Explore the vast range of titles available.

Perturbations in K+ have long been considered a key factor in skeletal muscle fatigue. However, the exercise-induced changes in K+ intra-to-extracellular gradient is by itself insufficiently large to be a major cause for the force decrease during fatigue unless combined to other ion gradient changes such as for Na+. Whilst several studies described K+-induced force depression at high extracellular [K+] ([K+]e), others reported that small increases in [K+]e induced potentiation during submaximal activation frequencies, a finding that has mostly been ignored. There is evidence for decreased Cl− ClC-1 channel activity at muscle activity onset, which may limit K+-induced force depression, and large increases in ClC-1 channel activity during metabolic stress that may enhance K+ induced force depression. The ATP-sensitive K+ channel (KATP channel) is also activated during metabolic stress to lower sarcolemmal excitability. Taking into account all these findings, we propose a revised concept in which K+ has two physiological roles: (1) K+-induced potentiation and (2) K+-induced force depression. During low-moderate intensity muscle contractions, the K+-induced force depression associated with increased [K+]e is prevented by concomitant decreased ClC-1 channel activity, allowing K+-induced potentiation of sub-maximal tetanic contractions to dominate, thereby optimizing muscle performance. When ATP demand exceeds supply, creating metabolic stress, both KATP and ClC-1 channels are activated. KATP channels contribute to force reductions by lowering sarcolemmal generation of action potentials, whilst ClC-1 channel enhances the force-depressing effects of K+, thereby triggering fatigue. The ultimate function of these changes is to preserve the remaining ATP to prevent damaging ATP depletion.

In 1970, the study of the pathomechanisms underlying myotonia in muscle fibers isolated from myotonic goats highlighted the importance of chloride conductance for skeletal muscle function; 20 years later, the human ClC-1 chloride channel has been cloned; last year, the crystal structure of human protein has been solved. Over the years, the efforts of many researchers led to significant advances in acknowledging the role of ClC-1 in skeletal muscle physiology and the mechanisms through which ClC-1 dysfunctions lead to impaired muscle function. The wide spectrum of pathophysiological conditions associated with modification of ClC-1 activity, either as the primary cause, such as in myotonia congenita, or as a secondary adaptive mechanism in other neuromuscular diseases, supports the idea that ClC-1 is relevant to preserve not only for skeletal muscle excitability, but also for skeletal muscle adaptation to physiological or harmful events. Improving this understanding could open promising avenues toward the development of selective and safe drugs targeting ClC-1, with the aim to restore normal muscle function. This review summarizes the most relevant research on ClC-1 channel physiology, associated diseases, and pharmacology.

Myotonia congenita (MC) is an inherited rare disease characterized by impaired muscle relaxation after contraction, resulting in muscle stiffness. It is caused by loss-of-function mutations in the skeletal muscle chloride channel ClC-1, important for the stabilization of resting membrane potential and for the repolarization phase of action potentials. Thanks to in vitro functional studies, the molecular mechanisms by which ClC-1 mutations alter chloride ion influx into the cell have been in part clarified, classifying them in “gating-defective” or “expression-defective” mutations. To date, the treatment of MC is only palliative because no direct ClC-1 activator is available. An ideal drug should be one which is able to correct biophysical defects of ClC-1 in the case of gating-defective mutations or a drug capable to recover ClC-1 protein expression on the plasma membrane for trafficking-defective ones. In this study, we tested the ability of niflumic acid (NFA), a commercial nonsteroidal anti-inflammatory drug, to act as a pharmacological chaperone on trafficking-defective MC mutants (A531V, V947E). Wild-type (WT) or MC mutant ClC-1 channels were expressed in HEK293 cells and whole-cell chloride currents were recorded with the patch-clamp technique before and after NFA incubation. Membrane biotinylation assays and western blot were performed to support electrophysiological results. A531V and V947E mutations caused a decrease in chloride current density due to a reduction of ClC-1 total protein level and channel expression on the plasma membrane. The treatment of A531V and V947E-transfected cells with 50 µM NFA restored chloride currents, reaching levels similar to those of WT. Furthermore, no significant difference was observed in voltage dependence, suggesting that NFA increased protein membrane expression without altering the function of ClC-1. Indeed, biochemical experiments confirmed that V947E total protein expression and its plasma membrane distribution were recovered after NFA incubation, reaching protein levels similar to WT. Thus, the use of NFA as a pharmacological chaperone in trafficking defective ClC-1 channel mutations could represent a good strategy in the treatment of MC. Because of the favorable safety profile of this drug, our study may easily open the way for confirmatory human pilot studies aimed at verifying the antimyotonic activity of NFA in selected patients carrying specific ClC-1 channel mutations.

