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1.  NaV1.4 mutations cause hypokalaemic periodic paralysis by disrupting IIIS4 movement during recovery 
Brain  2014;137(4):998-1008.
Cations leaking through the voltage sensor of mutant sodium or calcium channels underlie hypokalaemic periodic paralysis. Groome et al. use muscle fibre recordings, voltage clamp, and molecular dynamics, to investigate recently discovered Nav1.4 channel mutations. They identify a novel voltage sensor movement that may explain the muscle pathology.
Hypokalaemic periodic paralysis is typically associated with mutations of voltage sensor residues in calcium or sodium channels of skeletal muscle. To date, causative sodium channel mutations have been studied only for the two outermost arginine residues in S4 voltage sensor segments of domains I to III. These mutations produce depolarization of skeletal muscle fibres in response to reduced extracellular potassium, owing to an inward cation-selective gating pore current activated by hyperpolarization. Here, we describe mutations of the third arginine, R3, in the domain III voltage sensor i.e. an R1135H mutation which was found in two patients in separate families and a novel R1135C mutation identified in a third patient in another family. Muscle fibres from a patient harbouring the R1135H mutation showed increased depolarization tendency at normal and reduced extracellular potassium compatible with the diagnosis. Additionally, amplitude and rise time of action potentials were reduced compared with controls, even for holding potentials at which all NaV1.4 are fully recovered from inactivation. These findings may be because of an outward omega current activated at positive potentials. Expression of R1135H/C in mammalian cells indicates further gating defects that include significantly enhanced entry into inactivation and prolonged recovery that may additionally contribute to action potential inhibition at the physiological resting potential. After S4 immobilization in the outward position, mutant channels produce an inward omega current that most likely depolarizes the resting potential and produces the hypokalaemia-induced weakness. Gating current recordings reveal that mutations at R3 inhibit S4 deactivation before recovery, and molecular dynamics simulations suggest that this defect is caused by disrupted interactions of domain III S2 countercharges with S4 arginines R2 to R4 during repolarization of the membrane. This work reveals a novel mechanism of disrupted S4 translocation for hypokalaemic periodic paralysis mutations at arginine residues located below the gating pore constriction of the voltage sensor module.
doi:10.1093/brain/awu015
PMCID: PMC3959555  PMID: 24549961
hypokalaemic periodic paralysis; molecular dynamics; omega pore current; sodium channel; voltage sensor
2.  Functional and genetic characterization of clinical malignant hyperthermia crises: a multi-centre study 
Background
Malignant hyperthermia (MH) is a rare pharmacogenetic disorder which is characterized by life-threatening metabolic crises during general anesthesia. Classical triggering substances are volatile anesthetics and succinylcholine (SCh). The molecular basis of MH is excessive release of Ca2+ in skeletal muscle principally by a mutated ryanodine receptor type 1 (RyR1). To identify factors explaining the variable phenotypic presentation and complex pathomechanism, we analyzed proven MH events in terms of clinical course, muscle contracture, genetic factors and pharmocological triggers.
Methods
In a multi-centre study including seven European MH units, patients with a history of a clinical MH episode confirmed by susceptible (MHS) or equivocal (MHE) in vitro contracture tests (IVCT) were investigated. A test result is considered to be MHE if the muscle specimens develop pathological contractures in response to only one of the two test substances, halothane or caffeine. Crises were evaluated using a clinical grading scale (CGS), results of IVCT and genetic screening. The effects of SCh and volatile anesthetics on Ca2+ release from sarcoplasmic reticulum (SR) were studied in vitro.
Results
A total of 200 patients met the inclusion criteria. Two MH crises (1%) were triggered by SCh (1 MHS, 1 MHE), 18% by volatile anesthetics and 81% by a combination of both. Patients were 70% male and 50% were younger than 12 years old. Overall, CGS was in accord with IVCT results. Crises triggered by enflurane had a significantly higher CGS compared to halothane, isoflurane and sevoflurane. Of the 200 patients, 103 carried RyR1 variants, of which 14 were novel. CGS varied depending on the location of the mutation within the RyR1 gene. In contrast to volatile anesthetics, SCh did not evoke Ca2+ release from isolated rat SR vesicles.
