cl(-) conductance on the native muscle membrane, contradicting the findings from the recombinant CLC-1 channels in expressed systems. Here we show that in the presence of physiological concentrations of ATP, reduction of the intracellular pH indeed inhibits the expressed CLC-1, mostly by decreasing the open probability of the common gate of the channel.     

CLC chloride channels in Caenorhabditis elegans

The genome of the nematode Caenorhabditis elegans encodes six putative chloride channels (CeCLC-1 through CeCLC-6) that represent all three known branches of the mammalian CLC gene family. Using promoter fragments to drive the expression of the green fluorescent protein, CeCLC-2, -3, and -4 expression was studied in transgenic C. elegans. CeCLC-4 was specifically expressed in the large H-shaped excretory cell, where it was co-expressed with CeCLC-3, which is also expressed in other cells, including neurons, muscles, and epithelial cells. Also, CeCLC-2 was expressed in several cells of the nervous system, intestinal cells, and vulval muscle cells. Similar to mammalian CLC proteins, only two nematode CLC channels elicited detectable plasma membrane currents in Xenopus oocytes. CeCLC-3 currents were inwardly rectifying and were activated by positive prepulses. Its complex gating behavior can be explained by two gates, at least one of which depends on extracellular anions. In this respect it resembles some mammalian chloride channels with which it also shares a preference of chloride over iodide. C. elegans thus provides new opportunities to understand common mechanisms underlying structure and function in CLC channels and will allow for a genetic dissection of chloride channels in this simple model organism.     

Gating the glutamate gate of CLC-2 chloride channel by pore occupancy

CLC-2 channels are dimeric double-barreled chloride channels that open in response to hyperpolarization. Hyperpolarization activates protopore gates that independently regulate the permeability of the pore in each subunit and the common gate that affects the permeability through both pores. CLC-2 channels lack classic transmembrane voltage–sensing domains; instead, their protopore gates (residing within the pore and each formed by the side chain of a glutamate residue) open under repulsion by permeant intracellular anions or protonation by extracellular H⁺. Here, we show that voltage-dependent gating of CLC-2: (a) is facilitated when permeant anions (Cl⁻, Br⁻, SCN⁻, and I⁻) are present in the cytosolic side; (b) happens with poorly permeant anions fluoride, glutamate, gluconate, and methanesulfonate present in the cytosolic side; (c) depends on pore occupancy by permeant and poorly permeant anions; (d) is strongly facilitated by multi-ion occupancy; (e) is absent under likely protonation conditions (pH_e = 5.5 or 6.5) in cells dialyzed with acetate (an impermeant anion); and (f) was the same at intracellular pH 7.3 and 4.2; and (g) is observed in both whole-cell and inside-out patches exposed to increasing [Cl⁻]_i under unlikely protonation conditions (pH_e = 10). Thus, based on our results we propose that hyperpolarization activates CLC-2 mainly by driving intracellular anions into the channel pores, and that protonation by extracellular H⁺ plays a minor role in dislodging the glutamate gate.     

Activation by acidic pH of CLC-7 expressed in oocytes from Xenopus laevis

ClC chloride channels are important in diverse physiological functions such as transepithelial transport, cell volume regulation, excitability, and acidification of intracellular organelles. We have investigated the expression of CLC-7 in oocytes from Xenopus laevis with the two electrode voltage clamp technique and Western blot analysis. Using a specific antibody against CLC-7, we found an approximately 80 kDa protein in oocytes, previously injected with CLC-7-cRNA. In voltage clamp experiments on ClC-7-cRNA-injected oocytes, no current changes were detected at normal pH (7.4). However, acidification of the Ringer solution to pH values between 6 and 4 revealed strong currents which reversed at about -15 mV (30 mV positive to the normal resting potential) and showed strong outward rectification. We therefore suggest that ClC-7 in oocytes is a functional chloride current at acidic pH. Since ClC-7 is also found in neuronal tissues and was upregulated in a rat pain model, we suggest a role of CLC-7 also for nociception and pain.     

