A missense mutation in the skeletal muscle chloride channel 1 (CLCN1) as candidate causal mutation for congenital myotonia in a New Forest pony

A 7-month-old New Forest foal presented for episodes of recumbency and stiffness with myotonic discharges on electromyography. The observed phenotype resembled congenital myotonia caused by CLCN1 mutations in goats and humans. Mutation of the CLCN1 gene was considered as possible cause and mutation analysis was performed. The affected foal was homozygous for a missense mutation (c.1775A>C, p.D592A) located in a well conserved domain of the CLCN1 gene. The mutation showed a recessive mode of inheritance within the reported pony family. Therefore, this CLCN1 polymorphism is considered to be a possible cause of congenital myotonia.

A Spanish founder mutation in the chloride channel gene, CLCNKB, as a cause of atypical Bartter syndrome in adult age

BACKGROUND: Mutations in the chloride channel gene, CLCNKB, usually cause classic Bartter syndrome (cBS) or a mixed Bartter-Gitelman phenotype in the first years of life. METHODS: We report an adult woman with atypical BS caused by a homozygous missense mutation, A204T, in the CLCNKB gene, which has previously been described as the apparently unique cause of cBS in Spain. RESULTS: The evaluation of this patient revealed an overlap of phenotypic features ranging from severe biochemical and systemic disturbances typical of cBS to scarce symptoms and diagnosis in the adult age typical of Gitelman syndrome. The tubular disease caused a dramatic effect on mental, growth and puberal development leading to low IQ, final short stature and abnormal ovarian function. Furthermore, low serum PTH concentrations with concomitant nephrocalcinosis and normocalcaemia were observed. Both ovarian function and serum PTH levels were normalized after treatment with cyclooxygenase inhibitors. CONCLUSIONS: The present report confirms a weak genotype-phenotype correlation in patients with CLCNKB mutations and supports the founder effect of the A204T mutation in Spain. In our country, the genetic diagnosis of adult patients with hereditary hypokalaemic tubulopathies should include a screening of A204T mutation in the CLCNKB gene.

Patients with biallelic mutations in the chloride channel gene CLCNKB: long-term management and outcome

BACKGROUND: Little information on the management and long-term follow-up of patients with biallelic mutations in the chloride channel gene CLCNKB is available. METHODS: Long-term follow-up was evaluated from 5.0 to 24 years (median, 14 years) after diagnosis in 13 patients with homozygous (n = 10) or compound heterozygous (n = 3) mutations. RESULTS: Medical treatment at last follow-up control included supplementation with potassium in 12 patients and sodium in 2 patients and medical treatment with indomethacin in 9 patients. At the end of follow-up, body height was 2.0 standard deviation score or less in 6 patients; 2 of these patients had growth hormone deficiency. Body weight (chloride channel gene CLCNKB tend to present with pathological proteinuria and impaired kidney function after a median follow-up of 14 years, and growth retardation is common and sometimes related to growth hormone deficiency in these patients.

Novel chloride channel mutations leading to mild myotonia among Chinese

We describe two Chinese families with a mild form of the myotonia congenita due to novel chloride channel (ClCN1) mutations. In one case, heterozygous I553F and H555N mutations were found. The patient shared the I553F mutation with his healthy father, and his mother had a history of mild myotonia when she was younger. In another family, autosomal dominant myotonia congenita was due to a L844F change. The physiological effects of the mutations were examined by using the two-electrode voltage-clamp technique after expression of the channels in Xenopus oocytes. All mutations drastically shifted the voltage required for half-maximal activation, more under conditions mimicking the homozygous situation, than under conditions mimicking the heterozygous situation. The larger effect was seen in the compound heterozygous situation combining the I553F and the H555N mutations. Our data suggest that myotonia congenita caused by CLCN1 mutations in Chinese have similar variable features to those found in the West.