Conclusions
An MH event could depend on patient-related risk factors such as male gender, young age and causative RyR1 mutations as well as on the use of drugs lowering the threshold of myoplasmic Ca2+ release. SCh might act as an accelerant by promoting unspecific Ca2+ influx via the sarcolemma and indirect RyR1 activation. Most MH crises develop in response to the combined administration of SCh and volatile anesthetics.
doi:10.1186/1750-1172-9-8
PMCID: PMC3896768  PMID: 24433488
Malignant hyperthermia; Succinylcholine; Suxamethonium; Volatile anesthetics; RyR1 mutations; In vitro contracture test
3.  Transient compartment-like syndrome and normokalaemic periodic paralysis due to a Cav1.1 mutation 
Brain  2013;136(12):3775-3786.
We studied a two-generation family presenting with conditions that included progressive permanent weakness, myopathic myopathy, exercise-induced contracture before normokalaemic periodic paralysis or, if localized to the tibial anterior muscle group, transient compartment-like syndrome (painful acute oedema with neuronal compression and drop foot). 23Na and 1H magnetic resonance imaging displayed myoplasmic sodium overload, and oedema. We identified a novel familial Cav1.1 calcium channel mutation, R1242G, localized to the third positive charge of the domain IV voltage sensor. Functional expression of R1242G in the muscular dysgenesis mouse cell line GLT revealed a 28% reduced central pore inward current and a −20 mV shift of the steady-state inactivation curve. Both changes may be at least partially explained by an outward omega (gating pore) current at positive potentials. Moreover, this outward omega current of 27.5 nS/nF may cause the reduction of the overshoot by 13 mV and slowing of the upstroke of action potentials by 36% that are associated with muscle hypoexcitability (permanent weakness and myopathic myopathy). In addition to the outward omega current, we identified an inward omega pore current of 95 nS/nF at negative membrane potentials after long depolarizing pulses that shifts the R1242G residue above the omega pore constriction. A simulation reveals that the inward current might depolarize the fibre sufficiently to trigger calcium release in the absence of an action potential and therefore cause an electrically silent depolarization-induced muscle contracture. Additionally, evidence of the inward current can be found in 23Na magnetic resonance imaging-detected sodium accumulation and 1H magnetic resonance imaging-detected oedema. We hypothesize that the episodes are normokalaemic because of depolarization-induced compensatory outward potassium flux through both delayed rectifiers and omega pore. We conclude that the position of the R1242G residue before elicitation of the omega current is decisive for its conductance: if the residue is located below the gating pore as in the resting state then outward currents are observed; if the residue is above the gating pore because of depolarization, as in the inactivated state, then inward currents are observed. This study shows for the first time that functional characterization of omega pore currents is possible using a cultured cell line expressing mutant Cav1.1 channels. Likewise, it is the first calcium channel mutation for complicated normokalaemic periodic paralysis.
doi:10.1093/brain/awt300
PMCID: PMC3859226  PMID: 24240197
periodic paralyses; omega pore; voltage sensor; calcium channel
4.  The role of fibrosis in Duchenne muscular dystrophy 
Acta Myologica  2012;31(3):184-195.
Muscular dystrophies such as Duchenne muscular dystrophy (DMD) are usually approached as dysfunctions of the affected skeletal myofibres and their force transmission. Comparatively little attention has been given to the increase in connective tissue (fibrosis) which accompanies these muscular changes. Interestingly, an increase in endomysial tissue is apparent long before any muscular degeneration can be observed. Fibrosis is the result of a reactive or reparative process involving mechanical, humoral and cellular factors. Originating from vulnerable myofibres, muscle cell necrosis and inflammatory processes are present in DMD. Muscular recovery is limited due to the limited number and capacity of satellite cells. Hence, a proactive and multimodal approach is necessary in order to activate protective mechanisms and to hinder catabolic and tissue degrading pathways.
