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1.  Alternative mRNA splice variants of the rat ClC-2 chloride channel gene are expressed in lung: genomic sequence and organization of ClC-2. 
Nucleic Acids Research  1997;25(20):4153-4159.
The ClC-2 epithelial cell chloride channel is a voltage-, tonicity- and pH-regulated member of the ClC super family. We have previously shown that rat lung ClC-2 (rClC-2) is down-regulated at birth, and molecular diversity is generated by alternative splicing [Murray et al. (1995) Am. J. Respir. Cell Mol. Biol. 12, 597-604; Murray et al. (1996) Am. J. Physiol. 271, L829-L837; Chu et al . (1996) Nucleic Acids Res. 24, 3453-3457]. To investigate other possible mRNA splice variations, we sequenced the entire rClC-2 gene and found that ClC-2Sa (formerly ClC-2S) results from the deletion of exon 20. The preceding intron 19 has an unusually high CT content and a rare AAG acceptor site. Because both features were also found in intron 13, we next tested the hypothesis that intron 13 would be involved in alternative splicing. As predicted, a second splice product, ClC-2Sb, was found by RT-PCR, but only in lung. When we compared the genomic maps of rClC-2 and human ClC-1 (hClC-1), striking similarities were found in each exon except for rClC-2 exon 20, which is absent in hClC-1. These observations suggest that ClC-1 and ClC-2 may have evolved by gene duplication, mutation and DNA rearrangement.
PMCID: PMC147000  PMID: 9321672
2.  CLC channel function and dysfunction in health and disease 
CLC channels and transporters are expressed in most tissues and fulfill diverse functions. There are four human CLC channels, ClC-1, ClC-2, ClC-Ka, and ClC-Kb, and five CLC transporters, ClC-3 through −7. Some of the CLC channels additionally associate with accessory subunits. Whereas barttin is mandatory for the functional expression of ClC-K, GlialCam is a facultative subunit of ClC-2 which modifies gating and thus increases the functional variability within the CLC family. Isoform-specific ion conduction and gating properties optimize distinct CLC channels for their cellular tasks. ClC-1 preferentially conducts at negative voltages, and the resulting inward rectification provides a large resting chloride conductance without interference with the muscle action potential. Exclusive opening at voltages negative to the chloride reversal potential allows for ClC-2 to regulate intracellular chloride concentrations. ClC-Ka and ClC-Kb are equally suited for inward and outward currents to support transcellular chloride fluxes. Every human CLC channel gene has been linked to a genetic disease, and studying these mutations has provided much information about the physiological roles and the molecular basis of CLC channel function. Mutations in the gene encoding ClC-1 cause myotonia congenita, a disease characterized by sarcolemmal hyperexcitability and muscle stiffness. Loss-of-function of ClC-Kb/barttin channels impairs NaCl resorption in the limb of Henle and causes hyponatriaemia, hypovolemia and hypotension in patients suffering from Bartter syndrome. Mutations in CLCN2 were found in patients with CNS disorders but the functional role of this isoform is still not understood. Recent links between ClC-1 and epilepsy and ClC-Ka and heart failure suggested novel cellular functions of these proteins. This review aims to survey the knowledge about physiological and pathophysiological functions of human CLC channels in the light of recent discoveries from biophysical, physiological, and genetic studies.
doi:10.3389/fphys.2014.00378
PMCID: PMC4188032  PMID: 25339907
CLC channel; anion channel; patch clamp; myotonia congenita; Bartter syndrome; leukencephalopathy
3.  Chloride Channels and Transporters in Human Corneal Epithelium 
Experimental eye research  2010;90(6):771-779.
Transport of water and electrolytes is critical for corneal clarity. Recent studies indicate another important function of transport of ions and electrolytes - establishing wound electric fields that guide cell migration. We found chloride (Cl-) flux is a major component of the corneal wound electric current. In order to elucidate the mechanisms of Cl- transport, we studied Cl- channels and transporters in human corneal epithelial (HCE) cells.
We tested a transformed human corneal epithelial cell line (tHCE), primary cultures of human corneal epithelial cells (pHCE), and human donor corneas. We first used RT-PCR to determine expression levels of mRNA of CLC (Cl- channel/transporter) family members and CFTR (cystic fibrosis transmembrane conductance regulator) in HCE cells. We then confirmed protein expression and distribution of selected CLC family members and CFTR with Western blot and immuno-fluorescence confocal microscopy. Finally, Cl- currents were recorded with electrophysiological techniques.
The mRNAs of CLC-2, CLC-3, CLC-4, CLC-5, CLC-6, and CFTR were detected in the HCE cell line. CLC-1 and CLC-7 were not detectable. Western blot and immunostaining confirmed protein expression and distribution of CLC-2, CLC-3, CLC-4, CLC-6 and CFTR in human corneal epithelium. CLC-2 preferentially labeled the apical and basal layers, while CLC-3 and CLC-4 labeled only the superficial layer. CLC-6 and CFTR labeling showed a unique gradient with strong staining in apical layers which gradually decreased towards the basal layers. Corneal endothelium was positive for CLC-2, CLC-3, CLC-4, CLC-6 and possibly CFTR. Human corneal epithelial cells demonstrated voltage dependent Cl- currents.
HCE cells express functional Cl- channels and transporters. CLC-2, CLC-3, CLC-4, CLC-6, and CFTR had distinct expression patterns in human corneal epithelium. Those molecules and their distribution may play important roles in maintaining resting Cl- fluxes and in regulating Cl- flux at corneal wounds, which may be a major contributor to wound electrical signaling.
doi:10.1016/j.exer.2010.03.013
PMCID: PMC2873124  PMID: 20346358
4.  GlialCAM, a Protein Defective in a Leukodystrophy, Serves as a ClC-2 Cl− Channel Auxiliary Subunit 
Neuron  2012;73(5):951-961.