Chloride channels in myotonia congenita assessed by velocity recovery cycles

Introduction: Myotonia congenita (MC) is caused by congenital defects in the muscle chloride channel CLC-1. This study used muscle velocity recovery cycles (MVRCs) to investigate how membrane function is affected. Methods: MVRCs and responses to repetitive stimulation were compared between 18 patients with genetically confirmed MC (13 recessive, 7 dominant) and 30 age-matched normal controls. Results: MC patients exhibited increased early supernormality, but treatment with sodium channel blockers prevented this. After multiple conditioning stimuli, late supernormality was enhanced in all MC patients, indicating delayed repolarization. These abnormalities were similar between the MC subtypes, but recessive patients showed a greater drop in amplitude during repetitive stimulation. Discussion: MVRCs indicate that chloride conductance only becomes important when muscle fibers are depolarized. The differential responses to repetitive stimulation suggest that in dominant MC the affected chloride channels are activated by strong depolarization, consistent with a positive shift of the CLC-1 activation curve. © 2013 Wiley Periodicals, Inc.

The CFTR frameshift mutation 3905insT and its effect at transcript and protein level

Cystic fibrosis (CF) is one of the most common genetic diseases in the Caucasian population and is characterized by chronic obstructive pulmonary disease, exocrine pancreatic insufficiency, and elevation of sodium and chloride concentrations in the sweat and infertility in men. The disease is caused by mutations in the CF transmembrane conductance regulator (CFTR) gene, which encodes a protein that functions as chloride channel at the apical membrane of different epithelia. Owing to the high genotypic and phenotypic disease heterogeneity, effects and consequences of the majority of the CFTR mutations have not yet been studied. Recently, the frameshift mutation 3905insT was identified as the second most frequent mutation in the Swiss population and found to be associated with a severe phenotype. The frameshift mutation produces a premature termination codon (PTC) in exon 20, and transcripts bearing this PTC are potential targets for degradation through nonsense-mediated mRNA decay (NMD) and/or for exon skipping through nonsense-associated alternative splicing (NAS). Using RT-PCR analysis in lymphocytes and different tissue types from patients carrying the mutation, we showed that the PTC introduced by the mutation does neither elicit a degradation of the mRNA through NMD nor an alternative splicing through NAS. Moreover, immunocytochemical analysis in nasal epithelial cells revealed a significantly reduced amount of CFTR at the apical membrane providing a possible molecular explanation for the more severe phenotype observed in F508del/3905insT compound heterozygotes compared with F508del homozygotes. However, further experiments are needed to elucidate the fate of the 3905insT CFTR in the cell after its biosynthesis.

Characterization of the canine CLCN3 gene and evaluation as candidate for late-onset NCL

BACKGROUND: The neuronal ceroid lipofuscinoses (NCL) are a heterogenous group of inherited progressive neurodegenerative diseases in different mammalian species. Tibetan Terrier and Polish Owczarek Nizinny (PON) dogs show rare late-onset NCL variants with autosomal recessive inheritance, which can not be explained by mutations of known human NCL genes. These dog breeds represent animal models for human late-onset NCL. In mice the chloride channel 3 gene (Clcn3) encoding an intracellular chloride channel was described to cause a phenotype similar to NCL. RESULTS: Two full-length cDNA splice variants of the canine CLCN3 gene are reported. The current canine whole genome sequence assembly was used for gene structure analyses and revealed 13 coding CLCN3 exons in 52 kb of genomic sequence. Sequence analysis of the coding exons and flanking intron regions of CLCN3 using six NCL-affected Tibetan terrier dogs and an NCL-affected Polish Owczarek Nizinny (PON) dog, as well as eight healthy Tibetan terrier dogs revealed 13 SNPs. No consistent CLCN3 haplotype was associated with NCL. CONCLUSION: For the examined animals we excluded the complete coding region and adjacent intronic regions of canine CLCN3 to harbor disease-causing mutations. Therefore it seems to be unlikely that a mutation in this gene is responsible for the late-onset NCL phenotype in these two dog breeds.

