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1.  Muscle Chloride Channel Dysfunction in Two Mouse Models of Myotonic Dystrophy 
Muscle degeneration and myotonia are clinical hallmarks of myotonic dystrophy type 1 (DM1), a multisystemic disorder caused by a CTG repeat expansion in the 3′ untranslated region of the myotonic dystrophy protein kinase (DMPK) gene. Transgenic mice engineered to express mRNA with expanded (CUG)250 repeats (HSALR mice) exhibit prominent myotonia and altered splicing of muscle chloride channel gene (Clcn1) transcripts. We used whole-cell patch clamp recordings and nonstationary noise analysis to compare and biophysically characterize the magnitude, kinetics, voltage dependence, and single channel properties of the skeletal muscle chloride channel (ClC-1) in individual flexor digitorum brevis (FDB) muscle fibers isolated from 1–3-wk-old wild-type and HSALR mice. The results indicate that peak ClC-1 current density at −140 mV is reduced >70% (−48.5 ± 3.6 and −14.0 ± 1.6 pA/pF, respectively) and the kinetics of channel deactivation increased in FDB fibers obtained from 18–20- d-old HSALR mice. Nonstationary noise analysis revealed that the reduction in ClC-1 current density in HSALR FDB fibers results from a large reduction in ClC-1 channel density (170 ± 21 and 58 ± 11 channels/pF in control and HSALR fibers, respectively) and a modest decrease in maximal channel open probability(0.91 ± 0.01 and 0.75 ± 0.03, respectively). Qualitatively similar results were observed for ClC-1 channel activity in knockout mice for muscleblind-like 1 (Mbnl1ΔE3/ΔE3), a second murine model of DM1 that exhibits prominent myotonia and altered Clcn1 splicing (Kanadia et al., 2003). These results support a molecular mechanism for myotonia in DM1 in which a reduction in both the number of functional sarcolemmal ClC-1 and maximal channel open probability, as well as an acceleration in the kinetics of channel deactivation, results from CUG repeat–containing mRNA molecules sequestering Mbnl1 proteins required for proper CLCN1 pre-mRNA splicing and chloride channel function.
doi:10.1085/jgp.200609635
PMCID: PMC2151606  PMID: 17158949
2.  MBNL and CELF proteins regulate alternative splicing of the skeletal muscle chloride channel CLCN1 
Nucleic Acids Research  2009;37(19):6477-6490.
The expression and function of the skeletal muscle chloride channel CLCN1/ClC-1 is regulated by alternative splicing. Inclusion of the CLCN1 exon 7A is aberrantly elevated in myotonic dystrophy (DM), a genetic disorder caused by the expansion of a CTG or CCTG repeat. Increased exon 7A inclusion leads to a reduction in CLCN1 function, which can be causative of myotonia. Two RNA-binding protein families—muscleblind-like (MBNL) and CUG-BP and ETR-3-like factor (CELF) proteins—are thought to mediate the splicing misregulation in DM. Here, we have identified multiple factors that regulate the alternative splicing of a mouse Clcn1 minigene. The inclusion of exon 7A was repressed by MBNL proteins while promoted by an expanded CUG repeat or CELF4, but not by CUG-BP. Mutation analyses suggested that exon 7A and its flanking region mediate the effect of MBNL1, whereas another distinct region in intron 6 mediates that of CELF4. An exonic splicing enhancer essential for the inclusion of exon 7A was identified at the 5′ end of this exon, which might be inhibited by MBNL1. Collectively, these results provide a mechanistic model for the regulation of Clcn1 splicing, and reveal novel regulatory properties of MBNL and CELF proteins.
doi:10.1093/nar/gkp681
PMCID: PMC2770659  PMID: 19720736
3.  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
4.  Correction of ClC-1 splicing eliminates chloride channelopathy and myotonia in mouse models of myotonic dystrophy 
The Journal of Clinical Investigation  2007;117(12):3952-3957.