Summary
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.
Video Abstract
Highlights
► GlialCAM, which is defective in MLC disease, is a ClC-2 Cl− channel subunit ► GlialCAM modifies the targeting and the functional activity of the ClC-2 channel ► Mutations found in GLIALCAM in MLC affect the targeting of ClC-2 to cell junctions
Leukodystrophies are a group of genetic diseases affecting white matter. Jeworutzki et al. find that GlialCAM, a cell-adhesion molecule which is mutated in a leukodystrophy, serves as an auxiliary subunit of the chloride channel ClC-2.
doi:10.1016/j.neuron.2011.12.039
PMCID: PMC3334819  PMID: 22405205
5.  A Cytoplasmic Domain Mutation in ClC-Kb Affects Long-Distance Communication Across the Membrane 
PLoS ONE  2008;3(7):e2746.
Background
ClC-Kb and ClC-Ka are homologous chloride channels that facilitate chloride homeostasis in the kidney and inner ear. Disruption of ClC-Kb leads to Bartter's Syndrome, a kidney disease. A point mutation in ClC-Kb, R538P, linked to Bartter's Syndrome and located in the C-terminal cytoplasmic domain was hypothesized to alter electrophysiological properties due to its proximity to an important membrane-embedded helix.
Methodology/Principal Findings
Two-electrode voltage clamp experiments were used to examine the electrophysiological properties of the mutation R538P in both ClC-Kb and ClC-Ka. R538P selectively abolishes extracellular calcium activation of ClC-Kb but not ClC-Ka. In attempting to determine the reason for this specificity, we hypothesized that the ClC-Kb C-terminal domain had either a different oligomeric status or dimerization interface than that of ClC-Ka, for which a crystal structure has been published. We purified a recombinant protein corresponding to the ClC-Kb C-terminal domain and used multi-angle light scattering together with a cysteine-crosslinking approach to show that the dimerization interface is conserved between the ClC-Kb and ClC-Ka C-terminal domains, despite the fact that there are several differences in the amino acids that occur at this interface.
Conclusions
The R538P mutation in ClC-Kb, which leads to Bartter's Syndrome, abolishes calcium activation of the channel. This suggests that a significant conformational change – ranging from the cytoplasmic side of the protein to the extracellular side of the protein – is involved in the Ca2+-activation process for ClC-Kb, and shows that the cytoplasmic domain is important for the channel's electrophysiological properties. In the highly similar ClC-Ka (90% identical), the R538P mutation does not affect activation by extracellular Ca2+. This selective outcome indicates that ClC-Ka and ClC-Kb differ in how conformational changes are translated to the extracellular domain, despite the fact that the cytoplasmic domains share the same quaternary structure.
doi:10.1371/journal.pone.0002746
PMCID: PMC2447174  PMID: 18648499
6.  Identification of SNPs in the cystic fibrosis interactome influencing pulmonary progression in cystic fibrosis 
There is growing evidence that the great phenotypic variability in patients with cystic fibrosis (CF) not only depends on the genotype, but apart from a combination of environmental and stochastic factors predominantly also on modifier gene effects. It has been proposed that genes interacting with CF transmembrane conductance regulator (CFTR) and epithelial sodium channel (ENaC) are potential modifiers. Therefore, we assessed the impact of single-nucleotide polymorphisms (SNPs) of several of these interacters on CF disease outcome. SNPs that potentially alter gene function were genotyped in 95 well-characterized p.Phe508del homozygous CF patients. Linear mixed-effect model analysis was used to assess the relationship between sequence variants and the repeated measurements of lung function parameters. In total, we genotyped 72 SNPs in 10 genes. Twenty-five SNPs were used for statistical analysis, where we found strong associations for one SNP in PPP2R4 with the lung clearance index (P≤0.01), the specific effective airway resistance (P≤0.005) and the forced expiratory volume in 1 s (P≤0.005). In addition, we identified one SNP in SNAP23 to be significantly associated with three lung function parameters as well as one SNP in PPP2R1A and three in KRT19 to show a significant influence on one lung function parameter each. Our findings indicate that direct interacters with CFTR, such as SNAP23, PPP2R4 and PPP2R1A, may modify the residual function of p.Phe508del-CFTR while variants in KRT19 may modulate the amount of p.Phe508del-CFTR at the apical membrane and consequently modify CF disease.
doi:10.1038/ejhg.2012.181
PMCID: PMC3598332  PMID: 22892532
cystic fibrosis; interactome; modifier genes
7.  A regulatory calcium-binding site at the subunit interface of CLC-K kidney chloride channels 
The Journal of General Physiology  2010;136(3):311-323.
The two human CLC Cl− channels, ClC-Ka and ClC-Kb, are almost exclusively expressed in kidney and inner ear epithelia. Mutations in the genes coding for ClC-Kb and barttin, an essential CLC-K channel β subunit, lead to Bartter syndrome. We performed a biophysical analysis of the modulatory effect of extracellular Ca2+ and H+ on ClC-Ka and ClC-Kb in Xenopus oocytes. Currents increased with increasing [Ca2+]ext without full saturation up to 50 mM. However, in the absence of Ca2+, ClC-Ka currents were still 20% of currents in 10 mM [Ca2+]ext, demonstrating that Ca2+ is not strictly essential for opening. Vice versa, ClC-Ka and ClC-Kb were blocked by increasing [H+]ext with a practically complete block at pH 6. Ca2+ and H+ act as gating modifiers without changing the single-channel conductance. Dose–response analysis suggested that two protons are necessary to induce block with an apparent pK of ∼7.1. A simple four-state allosteric model described the modulation by Ca2+ assuming a 13-fold higher Ca2+ affinity of the open state compared with the closed state. The quantitative analysis suggested separate binding sites for Ca2+ and H+.