A role for CBS domain 2 in trafficking of chloride channel CLC-5

CLC-5 is a member of the CLC family of voltage-gated chloride channels. Mutations disrupting CLC-5 lead to Dent's disease, an X-linked renal tubular disorder, characterised by low molecular weight proteinuria, hypercalciuria, nephrocalcinosis, and renal stones. Sequence analysis of CLC-5 reveals a 746 amino acid protein with an intracellular amino-terminus, transmembrane spanning domains, and two CBS domains within its intracellular carboxy-terminus. CBS domains have been implicated in intracellular targetting and trafficking as well as protein-protein interactions. We investigate subcellular localisation of three naturally occurring CLC-5 mutants which all lead to a truncated protein, disrupting the second CBS domain. These mutants are unable to traffic normally to acidic endosomes but are retained in perinuclear compartments, colocalising with the Golgi complex. This is the first identification of the cellular pathogenesis of CBS domain mutations of CLC-5.     

Protein Kinase A Activation Phosphorylates the Rat ClC-2 Cl− Channel but Does Not Change Activity

Phosphorylation-dependent events have been shown to modulate the activity of several members of the mammalian CLC Cl⁻ channel gene family, including the inward rectifier ClC-2. In the present study we investigated the regulation of rat ClC-2 expressed in the TSA-201 cell line (a transformed HEK293 cell line that stably expresses the SV40 T-antigen) by protein kinases. Protein kinase A activation phosphorylated ClC-2 in vivo, whereas stimulation of protein kinase C with phorbol 12-myristate 13-acetate did not. In vitro labeling studies confirmed that protein kinase A could directly phosphorylate ClC-2, and that protein kinase C and Ca²⁺/calmodulin-dependent protein kinase II did not. Nevertheless, protein kinase A-dependent phosphorylation of CLC-2 failed to regulate either the magnitude or the kinetics of the hyperpolarization-activated Cl⁻ currents. Considered together, we demonstrate that protein kinase A activation results in the phosphorylation of rat ClC-2 in vivo, but this event is independent of Cl⁻ channel activity. Received: 20 November 2000/Revised: 28 March 2001     

Retracted: miR-942 promotes tumor migration,invasion, and angiogenesis by regulating EMT via BARX2 in non-small-cell lung cancer

Epithelial–mesenchymal transition (EMT) has an important function in cancer. Recently, microRNAs have been reported to be involved in EMT by regulating target genes. miR-942 is considered a novel oncogene in esophageal squamous cell carcinoma. However, its role in non-small-cell lung cancer (NSCLC) has not been investigated. In this study, the expression of miR-942 in NSCLC patients tumor and paired adjacent tissues were assessed by quantitative real-time polymerase chain reaction and in situ hybridization. Transwell, wound healing, tube formation, and tail vein xenograft assays were conducted to assess miR-942′s function in NSCLC. Potential miR-942 targets were confirmed using dual-luciferase reporter assays, immunohistochemistry, immunoblot, and rescue experiments. The results showed miR-942 is relatively highly expressed in human NSCLC tissues and cells. In vitro assays demonstrated that overexpression of miR-942 promoted cell migration, invasion, and angiogenesis. Tail vein xenograft assays suggested that miR-942 contributed to NSCLC metastasis in vivo. Three bioinformatics software was searched, and BARX2 was predicted as a downstream target of miR-942. Direct interaction between them was validated by dual-luciferase assays. Rescue experiments further confirmed that BARX2 overexpression could reverse functional changes caused by miR-942. Moreover, miR-942 increased EMT-associated proteins N-cadherin and vimentin by inhibiting BARX2, while E-cadherin expression is reduced. In summary, this study reveals that miR-942 induces EMT-related metastasis by directly targeting BARX2, which may provide a potential therapeutic strategy for NSCLC.