In myotonic dystrophy (dystrophia myotonica [DM]), an increase in the excitability of skeletal muscle leads to repetitive action potentials, stiffness, and delayed relaxation. This constellation of features, collectively known as myotonia, is associated with abnormal alternative splicing of the muscle-specific chloride channel (ClC-1) and reduced conductance of chloride ions in the sarcolemma. However, the mechanistic basis of the chloride channelopathy and its relationship to the development of myotonia are uncertain. Here we show that a morpholino antisense oligonucleotide (AON) targeting the 3′ splice site of ClC-1 exon 7a reversed the defect of ClC-1 alternative splicing in 2 mouse models of DM. By repressing the inclusion of this exon, the AON restored the full-length reading frame in ClC-1 mRNA, upregulated the level of ClC-1 mRNA, increased the expression of ClC-1 protein in the surface membrane, normalized muscle ClC-1 current density and deactivation kinetics, and eliminated myotonic discharges. These observations indicate that the myotonia and chloride channelopathy observed in DM both result from abnormal alternative splicing of ClC-1 and that antisense-induced exon skipping offers a powerful method for correcting alternative splicing defects in DM.
doi:10.1172/JCI33355
PMCID: PMC2075481  PMID: 18008009
5.  Dominantly-Inherited Myotonia Congenita Resulting from a Mutation That Increases Open Probability of the Muscle Chloride Channel CLC-1 
Neuromolecular medicine  2012;14(4):328-337.
Myotonia congenita-inducing mutations in the muscle chloride channel CLC-1 normally result in reduced open probability (Po) of this channel. One well-accepted mechanism of the dominant inheritance of this disease involves a dominant-negative effect of the mutation on the function of the common gate of this homodimeric, double-barreled molecule. We report here a family with myotonia congenita characterized by muscle stiffness and clinical and electrophysiologic myotonic phenomena transmitted in an autosomal dominant pattern. DNA sequencing of DMPK and ZNF9 genes for myotonic muscular dystrophy types I and II was normal, whereas sequencing of CLC-1 encoding gene, CLCN1, identified a single heterozygous missense mutation, G233S. Patch-clamp analyses of this mutant CLC-1 channel in Xenopus oocytes revealed an increased Po of the channel’s fast gate, from ~ 0.4 in the wild type to > 0.9 in the mutant at −90 mV. In contrast, the mutant exhibits a minimal effect on the Po of the common gate. These results are consistent with the structural prediction that the mutation site is adjacent to the fast gate of the channel. Overall, the mutant could lead to a significantly reduced dynamic response of CLC-1 to membrane depolarization, from a 5-fold increase in chloride conductance in the wild type to a 2-fold increase in the mutant—this might result in slower membrane repolarization during an action potential. Since expression levels of the mutant and wild-type subunits in artificial model cell systems were unable to explain the disease symptoms, the mechanism leading to dominant inheritance in this family remains to be determined.
doi:10.1007/s12017-012-8190-1
PMCID: PMC3508202  PMID: 22790975
myotonia congenita; muscle; chloride channel; CLCN1; dominant; gain of function
6.  Transcriptional and post-transcriptional impact of toxic RNA in myotonic dystrophy 
Human Molecular Genetics  2009;18(8):1471-1481.
Myotonic dystrophy type 1 (DM1) is an RNA dominant disease in which mutant transcripts containing an expanded CUG repeat (CUGexp) cause muscle dysfunction by interfering with biogenesis of other mRNAs. The toxic effects of mutant RNA are mediated partly through sequestration of splicing regulator Muscleblind-like 1 (Mbnl1), a protein that binds to CUGexp RNA. A gene that is prominently affected encodes chloride channel 1 (Clcn1), resulting in hyperexcitability of muscle (myotonia). To identify DM1-affected genes and study mechanisms for dysregulation, we performed global mRNA profiling in transgenic mice that express CUGexp RNA, when compared with Mbnl1 knockout and Clcn1 null mice. We found that the majority of changes induced by CUGexp RNA in skeletal muscle can be explained by reduced activity of Mbnl1, including many changes that are secondary to myotonia. The pathway most affected comprises genes involved in calcium signaling and homeostasis. Some effects of CUGexp RNA on gene expression are caused by abnormal alternative splicing or downregulation of Mbnl1-interacting mRNAs. However, several of the most highly dysregulated genes showed altered transcription, as indicated by parallel changes of the corresponding pre-mRNAs. These results support the idea that trans-dominant effects of CUGexp RNA on gene expression in this transgenic model may occur at the level of transcription, RNA processing and mRNA decay, and are mediated mainly but not entirely through sequestration of Mbnl1.