A mutagenic screen of a large number of extracellularly accessible amino acids identified a pair of acidic residues (E261 and D278 on the loop connecting helices I and J), which are close to each other but positioned on different subunits of the channel, as a likely candidate for forming an intersubunit Ca2+-binding site. Single mutants E261Q and D278N greatly diminished and the double mutant E261Q/D278N completely abolished modulation by Ca2+. Several mutations of a histidine residue (H497) that is homologous to a histidine that is responsible for H+ block in ClC-2 did not yield functional channels. However, the triple mutant E261Q/D278N/H497M completely eliminated H+ -induced current block. We have thus identified a protein region that is involved in binding these physiologically important ligands and that is likely undergoing conformational changes underlying the complex gating of CLC-K channels.
doi:10.1085/jgp.201010455
PMCID: PMC2931146  PMID: 20805576
8.  Inward Rectification in ClC-0 Chloride Channels Caused by Mutations in Several Protein Regions  
The Journal of General Physiology  1997;110(2):165-171.
Several cloned ClC-type Cl− channels open and close in a voltage-dependent manner. The Torpedo electric organ Cl− channel, ClC-0, is the best studied member of this gene family. ClC-0 is gated by a fast and a slow gating mechanism of opposite voltage direction. Fast gating is dependent on voltage and on the external and internal Cl− concentration, and it has been proposed that the permeant anion serves as the gating charge in ClC-0 (Pusch, M., U. Ludewig, A. Rehfeldt, and T.J. Jentsch. 1995. Nature (Lond.). 373:527–531). The deactivation at negative voltages of the muscular ClC-1 channel is similar but not identical to ClC-0. Different from the extrinsic voltage dependence suggested for ClC-0, an intrinsic voltage sensor had been proposed to underlie the voltage dependence in ClC-1 (Fahlke, C., R. Rüdel, N. Mitrovic, M. Zhou, and A.L. George. 1995. Neuron. 15:463–472; Fahlke, C., A. Rosenbohm, N. Mitrovic, A.L. George, and R. Rüdel. 1996. Biophys. J. 71:695–706). The gating model for ClC-1 was partially based on the properties of a point-mutation found in recessice myotonia (D136G). Here we investigate the functional effects of mutating the corresponding residue in ClC-0 (D70). Both the corresponding charge neutralization (D70G) and a charge conserving mutation (D70E) led to an inwardly rectifying phenotype resembling that of ClC-1 (D136G). Several other mutations at very different positions in ClC-0 (K165R, H472K, S475T, E482D, T484S, T484Q), however, also led to a similar phenotype. In one of these mutants (T484S) the typical wild-type gating, characterized by a deactivation at negative voltages, can be partially restored by using external perchlorate (ClO4−) solutions. We conclude that gating in ClC-0 and ClC-1 is due to similar mechanisms. The negative charge at position 70 in ClC-0 does not specifically confer the voltage sensitivity in ClC-channels, and there is no need to postulate an intrinsic voltage sensor in ClC-channels.
PMCID: PMC2233784  PMID: 9236209
gating; myotonia; voltage dependence; anion
9.  The Muscle Chloride Channel ClC-1 Is Not Directly Regulated by Intracellular ATP 
The Journal of General Physiology  2008;131(2):109-116.
ClC-1 belongs to the gene family of CLC Cl− channels and Cl−/H+ antiporters. It is the major skeletal muscle chloride channel and is mutated in dominant and recessive myotonia. In addition to the membrane-embedded part, all mammalian CLC proteins possess a large cytoplasmic C-terminal domain that bears two so-called CBS (from cystathionine-β-synthase) domains. Several studies indicate that these domains might be involved in nucleotide binding and regulation. In particular, Bennetts et al. (J. Biol. Chem. 2005. 280:32452–32458) reported that the voltage dependence of hClC-1 expressed in HEK cells is regulated by intracellular ATP and other nucleotides. Moreover, very recently, Bennetts et al. (J. Biol. Chem. 2007. 282:32780–32791) and Tseng et al. (J. Gen. Physiol. 2007. 130:217–221) reported that the ATP effect was enhanced by intracellular acidification. Here, we show that in striking contrast with these findings, human ClC-1, expressed in Xenopus oocytes and studied with the inside-out configuration of the patch-clamp technique, is completely insensitive to intracellular ATP at concentrations up to 10 mM, at neutral pH (pH 7.3) as well as at slightly acidic pH (pH 6.2). These results have implications for a general understanding of nucleotide regulation of CLC proteins and for the physiological role of ClC-1 in muscle excitation.
doi:10.1085/jgp.200709899
PMCID: PMC2213564  PMID: 18227271
10.  Nucleotide sequence and initial functional characterization of the clcR gene encoding a LysR family activator of the clcABD chlorocatechol operon in Pseudomonas putida. 
Journal of Bacteriology  1993;175(2):417-427.