The ClC-1 chloride channel 1 is important for muscle function as it stabilizes resting membrane potential and helps to repolarize the membrane after action potentials. We investigated the contribution of ClC-1 to adaptation of skeletal muscles to needs induced by the different stages of life. We analyzed the ClC-1 gene and protein expression as well as mRNA levels of protein kinase C (PKC) alpha and theta involved in ClC-1 modulation, in soleus (SOL) and extensor digitorum longus (EDL) muscles of rats in all stage of life. The cellular localization of ClC-1 in relation to age was also investigated. Our data show that during muscle development ClC-1 expression differs according to phenotype. In fast-twitch EDL muscles ClC-1 expression increased 10-fold starting at 7 days up to 8 months of life. Conversely, in slow-twitch SOL muscles ClC-1 expression remained constant until 33 days of life and subsequently increased fivefold to reach the adult value. Aging induced a downregulation of gene and protein ClC-1 expression in both muscle types analyzed. The mRNA of PKC-theta revealed the same trend as ClC-1 except in old age, whereas the mRNA of PKC-alpha increased only after 2 months of age. Also, we found that the ClC-1 is localized in both membrane and cytoplasm, in fibers of 12-day-old rats, becoming perfectly localized on the membrane in 2-month-old rats. This study could represent a point of comparison helpful for the identification of accurate pharmacological strategies for all the pathological situations in which ClC-1 protein is altered.

Myotonia congenita is a genetic disease characterized by impaired muscle relaxation after forceful contraction (myotonia) and caused by mutations in the chloride channel voltage-sensitive 1 (CLCN1) gene, encoding the voltage-gated chloride channel of skeletal muscle (ClC-1). In a large cohort of clinically diagnosed unrelated probands, we identified 75 different CLCN1 mutations in 106 individuals, among which 29 were novel mutations and 46 had already been reported. Despite the newly described mutations being scattered throughout the gene, in our patients, mutations were mostly found in exons 4 and 5. Most of the novel mutations located in the region comprising the intramembrane helices are involved in the ion-conducting pathway and predicted to affect channel function. We report for the first time that two mutations, inherited on the same allele as a heterozygous trait, abrogate disease expression, although when inherited singularly they were pathogenic. Such a mode of inheritance might explain the incomplete penetrance reported for autosomal dominant mutations in particular families.

In skeletal muscle, the resting chloride conductance (gCl), due to the ClC-1 chloride channel, controls the sarcolemma electrical stability. Indeed, loss-of-function mutations in ClC-1 gene are responsible of myotonia congenita. The ClC-1 channel can be phosphorylated and inactivated by protein kinases C (PKC), but the relative contribution of each PKC isoforms is unknown. Here, we investigated on the role of PKCθ in the regulation of ClC-1 channel expression and activity in fast- and slow-twitch muscles of mouse models lacking PKCθ. Electrophysiological studies showed an increase of gCl in the PKCθ-null mice with respect to wild type. Muscle excitability was reduced accordingly. However, the expression of the ClC-1 channel, evaluated by qRT-PCR, was not modified in PKCθ-null muscles suggesting that PKCθ affects the ClC-1 activity. Pharmacological studies demonstrated that although PKCθ appreciably modulates gCl, other isoforms are still active and concur to this role. The modification of gCl in PKCθ-null muscles has caused adaptation of the expression of phenotype-specific genes, such as calcineurin and myocyte enhancer factor-2, supporting the role of PKCθ also in the settings of muscle phenotype. Importantly, the lack of PKCθ has prevented the aging-related reduction of gCl, suggesting that its modulation may represent a new strategy to contrast the aging process.

The underlying mechanisms for statin-induced myopathy (SIM) are still equivocal. In this study, we employ Drosophila melanogaster to dissect possible underlying mechanisms for SIM. We observe that chronic fluvastatin treatment causes reduced general locomotion activity and climbing ability. In addition, transmission microscopy of dissected skeletal muscles of fluvastatin-treated flies reveals strong myofibrillar damage, including increased sarcomere lengths and Z-line streaming, which are reminiscent of myopathy, along with fragmented mitochondria of larger sizes, most of which are round-like shapes. Furthermore, chronic fluvastatin treatment is associated with impaired lipid metabolism and insulin signalling. Mechanistically, knockdown of the statin-target Hmgcr in the skeletal muscles recapitulates fluvastatin-induced mitochondrial phenotypes and lowered general locomotion activity; however, it was not sufficient to alter sarcomere length or elicit myofibrillar damage compared to controls or fluvastatin treatment. Moreover, we found that fluvastatin treatment was associated with reduced expression of the skeletal muscle chloride channel, ClC-a (Drosophila homolog of CLCN1), while selective knockdown of skeletal muscle ClC-a also recapitulated fluvastatin-induced myofibril damage and increased sarcomere lengths. Surprisingly, exercising fluvastatin-treated flies restored ClC-a expression and normalized sarcomere lengths, suggesting that fluvastatin-induced myofibrillar phenotypes could be linked to lowered ClC-a expression. Taken together, these results may indicate the potential role of ClC-a inhibition in statin-associated muscular phenotypes. This study underlines the importance of Drosophila melanogaster as a powerful model system for elucidating the locomotion and muscular phenotypes, promoting a better understanding of the molecular mechanisms underlying SIM.