doi:10.1093/hmg/ddp058
PMCID: PMC2664149  PMID: 19223393
7.  HTS-Compatible Patient-Derived Cell-Based Assay to Identify Small Molecule Modulators of Aberrant Splicing in Myotonic Dystrophy Type 1 
Myotonic dystrophy type 1 (DM1) is a genetic disorder characterized by muscle wasting, myotonia, cataracts, cardiac arrhythmia, hyperinsulinism and intellectual deficits, and is caused by expansion of a CTG repeat in the 3’UTR of the Dystrophia Myotonica-Protein Kinase (DMPK) gene. The DMPK transcripts containing expanded CUG repeats accumulate in nuclear foci and ultimately cause mis-splicing of secondary genes through the dysregulation of RNA-binding proteins including Muscleblind 1 (MBNL1) and CUG binding protein 1 (CUGBP1). Correction of mis-splicing of genes such as the Skeletal muscle-specific chloride channel 1 (CLCN1), Cardiac troponin T (TNNT2), Insulin receptor (INSR) and Sarcoplasmic/endoplasmic reticulum Ca2+ ATPase 1 (SERCA1) may alleviate some of the symptoms of DM1; hence identification of small molecule modulators is an important step towards a therapy for DM1 patients. Here we describe the generation of immortalized myoblast cell lines derived from healthy (DMPK CTG5) and DM1 patient (DMPK CTG1000) fibroblasts by constitutive overexpression of human telomerase reverse transcriptase (hTERT) and inducible overexpression of the Myoblast determination factor (MYOD). MBNL1-containing nuclear foci, mis-splicing events and defective myotube differentiation defects characteristic of DM1 were observed in these cells. A CLCN1 luciferase minigene construct (CLCN1-luc) was stably introduced to monitor intron 2 retention in the DM1 cellular context (a reported splicing defect in DM1). The assay was validated by performing a high-throughput screen (HTS) of ~13,000 low molecular weight compounds against the CLCN1-luc DM1 myoblast cell line, providing an ideal system for conducting HTS to better understand and treat DM1.
doi:10.2174/1875397301004010009
PMCID: PMC2874217  PMID: 20502647
Alternative splicing; HTS; myotonic dystrophy; DM1; cell-based assay.
8.  Novel brain expression of ClC-1 chloride channels and enrichment of CLCN1 variants in epilepsy 
Neurology  2013;80(12):1078-1085.
Objective:
To explore the potential contribution of genetic variation in voltage-gated chloride channels to epilepsy, we analyzed CLCN family (CLCN1-7) gene variant profiles in individuals with complex idiopathic epilepsy syndromes and determined the expression of these channels in human and murine brain.
Methods:
We used parallel exomic sequencing of 237 ion channel subunit genes to screen individuals with a clinical diagnosis of idiopathic epilepsy and evaluate the distribution of missense variants in CLCN genes in cases and controls. We examined regional expression of CLCN1 in human and mouse brain using reverse transcriptase PCR, in situ hybridization, and Western immunoblotting.
Results:
We found that in 152 individuals with sporadic epilepsy of unknown origin, 96.7% had at least one missense variant in the CLCN genes compared with 28.2% of 139 controls. Nonsynonymous single nucleotide polymorphisms in the “skeletal” chloride channel gene CLCN1 and in CLCN2, a putative human epilepsy gene, were detected in threefold excess in cases relative to controls. Among these, we report a novel de novo CLCN1 truncation mutation in a patient with pharmacoresistant generalized seizures and a dystonic writer's cramp without evidence of variants in other channel genes linked to epilepsy. Molecular localization revealed the unexpectedly widespread presence of CLCN1 mRNA transcripts and the ClC-1 subunit protein in human and murine brain, previously believed absent in neurons.