The 3-chlorocatechol operon clcABD is central to the biodegradative pathway of 3-chlorobenzoate. The clcR regulatory gene, which activates the clcABD operon, was cloned from the region immediately upstream of the operon and was shown to complement an insertion mutation for growth on 3-chlorobenzoate. ClcR activated the clcA promoter, which controls expression of the clcABD operon, in trans by 14-fold in an in vivo promoter probe assay in Pseudomonas putida when cells were incubated with 15 mM 3-chlorobenzoic acid. Specific binding of ClcR to the clcR-clcA intergenic promoter region was observed in a gel shift assay. Nucleotide sequence analysis of the clcR gene predicts a polypeptide of 32.5 kDa, which was confirmed by using specific in vivo 35S labeling of the protein from a T7 promoter-controlled ATG fusion construct. ClcR shares high sequence identity with the LysR family of bacterial regulator proteins and has especially high homology to a subgroup of the family consisting of TcbR (57% amino acid sequence identity), TfdS, CatR, and CatM. ClcR was shown to autoregulate its own production in trans to 35% of unrepressed levels but partially relieved this autorepression under conditions that induced transcription at the clcA promoter. Several considerations indicate that the clcR-clcABD locus is most similar to the tcbR-tcbCDEF regulon.
Images
PMCID: PMC196156  PMID: 8419291
11.  IL1B Polymorphisms Modulate Cystic Fibrosis Lung Disease 
Pediatric pulmonology  2009;44(6):580-593.
Summary
Rationale: Variability in pulmonary disease severity is found in patients with cystic fibrosis (CF) who have identical mutations in the CF transmembrane conductance regulator (CFTR) gene. We hypothesized that one factor accounting for heterogeneity in pulmonary disease severity is variation in the family of genes affecting the biology of interleukin-1 (IL-1), which impacts acquisition and maintenance of Pseudomonas aeruginosa infection in animal models of chronic infection. Methods: We genotyped 58 single nucleotide polymorphisms (SNPs) in the IL-1 gene cluster in 808 CF subjects from the University of North Carolina and Case Western Reserve University (UNC/CWRU) joint cohort. All were homozygous for ΔF508, and categories of “severe” (cases) or “mild” (control subjects) lung disease were defined by the lowest or highest quartile of forced expired volume (FEV1) for age in the CF population. After adjustment for age and gender, genotypic data were tested for association with lung disease severity. Odds ratios (ORs) comparing severe versus mild CF were also calculated for each genotype (with the homozygote major allele as the reference group) for all 58 SNPs. From these analyses, nine SNPs with a moderate effect size, OR ≤ 0.5or > 1.5, were selected for further testing. To replicate the case-control study results, we genotyped the same nine SNPs in a second population of CF parent-offspring trios (recruited from Children’s Hospital Boston), in which the offspring had similar pulmonary phenotypes. For the trio analysis, both family-based and population-based associations were performed. Results: SNPs rs1143634 and rs1143639 in the IL1B gene demonstrated a consistent association with lung disease severity categories (P < 0.10) and longitudinal analysis of lung disease severity (P < 0.10) in CF in both the case-control and family-based studies. In females, there was a consistent association (false discovery rate adjusted joint P-value < 0.06 for both SNPs) in both the analysis of lung disease severity in the UNC/CWRU cohort and the family-based analysis of affection status. Conclusion: Our findings suggest that IL1β is a clinically relevant modulator of CF lung disease.
doi:10.1002/ppul.21026
PMCID: PMC3716579  PMID: 19431193
gene modifiers; cystic fibrosis; CFTR; IL-1 gene family
12.  The swelling-activated chloride channel ClC-2, the chloride channel ClC-3, and ClC-5, a chloride channel mutated in kidney stone disease, are expressed in distinct subpopulations of renal epithelial cells. 
Journal of Clinical Investigation  1998;101(3):635-642.
The mammalian genome encodes at least nine different members of the ClC family of chloride channels. So far only two of them could be localized on a cellular level in the kidney. We now report on the precise intrarenal localization of the mRNAs coding for the chloride channels ClC-2, ClC-3 and ClC-5. Expression of ClC-2 mRNA, encoding a swelling-activated chloride channel, could be demonstrated in the S3 segment of the proximal tubule. The chloride channel ClC-3 mRNA and ClC-5 mRNA, coding for a chloride channel mutated in kidney stone disease, were both expressed in intercalated cells of the connecting tubule and collecting duct. Whereas ClC-3 mRNA expression was most prominent in the cortex of rat kidneys, ClC-5 mRNA was expressed from the cortex through the upper portion of the inner medulla. A detailed analysis revealed that ClC-3 was expressed by type B intercalated cells, whereas ClC-5 was expressed by type A intercalated cells. These findings have important implications for the pathogenesis of hereditary kidney stone disease caused by mutations in the CLCN5 gene.
PMCID: PMC508607  PMID: 9449697
13.  Expression and Localization of CLC Chloride Transport Proteins in the Avian Retina 
PLoS ONE  2011;6(3):e17647.
Members of the ubiquitously expressed CLC protein family of chloride channels and transporters play important roles in regulating cellular chloride and pH. The CLCs that function as Cl−/H+ antiporters, ClCs 3–7, are essential in particular for the acidification of endosomal compartments and protein degradation. These proteins are broadly expressed in the nervous system, and mutations that disrupt their expression are responsible for several human genetic diseases. Furthermore, knock-out of ClC3 and ClC7 in the mouse result in the degeneration of the hippocampus and the retina. Despite this evidence of their importance in retinal function, the expression patterns of different CLC transporters in different retinal cell types are as yet undescribed. Previous work in our lab has shown that in chicken amacrine cells, internal Cl− can be dynamic. To determine whether CLCs have the potential to participate, we used PCR and immunohistochemical techniques to examine CLC transporter expression in the chicken retina. We observed a high level of variation in the retinal expression levels and patterns among the different CLC proteins examined. These findings, which represent the first systematic investigation of CLC transporter expression in the retina, support diverse functions for the different CLCs in this tissue.
doi:10.1371/journal.pone.0017647
PMCID: PMC3049779  PMID: 21408174
14.  Transcriptional Regulation of the CLC-K1 Promoter by myc-Associated Zinc Finger Protein and Kidney-Enriched Krüppel-Like Factor, a Novel Zinc Finger Repressor 
Molecular and Cellular Biology  2000;20(19):7319-7331.