Several avenues are discussed in terms of potential antifibrotic therapy approaches. These include pharmaceutical, nutritional, exercise-based and other mechanostimulatory modalities (such as massage or yoga-like stretching) with the intention of exerting an anti-inflammatory and antifibrotic effect on the affected muscular tissues. A preventive intervention at an early age is crucial, based on the early and seemingly non-reversible nature of the fibrotic tissue changes. Since consistent assessment is essential, different measurement technologies are discussed.
PMCID: PMC3631802  PMID: 23620650
Duchenne muscular dystrophy; fibrosis; endo- and perimysium; extracellular matrix; TGF-β1; myostatin; antifibrotic therapy
5.  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
6.  The impact of permanent muscle weakness on quality of life in periodic paralysis: a survey of 66 patients 
Acta Myologica  2012;31(2):126-133.
The periodic paralyses are hereditary muscle diseases which cause both episodic and permanent weakness. Permanent weakness may include both reversible and fixed components, the latter caused by fibrosis and fatty replacement. To determine the degree of handicap and impact of permanent weakness on daily life, we conducted a 68-question online survey of 66 patients over 41 years (mean age, 60 ± 14 years). Permanent weakness occurred in 68%, muscle pain in 82% and muscle fatigue in 89%. Eighty-three percent of patients reported themselves as moderately to very active between ages 18-35. At the time of the survey only 14% reported themselves as moderately to very active. Contrary to the literature, only 21% of patients reported decreased frequency of episodic weakness with increased age. Sixty-seven percent had incurred injuries due to falls. Mobility aids were required by 49%. Strength increased in 49% of patients receiving professional physiotherapy and in 62% performing self-managed exercise routines. A decline of strength was observed by 40% with professional and by 16% with self-managed exercise routine, suggesting that overworking muscles may not be beneficial. There is an average of 26 years between age at onset and age at diagnosis indicating that diagnostic schemes can be improved. In summary our data suggests that permanent muscle weakness has a greater impact on the quality of life of patients than previously anticipated.
PMCID: PMC3476862  PMID: 23097604
periodic paralysis; myopathy; paramyotonia congenita
7.  Pathophysiological Role of Omega Pore Current in Channelopathies 
In voltage-gated cation channels, a recurrent pattern for mutations is the neutralization of positively charged residues in the voltage-sensing S4 transmembrane segments. These mutations cause dominant ion channelopathies affecting many tissues such as brain, heart, and skeletal muscle. Recent studies suggest that the pathogenesis of associated phenotypes is not limited to alterations in the gating of the ion-conducting alpha pore. Instead, aberrant so-called omega currents, facilitated by the movement of mutated S4 segments, also appear to contribute to symptoms. Surprisingly, these omega currents conduct cations with varying ion selectivity and are activated in either a hyperpolarized or depolarized voltage range. This review gives an overview of voltage sensor channelopathies in general and focuses on pathogenesis of skeletal muscle S4 disorders for which current knowledge is most advanced.
doi:10.3389/fphar.2012.00112
PMCID: PMC3372090  PMID: 22701429
epilepsy and neuromyotonia; long QT syndrome; familial hemiplegic migraine; myotonia and paramyotonia; hyperkalemic and hypokalemic periodic paralysis; sodium overload; cytotoxic edema; degeneration
8.  Rationale for treating oedema in Duchenne muscular dystrophy with eplerenone 
Acta Myologica  2012;31(1):31-39.
Recently we reported a cytoplasmic sodium overload to cause a severe osmotic oedema in Duchenne muscular dystrophy (DMD). Our results suggested that this dual overload of sodium ions and water precedes the dystrophic process and persists until fatty muscle degeneration is complete. The present paper addresses the questions as to whether these overloads are important for the pathogenesis of the disease, and if so, whether they can be treated. As a first step, we investigated the effects of various diuretic drugs on a cell model of DMD, i.e. rat diaphragm strips previously exposed to amphotericin B. We found that both carbonic anhydrase inhibitors and aldosterone antagonists were able to repolarise depolarised muscle fibres. Since carbonic anhydrase inhibitors are known to have acidifying effects and this might be detrimental to the ventilation of DMD patients, we mainly concentrated on the modern spironolactone derivative, eplerenone. This drug had a very high repolarizing power, the parameter considered by us as being most relevant for a beneficial effect. In a pilot study we administered this drug to a 22-yr-old female DMD patient who was bound to an electric wheelchair and has had no corticosteroid therapy before. Eplerenone decreased both cytoplasmic sodium and water overload and increased muscle strength and mobility. We conclude that eplerenone has beneficial effects on DMD muscle. In our opinion the cytoplasmic oedema is cytotoxic and should be treated before fatty degeneration takes place.