Conclusions:
Our findings support a possible comorbid contribution of the “skeletal” chloride channel ClC-1 to the regulation of brain excitability and the need for further elucidation of the roles of CLCN genes in neuronal network excitability disorders.
doi:10.1212/WNL.0b013e31828868e7
PMCID: PMC3662306  PMID: 23408874
9.  CLCN1 Mutations in Czech Patients with Myotonia Congenita, In Silico Analysis of Novel and Known Mutations in the Human Dimeric Skeletal Muscle Chloride Channel 
PLoS ONE  2013;8(12):e82549.
Myotonia congenita (MC) is a genetic disease caused by mutations in the skeletal muscle chloride channel gene (CLCN1) encoding the skeletal muscle chloride channel (ClC-1). Mutations of CLCN1 result in either autosomal dominant MC (Thomsen disease) or autosomal recessive MC (Becker disease). The ClC-1 protein is a homodimer with a separate ion pore within each monomer. Mutations causing recessive myotonia most likely affect properties of only the mutant monomer in the heterodimer, leaving the wild type monomer unaffected, while mutations causing dominant myotonia affect properties of both subunits in the heterodimer. Our study addresses two points: 1) molecular genetic diagnostics of MC by analysis of the CLCN1 gene and 2) structural analysis of mutations in the homology model of the human dimeric ClC-1 protein. In the first part, 34 different types of CLCN1 mutations were identified in 51 MC probands (14 mutations were new). In the second part, on the basis of the homology model we identified the amino acids which forming the dimer interface and those which form the Cl- ion pathway. In the literature, we searched for mutations of these amino acids for which functional analyses were performed to assess the correlation between localisation of a mutation and occurrence of a dominant-negative effect (corresponding to dominant MC). This revealed that both types of mutations, with and without a dominant-negative effect, are localised at the dimer interface while solely mutations without a dominant-negative effect occur inside the chloride channel. This work is complemented by structural analysis of the homology model which provides elucidation of the effects of mutations, including a description of impacts of newly detected missense mutations.
doi:10.1371/journal.pone.0082549
PMCID: PMC3859631  PMID: 24349310
10.  Functional characterization of ClC-1 mutations from patients affected by recessive myotonia congenita presenting with different clinical phenotypes☆ 
Experimental Neurology  2013;248(100):530-540.
Myotonia congenita (MC) is caused by loss-of-function mutations of the muscle ClC-1 chloride channel. Clinical manifestations include the variable association of myotonia and transitory weakness. We recently described a cohort of recessive MC patients showing, at a low rate repetitive nerves stimulation protocol, different values of compound muscle action potential (CMAP) transitory depression, which is considered the neurophysiologic counterpart of transitory weakness. From among this cohort, we studied the chloride currents generated by G190S (associated with pronounced transitory depression), F167L (little or no transitory depression), and A531V (variable transitory depression) hClC-1 mutants in transfected HEK293 cells using patch-clamp. While F167L had no effect on chloride currents, G190S dramatically shifts the voltage dependence of channel activation and A531V reduces channel expression. Such variability in molecular mechanisms observed in the hClC-1 mutants may help to explain the different clinical and neurophysiologic manifestations of each ClCN1 mutation. In addition we examined five different mutations found in compound heterozygosis with F167L, including the novel P558S, and we identified additional molecular defects. Finally, the G190S mutation appeared to impair acetazolamide effects on chloride currents in vitro.
Highlights
•Myotonia congenita is a muscle disorder due to mutations in ClC-1 chloride channel.•Eight ClC-1 channel mutants were studied using patch-clamp technique.•Mutations induce a variety of molecular defects in ClC-1 channel function.•We discuss the relationship between genotype and clinical phenotype.
doi:10.1016/j.expneurol.2013.07.018
PMCID: PMC3781327  PMID: 23933576
Acetazolamide; Chloride channel mutation; ClC-1 chloride channel; Genotype–phenotype relationship; Myotonia congenita; Non-dystrophic myotonia; Patch-clamp; Transitory weakness
11.  Ultrasound-enhanced delivery of Morpholino with Bubble liposomes ameliorates the myotonia of myotonic dystrophy model mice 
Scientific Reports  2013;3:2242.