The expression of CLC-K1 and CLC-K2, two kidney-specific CLC chloride channels, is transcriptionally regulated on a tissue-specific basis. Previous studies have shown that a GA element near their transcriptional start sites is important for basal and cell-specific activities of the CLC-K1 and CLC-K2 gene promoters. To identify the GA-binding proteins, the human kidney cDNA library was screened by a yeast one-hybrid system. A novel member of the Cys2-His2 zinc finger gene designated KKLF (for “kidney-enriched Krüppel-like factor”) and the previously isolated MAZ (for “myc-associated zinc finger protein”) were cloned. KKLF was found to be abundantly expressed in the liver, kidneys, heart, and skeletal muscle, and immunohistochemistry revealed the nuclear localization of KKLF protein in interstitial cells in heart and skeletal muscle, stellate cells, and fibroblasts in the liver. In the kidneys, KKLF protein was localized in interstitial cells, mesangial cells, and nephron segments, where CLC-K1 and CLC-K2 were not expressed. A gel mobility shift assay revealed sequence-specific binding of recombinant KKLF and MAZ proteins to the CLC-K1 GA element, and the fine-mutation assay clarified that the consensus sequence for the KKLF binding site was GGGGNGGNG. In a transient-transfection experiment, MAZ had a strong activating effect on transcription of the CLC-K1–luciferase reporter gene. On the other hand, KKLF coexpression with MAZ appeared to block the activating effect of MAZ. These results suggest that a novel set of zinc finger proteins may help regulate the strict tissue- and nephron segment-specific expression of the CLC-K1 and CLC-K2 channel genes through their GA cis element.
PMCID: PMC86286  PMID: 10982849
15.  ClC1 chloride channel in myotonic dystrophy type 2 and ClC1 splicing in vitro 
Acta Myologica  2012;31(2):144-153.
Myotonic dystrophy type 2 (DM2) is caused by CCTG-repeat expansions. Occurrence of splicing and mutations in the muscle chloride channel gene CLCN1 have been reported to contribute to the phenotype. To examine the effect of CLCN1 in DM2 in Germany, we determined the frequency of a representative ClC1 mutation, R894X, and its effect on DM2 clinical features. Then, we examined CLCN1 mRNA splice variants in patient muscle functionally expressed the most abundant variant, and determined its subcellular localization. Finally, we established a cellular system for studying mouse clcn1 pre-mRNA splicing and tested effects of expression of (CCUG)18, (CUG)24 and (AAG)24 RNAs. The R894X mutation was present in 7.7% of DM2 families. DM2 R894X-carriers had more myotonia and myalgia than non-carriers. The most abundant CLCN1 splice variant in DM2 (80% of all transcripts) excluded exons 6-7 and lead to a truncated ClC1236X protein. Heterologous ClC1236X expression did not yield functional channels. Co-expression with ClC1 did not show a dominant negative effect, but a slightly suppressive effect. In C2C12 cells, the clc1 splice variants generated by (CCUG)18-RNA resembled those in DM2 muscle and differed from those generated by (CUG)24 and (AAG)24. We conclude that ClC1 mutations exert gene dose effects and enhance myotonia and pain in DM2 in Germany. Additionally, the ClC1236X splice variant may contribute to myotonia in DM2. Since splice variants depend on the types of repeats expressed in the cellular C2C12 model, similar cell models of other tissues may be useful for studying repeatdependent pathogenetic mechanisms more easily than in transgenic animals.
PMCID: PMC3476861  PMID: 23097607
PROMM; myotonic dystrophy; chloride channel
16.  Regulation of the ClC-2 Lung Epithelial Chloride Channel by Glycosylation of SP1 
Chloride channel–2 (ClC-2) is a pH- and voltage-activated chloride channel that is highly expressed in mammalian fetal airway epithelia during the period of maximal fluid secretion. A high level of luminal ClC-2 protein expression is maintained by the SP1 transcription factor until SP1 and ClC-2 decline rapidly at birth. Using fetal (preII-19) and adult (L2) rat lung Type 2 cell lines, we demonstrate that the active higher-molecular-weight 105-kD isoform of SP1 is phosphorylated and glycosylated. Exposure of either cell line to high-dose glutamine is sufficient to induce glycosylation of SP1 and to induce and maintain ClC-2. Exposure to tunicamycin to inhibit SP1 glycosylation reduces ClC-2 expression. We also demonstrate that in vivo ClC-2 expression is similarly regulated. SP1 from 6-wk-old murine lung (high ClC-2 expression) is hyperphosphorylated and hyperglycosylated compared with SP1 from 16–wk-old lung (low ClC-2 expression). Our results support the hypothesis that glycosylation of SP1 produces the 105-kD isoform of SP1 and is involved in regulating ClC-2 gene expression.
doi:10.1165/rcmb.2005-0442OC
PMCID: PMC2644236  PMID: 16456185
chloride channel; transcription factor; cystic fibrosis; mouse; lung development
17.  Sarcolemmal-restricted localization of functional ClC-1 channels in mouse skeletal muscle 
The Journal of General Physiology  2010;136(6):597-613.