Exon 17 skipping in CLCN1 leads to recessive myotonia congenita

Mutations in CLCN1, the gene encoding the ClC-1 chloride channel in skeletal muscle, lead to myotonia congenita. The effects on the intramembranous channel forming domains have been investigated more than that at the intracellular C-terminus. We have performed a mutation screen involving the whole CLCN1 gene of patients with myotonia congenita by polymerase chain reaction (PCR), single-strand conformation polymorphism studies, and sequencing. Two unrelated patients harbored the same homozygous G-to-T mutation on the donor splice site of intron 17. This led to the skipping of exon 17, as evidenced by the reverse transcriptase PCR. When the exon 17-deleted CLCN1 was expressed in Xenopus oocytes, no chloride current was measurable. This function could be restored by coexpression with the wild-type channel. Our data suggest an important role of this C-terminal region and that exon 17 skipping resulting from a homozygous point mutation in CLCN1 can lead to recessive myotonia congenita.

Differential association of MUC5AC and CLCA1 expression in small cartilaginous airways of RAO-affected and control horses

REASONS FOR PERFORMING STUDY: Airway mucus accumulation is associated with indoor irritant and allergen exposure in horses with recurrent airway obstruction (RAO). Epidermal growth factor receptor (EGFR) and a chloride channel (calcium activated, family member 1; CLCA1) are key signalling molecules involved in mucin gene expression. OBJECTIVES: We hypothesised that exposure to irritants and aeroallergens would lead to increased expression of the mucin gene eqMUC5AC and increased stored mucosubstance in the airways of RAO-affected horses, associated with increased neutrophils and CLCA1 and EGFR mRNA levels. METHODS: We performed quantitative RT-PCR of eqMUC5AC, CLCA1 and EGFR; volume density measurements of intraepithelial mucosubstances; and cytological differentiation of intraluminal inflammatory cells in small cartilaginous airways from cranial left and right and caudal left and right lung lobes of 5 clinically healthy and 5 RAO-affected horses that had been exposed to indoor stable environment for 5 days before euthanasia. RESULTS: Neutrophils were increased in RAO-affected horses compared to clinically healthy controls. EqMUC5AC mRNA levels were positively correlated with both CLCA1 and EGFR mRNA levels in RAO-affected horses but only with CLCA1 in controls. The relationship between eqMUC5AC and CLCA1 differed in the 2 groups of horses with RAO-affected animals overexpressing CLCA1 in relation to eqMUC5AC. CONCLUSIONS: These data implicate CLCA1 as a signalling molecule in the expression of eqMUC5AC in horses but also suggest differential regulation by CLCA1 and EGFR between horses with RAO and those with milder degrees of airway inflammation.

Biokinetics of nanoparticles and susceptibility to particulate exposure in a murine model of cystic fibrosis.

BACKGROUND Persons with cystic fibrosis (CF) are at-risk for health effects from ambient air pollution but little is known about the interaction of nanoparticles (NP) with CF lungs. Here we study the distribution of inhaled NP in a murine CF model and aim to reveal mechanisms contributing to adverse effects of inhaled particles in susceptible populations. METHODS Chloride channel defective CftrTgH (neoim) Hgu mice were used to analyze lung function, lung distribution and whole body biokinetics of inhaled NP, and inflammatory responses after intratracheal administration of NP. Distribution of 20-nm titanium dioxide NP in lungs was assessed on ultrathin sections immediately and 24 h after a one-hour NP inhalation. NP biokinetics was deduced from total and regional lung deposition and from whole body translocation of inhaled 30-nm iridium NP within 24 h after aerosol inhalation. Inflammatory responses were assessed within 7 days after carbon NP instillation. RESULTS Cftr mutant females had moderately reduced lung compliance and slightly increased airway resistance compared to wild type mice. We found no genotype dependent differences in total, regional and head deposition or in secondary-organ translocation of inhaled iridium NP. Titanium dioxide inhalation resulted in higher NP uptake by alveolar epithelial cells in Cftr mutants. Instillation of carbon NP induced a comparable acute and transient inflammatory response in both genotypes. The twofold increase of bronchoalveolar lavage (BAL) neutrophils in Cftr mutant compared to wild type mice at day 3 but not at days 1 and 7, indicated an impaired capacity in inflammation resolution in Cftr mutants. Concomitant to the delayed decline of neutrophils, BAL granulocyte-colony stimulating factor was augmented in Cftr mutant mice. Anti-inflammatory 15-hydroxyeicosatetraenoic acid was generally significantly lower in BAL of Cftr mutant than in wild type mice. CONCLUSIONS Despite lacking alterations in lung deposition and biokinetics of inhaled NP, and absence of significant differences in lung function, higher uptake of NP by alveolar epithelial cells and prolonged, acute inflammatory responses to NP exposure indicate a moderately increased susceptibility of lungs to adverse effects of inhaled NP in Cftr mutant mice and provides potential mechanisms for the increased susceptibility of CF patients to air pollution.