The chloride channel (CLC) family of proteins consists of channels and transporters that share similarities in architecture and play essential roles in physiological functions. Among the CLC family, CLC-1 channels have the representative homodimeric double-barreled structure carrying two gating processes. One is protopore gating that acts on each pore independently by glutamate residue (E ext ). The other is common gating that closes both pores simultaneously in association with large conformational changes across each subunit. In skeletal muscle, CLC-1 is associated with maintaining normal sarcolemmal excitability, and a number of myotonic mutants were reported to modify the channel gating of CLC-1. In this study, we characterized highly conserved helix O as a key determinant of structural stability in CLC-1. Supporting this hypothesis, myotonic mutant (G523D) at N-terminal of helix O showed the activation at hyperpolarizing membrane potentials with a reversed voltage dependency. However, introducing glutamate at serine residue (S537) at the C-terminal of the helix O on G523D restored WT-like voltage dependency of the common gate and showed proton insensitive voltage dependency. To further validate this significant site, site-specific mutagenesis experiments was performed on V292 that is highly conserved as glutamate in antiporter and closely located to S537 and showed that this area is essential for channel function. Taken together, the results of our study suggest the importance of helix O as the main contributor for stable structure of evolutionary conserved CLC proteins and its key role in voltage dependency of the CLC-1. Furthermore, the C-terminal of the helix O can offer a clue for possible proton involvement in CLC-1 channel.

Pure non‐syndromic, non‐dystrophic myotonia in humans is caused by mutations in the genes coding for the skeletal muscle sodium channel (SCN5A) or the skeletal muscle chloride channel (CLCN1) with similar phenotypes. Chloride‐channel myotonia can be dominant (Thomsen‐type myotonia) or recessive (Becker‐type myotonia). More than 60 myotonia‐causing mutations in the CLCN1 gene have been identified, with only a few of them being dominant. A common phenotype of dominant mutations is a dominant negative effect of mutant subunits in mutant‐WT heterodimers, causing a large shift of the steady‐state open probability voltage‐dependence towards more positive, unphysiological voltages. The study of the properties of disease causing mutations has helped in understanding the functional properties of the CLC‐1 channel that is part of a nine‐member gene family of chloride channels. The large body of knowledge obtained for CLC‐1 may also help to better understand the other CLC channels, three of which are also involved in genetic diseases. Hum Mutat 19:423–434, 2002. © 2002 Wiley‐Liss, Inc.

Background and purpose: Given the crucial role of the skeletal muscle chloride conductance (gCl), supported by the voltage‐gated chloride channel CLC‐1, in controlling muscle excitability, the availability of ligands modulating CLC‐1 are of potential medical as well as toxicological importance. Here, we focused our attention on niflumic acid (NFA), a molecule belonging to the fenamates group of non‐steroidal anti‐inflammatory drugs (NSAID). Experimental approach: Rat muscle Cl− conductance (gCl) and heterologously expressed CLC‐1 currents were evaluated by means of current‐clamp (using two‐microelectrodes) and patch‐clamp techniques, respectively. Fura‐2 fluorescence was used to determine intracellular calcium concentration, [Ca2+]i, in native muscle fibres. Key results: NFA inhibited native gCl with an IC50 of 42 μM and blocked CLC‐1 by interacting with an intracellular binding site. Additionally, NFA increased basal [Ca2+]i in myofibres by promoting a mitochondrial calcium efflux that was not dependent on cyclooxygenase or CLC‐1. A structure‐activity study revealed that the molecular conditions that mediate the two effects are different. Pretreatment with the Ca‐dependent protein kinase C (PKC) inhibitor chelerythrine partially inhibited the NFA effect. Therefore, in addition to direct channel block, NFA also inhibits gCl indirectly by promoting PKC activation. Conclusions and Implications: These cellular effects of NFA on skeletal muscle demonstrate that it is possible to modify CLC‐1 and consequently gCl directly by interacting with channel proteins and indirectly by interfering with the calcium‐dependent regulation of the channel. The effect of NFA on mitochondrial calcium stores suggests that NSAIDs, widely used drugs, could have potentially dangerous side‐effects. British Journal of Pharmacology (2007) 150, 235–247. doi:10.1038/sj.bjp.0706954