Skeletal muscle fibers exhibit a high resting chloride conductance primarily determined by ClC-1 chloride channels that stabilize the resting membrane potential during repetitive stimulation. Although the importance of ClC-1 channel activity in maintaining normal muscle excitability is well appreciated, the subcellular location of this conductance remains highly controversial. Using a three-pronged multidisciplinary approach, we determined the location of functional ClC-1 channels in adult mouse skeletal muscle. First, formamide-induced detubulation of single flexor digitorum brevis (FDB) muscle fibers from 15–16-day-old mice did not significantly alter macroscopic ClC-1 current magnitude (at −140 mV; −39.0 ± 4.5 and −42.3 ± 5.0 nA, respectively), deactivation kinetics, or voltage dependence of channel activation (V1/2 was −61.0 ± 1.7 and −64.5 ± 2.8 mV; k was 20.5 ± 0.8 and 22.8 ± 1.2 mV, respectively), despite a 33% reduction in cell capacitance (from 465 ± 36 to 312 ± 23 pF). In paired whole cell voltage clamp experiments, where ClC-1 activity was measured before and after detubulation in the same fiber, no reduction in ClC-1 activity was observed, despite an ∼40 and 60% reduction in membrane capacitance in FDB fibers from 15–16-day-old and adult mice, respectively. Second, using immunofluorescence and confocal microscopy, native ClC-1 channels in adult mouse FDB fibers were localized within the sarcolemma, 90° out of phase with double rows of dihydropyridine receptor immunostaining of the T-tubule system. Third, adenoviral-mediated expression of green fluorescent protein–tagged ClC-1 channels in adult skeletal muscle of a mouse model of myotonic dystrophy type 1 resulted in a significant reduction in myotonia and localization of channels to the sarcolemma. Collectively, these results demonstrate that the majority of functional ClC-1 channels localize to the sarcolemma and provide essential insight into the basis of myofiber excitability in normal and diseased skeletal muscle.
doi:10.1085/jgp.201010526
PMCID: PMC2995150  PMID: 21078869
18.  A missense mutation accelerating the gating of the lysosomal Cl−/H+-exchanger ClC-7/Ostm1 causes osteopetrosis with gingival hamartomas in cattle 
Disease Models & Mechanisms  2013;7(1):119-128.
Chloride-proton exchange by the lysosomal anion transporter ClC-7/Ostm1 is of pivotal importance for the physiology of lysosomes and bone resorption. Mice lacking either ClC-7 or Ostm1 develop a lysosomal storage disease and mutations in either protein have been found to underlie osteopetrosis in mice and humans. Some human disease-causing CLCN7 mutations accelerate the usually slow voltage-dependent gating of ClC-7/Ostm1. However, it has remained unclear whether the fastened kinetics is indeed causative for the disease. Here we identified and characterized a new deleterious ClC-7 mutation in Belgian Blue cattle with a severe symptomatology including perinatal lethality and in most cases gingival hamartomas. By autozygosity mapping and genome-wide sequencing we found a handful of candidate variants, including a cluster of three private SNPs causing the substitution of a conserved tyrosine in the CBS2 domain of ClC-7 by glutamine. The case for ClC-7 was strengthened by subsequent examination of affected calves that revealed severe osteopetrosis. The Y750Q mutation largely preserved the lysosomal localization and assembly of ClC-7/Ostm1, but drastically accelerated its activation by membrane depolarization. These data provide first evidence that accelerated ClC-7/Ostm1 gating per se is deleterious, highlighting a physiological importance of the slow voltage-activation of ClC-7/Ostm1 in lysosomal function and bone resorption.
doi:10.1242/dmm.012500
PMCID: PMC3882054  PMID: 24159188
CLCN7; Hamartomas; Osteopetrosis; Lysosomal storage; Ion homeostasis; Belgian Blue cattle
19.  Temperature Dependence of Fast and Slow Gating Relaxations of ClC-0 Chloride Channels  
The Journal of General Physiology  1997;109(1):105-116.
The chloride channel from the Torpedo electric organ, ClC-0, is the best studied member of a large gene-family (Jentsch, T.J. 1996. Curr. Opin. Neurobiol. 6:303–310.). We investigate the temperature dependence of both the voltage- and chloride-dependent fast gate and of the slow gate of the “double-barreled” ClC-0 expressed in Xenopus oocytes. Kinetics of the fast gate exhibit only a moderate temperature dependence with a Q10 of 2.2. Steady-state popen of the fast gate is relatively independent of temperature. The slow gate, in contrast, is highly temperature sensitive. Deactivation kinetics at positive voltages are associated with a Q10 of ∼40. Steady-state open probability of the slow gate (popenslow(V)) can be described by a Boltzmann distribution with an apparent gating valence of ≈2 and a variable “offset” at positive voltages. We note a positive correlation of this offset (i.e., the fraction of channels that are not closed by the slow gate) with the amount of expression. This offset is also highly temperature sensitive, being drastically decreased at high temperatures. Paradoxically, the maximum degree of activation of the slow gate also decreases at higher temperatures. The strong temperature dependence of the slow gate was also observed at the single channel level in inside-out patches. The results imply that within a Markovian-type description at least two open and two closed states are needed to describe slow gating. The strong temperature dependence of the slow gate explains the phenotype of several ClC-0 point-mutants described recently by Ludewig et al. (Ludewig, U., T.J. Jentsch, and M. Pusch. 1996. J. Physiol. (Lond.). In press). The large Q10 of slow gating kinetics points to a complex rearrangement. This, together with the correlation of the fraction of noninactivating channels with the amount of expression and the fact that the slow gate closes both protochannels simultaneously suggests that the slow gate is coupled to subunit interaction of the multimeric ClC-0 channel.