Calcium-modulated chloride pathways contribute to chloride flux in murine cystic fibrosis-affected macrophages

Cystic fibrosis (CF), a common lethal inherited disorder defined by ion transport abnormalities, chronic infection, and robust inflammation, is the result of mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) protein, a cAMP-activated chloride (Cl-) channel. Macrophages are reported to have impaired activity in CF. Previous studies suggest that Cl- transport is important for macrophage function; therefore, impaired Cl- secretion may underlie CF macrophage dysfunction. To determine whether alterations in Cl- transport exist in CF macrophages, Cl- efflux was measured using N-[ethoxycarbonylmethyl]- 6-methoxy-quinolinium bromide (MQAE), a fluorescent indicator dye. The contribution of CFTR was assessed by calculating Cl- flux in the presence and absence of cftr(inh)-172. The contribution of calcium (Ca(2+))-modulated Cl- pathways was assessed by examining Cl- flux with varied extracellular Ca(2+) concentrations or after treatment with carbachol or thapsigargin, agents that increase intracellular Ca(2+) levels. Our data demonstrate that CFTR contributed to Cl- efflux only in WT macrophages, while Ca(2+)-mediated pathways contributed to Cl- transport in CF and WT macrophages. Furthermore, CF macrophages demonstrated augmented Cl- efflux with increases in extracellular Ca(2+). Taken together, this suggests that Ca(2+)-mediated Cl- pathways are enhanced in CF macrophages compared with WT macrophages.

A novel open channel blocker of GABA-A receptors

GABA-A receptors are chloride ion channels composed of five subunits, mediating fast synaptic and tonic inhibition in the mammalian brain. 19 different subunit isoforms have been identified, with the major receptor type in mammalian adult brain consisting of α1, β2, and γ2 subunits. GABA-A receptors are the target of numerous sedating and anxiolytic drugs such as benzodiazepines. The currently known endogenous ligands are GABA, neurosteroids and the endocannabinoid 2- arachidonoyl glycerol (2-AG). The pharmacological properties of this chloride ion channel strictly depend on receptor subunit composition and arrangement. GABA-A receptors bind and are inhibited by epileptogenic agents such as picrotoxin, and cyclodiene insecticides such as dieldrin. We screened aromatic monovalent anions with five-fold symmetry for inhibition of GABA-A receptors. One of the anions, PCCPinhibited currents elicited by GABA with comparable potency as picrotoxin. This inhibition showed all characteristics of an open channel block. The GABA-A receptor ion channel is lined by residues from the M2 membrane-spanning segment. To identify important residues of the pore involved in the interaction with the blocking molecules PCCP-, a mutation scan was performed in combination with subsequent analysis of the expressed mutant proteins using electrophysiological techniques. In a second project we characterised a light-switchable modulator of GABA-A receptors based on propofol. It was my responsibility to investigate the switching kinetics in patch clamp experiments. After its discovery in 1980, propofol has become the most widely used intravenous general anaesthetic. It is commonly accepted that the anaesthesia induced by this unusually lipophilic drug mostly results from potentiation of GABA induced currents. While GABA-A receptors respond to a variety of ligands, they are normally not sensitive towards light. This light sensitivity could be indirectly achieved by using modulators that can be optically switched between an active and an inactive form. We tested an azobenzene derivative of propofol where an aryldiazene unit is directly coupled to the pharmacophore. This molecule was termed azopropofol (AP2). The effect of AP2 on Cl- currents was investigated with electrophysiological techniques using α1β2γ2 GABA-A receptors expressed in Xenopus oocytes and HEK-cells. In the third project we wanted to investigate the functional role of GABA-A receptors in the liver, and their possible involvement in cell proliferation. GABA-A receptors are also found in a wide range of peripheral tissues, including parts of the peripheral nervous system and non-neural tissues such as smooth muscle, the female reproductive system, liver and several cancer tissues. However their precise function in non neuronal or cancerous cells is still unknown. For this purpose we investigated expression, localization and function of the hepatocytes GABA-A receptors in model cell lines and healthy and cancerous hepatocytes.