Phosphorodiamidate morpholino oligonucleotide (PMO)-mediated control of the alternative splicing of the chloride channel 1 (CLCN1) gene is a promising treatment for myotonic dystrophy type 1 (DM1) because the abnormal splicing of this gene causes myotonia in patients with DM1. In this study, we optimised a PMO sequence to correct Clcn1 alternative splicing and successfully remedied the myotonic phenotype of a DM1 mouse model, the HSALR mouse. To enhance the efficiency of delivery of PMO into HSALR mouse muscles, Bubble liposomes, which have been used as a gene delivery tool, were applied with ultrasound exposure. Effective delivery of PMO led to increased expression of Clcn1 protein in skeletal muscle and the amelioration of myotonia. Thus, PMO-mediated control of the alternative splicing of the Clcn1 gene must be important target of antisense therapy of DM1.
doi:10.1038/srep02242
PMCID: PMC3718203  PMID: 23873129
12.  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
13.  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
14.  CLC-5 and KIF3B interact to facilitate CLC-5 plasma membrane expression, endocytosis, and microtubular transport: relevance to pathophysiology of Dent's disease 
Renal tubular reabsorption is important for extracellular fluid homeostasis and much of this occurs via the receptor-mediated endocytic pathway. This pathway is disrupted in Dent’s disease, an X-linked renal tubular disorder that is characterized by low-molecular-weight proteinuria, hypercalciuria, nephrolithiasis, and renal failure. Dent's disease is due to mutations of CLC-5, a chloride/proton antiporter, expressed in endosomes and apical membranes of renal tubules. Loss of CLC-5 function alters receptor-mediated endocytosis and trafficking of megalin and cubilin, although the underlying mechanisms remain to be elucidated. Here, we report that CLC-5 interacts with kinesin family member 3B (KIF3B), a heterotrimeric motor protein that facilitates fast anterograde translocation of membranous organelles. Using yeast two-hybrid, glutathione-S-transferase pull-down and coimmunoprecipitation assays, the COOH terminus of CLC-5 and the coiled-coil and globular domains of KIF3B were shown to interact. This was confirmed in vivo by endogenous coimmunoprecipitation of CLC-5 and KIF3B and codistribution with endosomal markers in mouse kidney fractions. Confocal live cell imaging in kidney cells further demonstrated association of CLC-5 and KIF3B, and transport of CLC-5-containing vesicles along KIF3B microtubules. KIF3B overexpression and underexpression, using siRNA, had reciprocal effects on whole cell chloride current amplitudes, CLC-5 cell surface expression, and endocytosis of albumin and transferrin. Clcn5Y/− mouse kidneys and isolated proximal tubular polarized cells showed increased KIF3B expression, whose effects on albumin endocytosis were dependent on CLC-5 expression. Thus, the CLC-5 and KIF3B interaction is important for CLC-5 plasma membrane expression and for facilitating endocytosis and microtubular transport in the kidney.
doi:10.1152/ajprenal.00038.2009
PMCID: PMC2822520  PMID: 19940036
chloride/proton antiporter; kinesin family; proximal tubular reabsorption; hypercalciuric nephrolithiasis; receptor-mediated endocytosis
15.  A Novel Mutation in CLCN1 Associated with Feline Myotonia Congenita 
PLoS ONE  2014;9(10):e109926.
Myotonia congenita (MC) is a skeletal muscle channelopathy characterized by inability of the muscle to relax following voluntary contraction. Worldwide population prevalence in humans is 1∶100,000. Studies in mice, dogs, humans and goats confirmed myotonia associated with functional defects in chloride channels and mutations in a skeletal muscle chloride channel (CLCN1). CLCN1 encodes for the most abundant chloride channel in the skeletal muscle cell membrane. Five random bred cats from Winnipeg, Canada with MC were examined. All cats had a protruding tongue, limited range of jaw motion and drooling with prominent neck and proximal limb musculature. All cats had blepharospasm upon palpebral reflex testing and a short-strided gait. Electromyograms demonstrated myotonic discharges at a mean frequency of 300 Hz resembling the sound of a ‘swarm of bees’. Muscle histopathology showed hypertrophy of all fiber types. Direct sequencing of CLCN1 revealed a mutation disrupting a donor splice site downstream of exon 16 in only the affected cats. In vitro translation of the mutated protein predicted a premature truncation and partial lack of the highly conserved CBS1 (cystathionine β-synthase) domain critical for ion transport activity and one dimerization domain pivotal in channel formation. Genetic screening of the Winnipeg random bred population of the cats' origin identified carriers of the mutation. A genetic test for population screening is now available and carrier cats from the feral population can be identified.
doi:10.1371/journal.pone.0109926
PMCID: PMC4214686  PMID: 25356766
16.  Complexity of the 5′UTR region of the CLCN5 gene: eleven 5′UTR ends are differentially expressed in the human kidney 
BMC Medical Genomics  2014;7:41.