PMCID: PMC2217054  PMID: 8997669
anion channel; multimer; conformational change; double-barrel
20.  Physiology and Pathophysiology of ClC-K/barttin Channels 
ClC-K channels form a subgroup of anion channels within the ClC family of anion transport proteins. They are expressed predominantly in the kidney and in the inner ear, and are necessary for NaCl resorption in the loop of Henle and for K+ secretion by the stria vascularis. Subcellular distribution as well as the function of these channels are tightly regulated by an accessory subunit, barttin. Barttin improves the stability of ClC-K channel protein, stimulates the exit from the endoplasmic reticulum and insertion into the plasma membrane and changes its function by modifying voltage-dependent gating processes. The importance of ClC-K/barttin channels is highlighted by several genetic diseases. Dysfunctions of ClC-K channels result in Bartter syndrome, an inherited human condition characterized by impaired urinary concentration. Mutations in the gene encoding barttin, BSND, affect the urinary concentration as well as the sensory function of the inner ear. Surprisingly, there is one BSND mutation that causes deafness without affecting renal function, indicating that kidney function tolerates a reduction of anion channel activity that is not sufficient to support normal signal transduction in inner hair cells. This review summarizes recent work on molecular mechanisms, physiology, and pathophysiology of ClC-K/barttin channels.
doi:10.3389/fphys.2010.00155
PMCID: PMC3059957  PMID: 21423394
ClC channels; barttin; epithelial transport; loop of Henle; stria vascularis
21.  Regulation of ClC-2 gating by intracellular ATP 
Pflugers Archiv  2013;465(10):1423-1437.
ClC-2 is a voltage-dependent chloride channel that activates slowly at voltages negative to the chloride reversal potential. Adenosine triphosphate (ATP) and other nucleotides have been shown to bind to carboxy-terminal cystathionine-ß-synthase (CBS) domains of ClC-2, but the functional consequences of binding are not sufficiently understood. We here studied the effect of nucleotides on channel gating using single-channel and whole-cell patch clamp recordings on transfected mammalian cells. ATP slowed down macroscopic activation and deactivation time courses in a dose-dependent manner. Removal of the complete carboxy-terminus abolishes the effect of ATP, suggesting that CBS domains are necessary for ATP regulation of ClC-2 gating. Single-channel recordings identified long-lasting closed states of ATP-bound channels as basis of this gating deceleration. ClC-2 channel dimers exhibit two largely independent protopores that are opened and closed individually as well as by a common gating process. A seven-state model of common gating with altered voltage dependencies of opening and closing transitions for ATP-bound states correctly describes the effects of ATP on macroscopic and microscopic ClC-2 currents. To test for a potential pathophysiological impact of ClC-2 regulation by ATP, we studied ClC-2 channels carrying naturally occurring sequence variants found in patients with idiopathic generalized epilepsy, G715E, R577Q, and R653T. All naturally occurring sequence variants accelerate common gating in the presence but not in the absence of ATP. We propose that ClC-2 uses ATP as a co-factor to slow down common gating for sufficient electrical stability of neurons under physiological conditions.
Electronic supplementary material
The online version of this article (doi:10.1007/s00424-013-1286-0) contains supplementary material, which is available to authorized users.
doi:10.1007/s00424-013-1286-0
PMCID: PMC3778897  PMID: 23632988
Chloride channel; Single-channel recording; Channel gating; Epilepsy
22.  An association study on contrasting cystic fibrosis endophenotypes recognizes KRT8 but not KRT18 as a modifier of cystic fibrosis disease severity and CFTR mediated residual chloride secretion 
BMC Medical Genetics  2011;12:62.
Background
F508del-CFTR, the most frequent disease-causing mutation among Caucasian cystic fibrosis (CF) patients, has been characterised as a mutant defective in protein folding, processing and trafficking. We have investigated the two neighbouring cytokeratin genes KRT8 and KRT18 in a candidate gene approach to ask whether variants in KRT8 and/or KRT18 modify the impaired ion conductance known as the CF basic defect, and whether they are associated with correct trafficking of mutant CFTR and disease severity of CF.
Methods
We have selected contrasting F508del-CFTR homozygous patient subpopulations stratified for disease severity, comparing 13 concordant mildly affected sib pairs vs. 12 concordant severely affected sib pairs, or manifestation of the CF basic defect in intestinal epithelium, comparing 22 individuals who exhibit CFTR-mediated residual chloride secretion vs. 14 individuals who do not express any chloride secretion, for an association. The KRT8/KRT18 locus was initially interrogated with one informative microsatellite marker. Subsequently, a low density SNP map with four SNPs in KRT8 and two SNPs in KRT18, each selected for high polymorphism content, was used to localize the association signal.
Results
KRT8, but not KRT18, showed an association with CF disease severity (Pbest = 0.00131; Pcorr = 0.0185) and CFTR mediated residual chloride secretion (Pbest = 0.0004; Pcorr = 0.0069). Two major four-marker-haplotypes spanning 13 kb including the entire KRT8 gene accounted for 90% of chromosomes, demonstrating strong linkage disequilibrium at that locus. Absence of chloride secretion was associated with the recessive haplotype 1122 at rs1907671, rs4300473, rs2035878 and rs2035875. The contrasting haplotype 2211 was dominant for the presence of CFTR mediated residual chloride secretion. In consistency, the KRT8 haplotype 2211 was associated with mild CF disease while 1122 was observed as risk haplotype. Analysis of microsatellite allele distributions on the SNP background suggests that the mild KRT8 haplotype 2211 is phylogenetically older than its severe counterpart.