PMCID: PMC3440802  PMID: 22655515
Duchenne muscular dystrophy; eplerenone; cytotoxic oedema
9.  Sodium channelopathies of skeletal muscle result from gain or loss of function 
Pflugers Archiv   2010;460(2):239-248.
Five hereditary sodium channelopathies of skeletal muscle have been identified. Prominent symptoms are either myotonia or weakness caused by an increase or decrease of muscle fiber excitability. The voltage-gated sodium channel NaV1.4, initiator of the muscle action potential, is mutated in all five disorders. Pathogenetically, both loss and gain of function mutations have been described, the latter being the more frequent mechanism and involving not just the ion-conducting pore, but aberrant pores as well. The type of channel malfunction is decisive for therapy which consists either of exerting a direct effect on the sodium channel, i.e., by blocking the pore, or of restoring skeletal muscle membrane potential to reduce the fraction of inactivated channels.
doi:10.1007/s00424-010-0814-4
PMCID: PMC2883924  PMID: 20237798
Myotonia; Paramyotonia congenita; Hyperkalemic periodic paralysis; Hypokalemic periodic paralysis; Congenital myasthenic syndrome; Excitability; Muscle; Channels; Sodium channel; Muscle strength
11.  Muscle channelopathies and critical points in functional and genetic studies 
Journal of Clinical Investigation  2005;115(8):2000-2009.
Muscle channelopathies are caused by mutations in ion channel genes, by antibodies directed against ion channel proteins, or by changes of cell homeostasis leading to aberrant splicing of ion channel RNA or to disturbances of modification and localization of channel proteins. As ion channels constitute one of the only protein families that allow functional examination on the molecular level, expression studies of putative mutations have become standard in confirming that the mutations cause disease. Functional changes may not necessarily prove disease causality of a putative mutation but could be brought about by a polymorphism instead. These problems are addressed, and a more critical evaluation of the underlying genetic data is proposed.
doi:10.1172/JCI25525
PMCID: PMC1180551  PMID: 16075040
12.  Impairment of skeletal muscle adenosine triphosphate–sensitive K+ channels in patients with hypokalemic periodic paralysis 
Journal of Clinical Investigation  1999;103(5):675-682.
The adenosine triphosphate (ATP)–sensitive K+ (KATP) channel is the most abundant K+ channel active in the skeletal muscle fibers of humans and animals. In the present work, we demonstrate the involvement of the muscular KATP channel in a skeletal muscle disorder known as hypokalemic periodic paralysis (HOPP), which is caused by mutations of the dihydropyridine receptor of the Ca2+ channel. Muscle biopsies excised from three patients with HOPP carrying the R528H mutation of the dihydropyridine receptor showed a reduced sarcolemma KATP current that was not stimulated by magnesium adenosine diphosphate (MgADP; 50–100 μM) and was partially restored by cromakalim. In contrast, large KATP currents stimulated by MgADP were recorded in the healthy subjects. At channel level, an abnormal KATP channel showing several subconductance states was detected in the patients with HOPP. None of these were surveyed in the healthy subjects. Transitions of the KATP channel between subconductance states were also observed after in vitro incubation of the rat muscle with low-K+ solution. The lack of the sarcolemma KATP current observed in these patients explains the symptoms of the disease, i.e., hypokalemia, depolarization of the fibers, and possibly the paralysis following insulin administration.
PMCID: PMC408119  PMID: 10074484

Results 1-12 (12)