Background
Dent disease 1 represents a hereditary disorder of renal tubular epithelial function associated with mutations in the CLCN5 gene that encoded the ClC-5 Cl-/H+ antiporter. All of the reported disease-causing mutations are localized in the coding region except for one recently identified in the 5’UTR region of a single patient. This finding highlighted the possible role for genetic variability in this region in the pathogenesis of Dent disease 1.
The structural complexity of the CLCN5 5’UTR region has not yet been fully characterized. To date 6 different 5’ alternatively used exons - 1a, 1b, 1b1 and I-IV with an alternatively spliced exon II (IIa, IIb) - have been described, but their significance and differential expression in the human kidney have not been investigated. Therefore our aim was to better characterize the CLCN5 5’UTR region in the human kidney and other tissues.
Methods
To clone more of the 5’ end portion of the human CLCN5 cDNA, total human kidney RNA was utilized as template and RNA ligase-mediated rapid amplification of cDNA 5’ ends was applied.
The expression of the different CLCN5 isoforms was studied in the kidney, leucocytes and in different tissues by quantitative comparative RT/PCR and Real -Time RT/PCR.
Results
Eleven transcripts initiating at 3 different nucleotide positions having 3 distinct promoters of varying strength were identified. Previously identified 5’UTR isoforms were confirmed, but their ends were extended. Six additional 5’UTR ends characterized by the presence of new untranslated exons (c, V and VI) were also identified. Exon c originates exon c.1 by alternative splicing. The kidney uniquely expresses all isoforms, and the isoform containing exon c appears kidney specific. The most abundant isoforms contain exon 1a, exon IIa and exons 1b1 and c. ORF analysis predicts that all isoforms except 3 encode for the canonical 746 amino acid ClC-5 protein.
Conclusions
Our results confirm the structural complexity of the CLCN5 5’UTR region. Characterization of this crucial region could allow a clear genetic classification of a greater number of Dent disease patients, but also provide the basis for highlighting some as yet unexplored functions of the ClC-5 proton exchanger.
doi:10.1186/1755-8794-7-41
PMCID: PMC4105828  PMID: 25001568
Dent disease; CLCN5 gene; 5’UTR isoforms; mRNA variant; Gene expression
17.  Manumycin A corrects aberrant splicing of Clcn1 in myotonic dystrophy type 1 (DM1) mice 
Scientific Reports  2013;3:2142.
Myotonic dystrophy type 1 (DM1) is the most common muscular dystrophy in adults and as yet no cure for DM1. Here, we report the potential of manumycin A for a novel DM1 therapeutic reagent. DM1 is caused by expansion of CTG repeat. Mutant transcripts containing expanded CUG repeats lead to aberrant regulation of alternative splicing. Myotonia (delayed muscle relaxation) is the most commonly observed symptom in DM1 patients and is caused by aberrant splicing of the skeletal muscle chloride channel (CLCN1) gene. Identification of small-molecule compounds that correct aberrant splicing in DM1 is attracting much attention as a way of improving understanding of the mechanism of DM1 pathology and improving treatment of DM1 patients. In this study, we generated a reporter screening system and searched for small-molecule compounds. We found that manumycin A corrects aberrant splicing of Clcn1 in cell and mouse models of DM1.
doi:10.1038/srep02142
PMCID: PMC3701899  PMID: 23828222
18.  New immunohistochemical method for improved myotonia and chloride channel mutation diagnostics 
Neurology  2012;79(22):2194-2200.
Objective:
The objective of this study was to validate the immunohistochemical assay for the diagnosis of nondystrophic myotonia and to provide full clarification of clinical disease to patients in whom basic genetic testing has failed to do so.