A pentasymmetric open channel blocker for Cys-loop receptor channels.

γ-Aminobutyric acid type A receptors (GABAA receptors) are chloride ion channels composed of five subunits, mediating fast synaptic and tonic inhibition in the mammalian brain. These receptors show near five-fold symmetry that is most pronounced in the second trans-membrane domain M2 lining the Cl- ion channel. To take advantage of this inherent symmetry, we screened a variety of aromatic anions with matched symmetry and found an inhibitor, pentacyanocyclopentdienyl anion (PCCP-) that exhibited all characteristics of an open channel blocker. Inhibition was strongly dependent on the membrane potential. Through mutagenesis and covalent modification, we identified the region α1V256-α1T261 in the rat recombinant GABAA receptor to be important for PCCP- action. Introduction of positive charges into M2 increased the affinity for PCCP- while PCCP- prevented the access of a positively charged molecule into M2. Interestingly, other anion selective cys-loop receptors were also inhibited by PCCP-, among them the Drosophila RDL GABAA receptor carrying an insecticide resistance mutation, suggesting that PCCP- could serve as an insecticide.

Cardiac sodium channel Na(v)1.5 and interacting proteins: Physiology and pathophysiology

The cardiac voltage-gated Na(+) channel Na(v)1.5 generates the cardiac Na(+) current (INa). Mutations in SCN5A, the gene encoding Na(v)1.5, have been linked to many cardiac phenotypes, including the congenital and acquired long QT syndrome, Brugada syndrome, conduction slowing, sick sinus syndrome, atrial fibrillation, and dilated cardiomyopathy. The mutations in SCN5A define a sub-group of Na(v)1.5/SCN5A-related phenotypes among cardiac genetic channelopathies. Several research groups have proposed that Na(v)1.5 may be part of multi-protein complexes composed of Na(v)1.5-interacting proteins which regulate channel expression and function. The genes encoding these regulatory proteins have also been found to be mutated in patients with inherited forms of cardiac arrhythmias. The proteins that associate with Na(v)1.5 may be classified as (1) anchoring/adaptor proteins, (2) enzymes interacting with and modifying the channel, and (3) proteins modulating the biophysical properties of Na(v)1.5 upon binding. The aim of this article is to review these Na(v)1.5 partner proteins and to discuss how they may regulate the channel's biology and function. These recent investigations have revealed that the expression level, cellular localization, and activity of Na(v)1.5 are finely regulated by complex molecular and cellular mechanisms that we are only beginning to understand.

Radiological stab wound channel depiction with instillation of contrast medium

The radiological depiction of stab wounds in soft-tissues using multislice computed tomography is difficult and the hereby obtained images often lack contrast. To overcome these shortcomings we tried a new method. We gently instilled the contrast medium Lipiodol((R)) Ultra-fluide into five experimentally induced stab wounds on a pork haunch. Subsequent MSCT reconstructions of the wounds delivered clear, for medical laymen easily appreciable images regarding the stab direction and the stab depth. We believe that this easy and rapid technique can be useful in the examination of stab wounds in living and dead victims of sharp trauma.