Conclusions
The two opposing KRT8 alleles which have been identified as a benign and as a risk allele in this work are likely effective in the context of epithelial cell differentiation. As the mild KRT8 allele is associated with CFTR mediated residual chloride secretion among F508del-CFTR homozygotes, the KRT8/KRT18 heterodimeric intermediary filaments of the cytoskeleton apparently are an essential component for the proper targeting of CFTR to the apical membrane in epithelial cells.
doi:10.1186/1471-2350-12-62
PMCID: PMC3107781  PMID: 21548936
23.  Asthma and COPD in cystic fibrosis intron-8 5T carriers. A population-based study 
Respiratory Research  2005;6(1):113.
Background
Carriers of cystic fibrosis intron-8 5T alleles with high exon-9 skipping could have increased annual lung function decline and increased risk for asthma or chronic obstructive pulmonary disease (COPD).
Methods
We genotyped 9131 individuals from the adult Danish population for cystic fibrosis 5T, 7T, 9T, and F508del alleles, and examined associations between 11 different genotype combinations, and annual FEV1 decline and risk of asthma or COPD.
Results
5T heterozygotes vs. 7T homozygous controls had no increase in annual FEV1 decline, self-reported asthma, spirometry-defined COPD, or incidence of hospitalization from asthma or COPD. In 5T/7T heterozygotes vs. 7T homozygous controls we had 90% power to detect an increase in FEV1 decline of 8 ml, an odds ratio for self-reported asthma and spirometry-defined COPD of 1.9 and 1.7, and a hazard ratio for asthma and COPD hospitalization of 1.8 and 1.6, respectively. Both 5T homozygotes identified in the study showed evidence of asthma, while none of four 5T/F508del compound heterozygotes had severe pulmonary disease. 7T/9T individuals had annual decline in FEV1 of 19 ml compared with 21 ml in 7T homozygous controls (t-test:P = 0.03). 6.7% of 7T homozygotes without an F508del allele in the cystic fibrosis transmembrane conductance regulator gene reported asthma vs. 11% of 7T/9T individuals with an F508del allele (χ2:P = 0.01) and 40% of 7T homozygotes with an F508del allele (P = 0.04). 7T homozygotes with vs. without an F508del allele also had higher incidence of asthma hospitalization (log-rank:P = 0.003); unadjusted and adjusted equivalent hazard ratios for asthma hospitalization were 11 (95%CI:1.5–78) and 6.3 (0.84–47) in 7T homozygotes with vs. without an F508del allele.
Conclusion
Polythymidine 5T heterozygosity is not associated with pulmonary dysfunction or disease in the adult Caucasian population. Furthermore, our results support that F508del heterozygosity is associated with increased asthma risk independently of the 5T allele.
doi:10.1186/1465-9921-6-113
PMCID: PMC1277850  PMID: 16212675
24.  Phenomics of Cardiac Chloride Channels 
Comprehensive Physiology  2013;3(2):667-692.
Forward genetic studies have identified several chloride (Cl−) channel genes, including CFTR, ClC-2, ClC-3, CLCA, Bestrophin, and Ano1, in the heart. Recent reverse genetic studies using gene targeting and transgenic techniques to delineate the functional role of cardiac Cl− channels have shown that Cl− channels may contribute to cardiac arrhythmogenesis, myocardial hypertrophy and heart failure, and cardioprotection against ischemia reperfusion. The study of physiological or pathophysiological phenotypes of cardiac Cl− channels, however, is complicated by the compensatory changes in the animals in response to the targeted genetic manipulation. Alternatively, tissue-specific conditional or inducible knockout or knockin animal models may be more valuable in the phenotypic studies of specific Cl− channels by limiting the effect of compensation on the phenotype. The integrated function of Cl− channels may involve multiprotein complexes of the Cl− channel subproteome. Similar phenotypes can be attained from alternative protein pathways within cellular networks, which are influenced by genetic and environmental factors. The phenomics approach, which characterizes phenotypes as a whole phenome and systematically studies the molecular changes that give rise to particular phenotypes achieved by modifying the genotype under the scope of genome/proteome/phenome, may provide more complete understanding of the integrated function of each cardiac Cl− channel in the context of health and disease.
doi:10.1002/cphy.c110014
PMCID: PMC3966620  PMID: 23720326
25.  The CLC-5 2Cl−/H+ exchange transporter in endosomal function and Dent's disease 
CLC-5 plays a critical role in the process of endocytosis in the proximal tubule of the kidney and mutations that alter protein function are the cause of Dent's I disease. In this X-linked disorder impaired reabsorption results in the wasting of calcium and low molecular weight protein to the urine, kidney stones, and progressive renal failure. Several different ion-transporting and protein clustering roles have been proposed as the physiological function of CLC-5 in endosomal membranes. At the time of its discovery, nearly 20 years ago, it was understandably assumed to be a chloride channel similar to known members of the CLC family, such as CLC-1, suggesting that chloride transport by CLC-5 was critical for endosomal function. Since then CLC-5 was found instead to be a 2Cl−/H+ exchange transporter with voltage-dependent activity. Recent studies have determined that it is this coupled exchange of protons for chloride, and not just chloride transport, which is critical for endosomal and kidney function. This review discusses the recent ideas that describe how CLC-5 might function in endosomal membranes, the aspects that we still do not understand, and where controversies remain.
doi:10.3389/fphys.2012.00449
PMCID: PMC3510460  PMID: 23226131
CLC-5; Dent's disease; endocytosis; endosomal acidification; chloride transport

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