Methods:
An immunohistochemical assay of sarcolemmal chloride channel abundance using 2 different ClC1-specific antibodies.
Results:
This method led to the identification of new mutations, to the reclassification of W118G in CLCN1 as a moderately pathogenic mutation, and to confirmation of recessive (Becker) myotonia congenita in cases when only one recessive CLCN1 mutation had been identified by genetic testing.
Conclusions:
We have developed a robust immunohistochemical assay that can detect loss of sarcolemmal ClC-1 protein on muscle sections. This in combination with gene sequencing is a powerful approach to achieving a final diagnosis of nondystrophic myotonia.
doi:10.1212/WNL.0b013e31827595e2
PMCID: PMC3570820  PMID: 23152584
19.  Zebrafish deficient for Muscleblind-like 2 exhibit features of myotonic dystrophy 
Disease Models & Mechanisms  2011;4(3):381-392.
SUMMARY
Myotonic dystrophy (DM; also known as dystrophia myotonica) is an autosomal dominant disorder that affects the heart, eyes, brain and endocrine system, but the predominant symptoms are neuromuscular, with progressive muscle weakness and wasting. DM presents in two forms, DM1 and DM2, both of which are caused by nucleotide repeat expansions: CTG in the DMPK gene for DM1 and CCTG in ZNF9 (CNBP) for DM2. Previous studies have shown that the mutant mRNAs containing the transcribed CUG or CCUG repeats are retained within the nuclei of cells from individuals with DM, where they bind and sequester the muscleblind-like proteins MBNL1, MBNL2 and MBNL3. It has been proposed that the sequestration of these proteins plays a key role in determining the classic features of DM. However, the functions of each of the three MBNL genes are not completely understood. We have generated a zebrafish knockdown model in which we demonstrate that a lack of mbnl2 function causes morphological abnormalities at the eye, heart, brain and muscle levels, supporting an essential role for mbnl2 during embryonic development. Major features of DM are replicated in our model, including muscle defects and splicing abnormalities. We found that the absence of mbnl2 causes disruption to the organization of myofibrils in skeletal and heart muscle of zebrafish embryos, and a reduction in the amount of both slow and fast muscle fibres. Notably, our findings included altered splicing patterns of two transcripts whose expression is also altered in DM patients: clcn1 and tnnt2. The studies described herein provide broader insight into the functions of MBNL2. They also lend support to the hypothesis that the sequestration of this protein is an important determinant in DM pathophysiology, and imply a direct role of MBNL2 in splicing regulation of specific transcripts, which, when altered, contributes to the DM phenotype.
doi:10.1242/dmm.004150
PMCID: PMC3097459  PMID: 21303839
20.  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
21.  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
22.  The Muscle Chloride Channel ClC-1 Has a Double-Barreled Appearance that Is Differentially Affected in Dominant and Recessive Myotonia  
The Journal of General Physiology  1999;113(3):457-468.
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 ≈ 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.
PMCID: PMC2222904  PMID: 10051520
ClC-0; fast gate; slow gate; subconductance
23.  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
24.  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
25.  Novel Mutations in the CLCN1 Gene of Myotonia Congenita: 2 Case Reports 
Introduction: Myotonia Congenita is an inherited myotonia that is due to a mutation in the skeletal muscle chloride channel CLCN1. These mutations lead to reduced sarcolemmal chloride conductance, causing delayed muscle relaxation that is evident as clinical and electrical myotonia.
Methods: We report the clinical presentations of two individuals with Myotonia Congenita (MC).
Results: Patient 1 has been diagnosed with the recessive form of MC, known as the Becker variant, and Patient 2 has been diagnosed with the dominant form of MC, known as the Thomsen variant. In both patients, the diagnosis was made based on the clinical presentation, EMG and CLCN1 gene sequencing. Patient 1 also had a muscle biopsy.
Conclusions: Genetic testing in both patients reveals previously unidentified mutations in the CLCN1 gene specific to Myotonia Congenita. We report the salient clinical features of each patient and discuss the effects and common types of CLCN1 mutations and review the literature.
PMCID: PMC3584487  PMID: 23483815
Myotonia Congenita; Becker variant; Thomsen variant; CLCN1 mutation

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