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1.  A gating model for wildtype and R1448H Nav1.4 channels in paramyotonia 
Acta Myologica  2014;33(1):22-33.
We studied the consequences of the Nav1.4 mutation R1448H that is situated in the fourth voltage sensor of the channel and causes paramyotonia, a cold-induced myotonia followed by weakness. Previous work showed that the mutation uncouples inactivation from activation. We measured whole-cell Na+ currents at 10, 15, 20, and 25°C using HEK293 cells stably transfected with wildtype (WT) and R1448H Na+ channels. A Markov model was developed the parameters of which reproduced the data measured on WT and R1448H channels in the whole voltage and temperature range. It required an additional transient inactivated state and an additional closed-state inactivation transition not previously described. The model was used to predict single-channel properties, free energy barriers and temperature dependence of rates. It allowed us to draw the following conclusions: i) open-state inactivation results from a two-step process; ii) the channel re-openings that cause paramyotonia originate from enhanced deactivation/reactivation and not from destabilized inactivation; iii) the closed-state inactivation of R1448H is strikingly enhanced. We assume that latter explains the episodic weakness following cold-induced myotonia.
PMCID: PMC4021628  PMID: 24843232
Paramyotonia; markov model; sodium channel; closed-state inactivation; channelopathy; skeletal muscle
2.  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.
PMCID: PMC3959555  PMID: 24549961
hypokalaemic periodic paralysis; molecular dynamics; omega pore current; sodium channel; voltage sensor
3.  Functional and genetic characterization of clinical malignant hyperthermia crises: a multi-centre study 
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.
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.
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.
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.
PMCID: PMC3896768  PMID: 24433488
Malignant hyperthermia; Succinylcholine; Suxamethonium; Volatile anesthetics; RyR1 mutations; In vitro contracture test
4.  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.
PMCID: PMC3859226  PMID: 24240197
periodic paralyses; omega pore; voltage sensor; calcium channel
5.  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
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.
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.  Open- and closed-state fast inactivation in sodium channels 
Channels  2011;5(1):65-78.
The role of sodium channel closed-state fast inactivation in membrane excitability is not well understood. We compared open- and closed-state fast inactivation, and the gating charge immobilized during these transitions, in skeletal muscle channel hNaV1.4. A significant fraction of total charge movement and its immobilization occurred in the absence of channel opening. Simulated action potentials in skeletal muscle fibers were attenuated when pre-conditioned by subthreshold depolarization. Anthopleurin A, a site-3 toxin that inhibits gating charge associated with the movement of DIVS4, was used to assess the role of this voltage sensor in closed-state fast inactivation. Anthopleurin elicited opposing effects on the gating mode, kinetics and charge immobilized during open- versus closed-state fast inactivation. This same toxin produced identical effects on recovery of channel availability and remobilization of gating charge, irrespective of route of entry into fast inactivation. Our findings suggest that depolarization promoting entry into fast inactivation from open versus closed states provides access to the IFMT receptor via different rate-limiting conformational translocations of DIVS4.
PMCID: PMC3052208  PMID: 21099342
anthopleurin; charge immobilization; fast inactivation; sodium channel; voltage-gated
10.  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.
PMCID: PMC2883924  PMID: 20237798
Myotonia; Paramyotonia congenita; Hyperkalemic periodic paralysis; Hypokalemic periodic paralysis; Congenital myasthenic syndrome; Excitability; Muscle; Channels; Sodium channel; Muscle strength
11.  Letter to the Editor concerning “A hypothesis of chronic back pain: ligament subfailure injuries lead to muscle control dysfunction” (M. Panjabi) 
European Spine Journal  2007;16(10):1733-1735.
PMCID: PMC2078289  PMID: 17342512
Low back pain; Biomechanics; Hypothesis; Thoracolumbar fascia; Proprioception
12.  GLUT1 mutations are a cause of paroxysmal exertion-induced dyskinesias and induce hemolytic anemia by a cation leak 
The Journal of Clinical Investigation  2008;118(6):2157-2168.
Paroxysmal dyskinesias are episodic movement disorders that can be inherited or are sporadic in nature. The pathophysiology underlying these disorders remains largely unknown but may involve disrupted ion homeostasis due to defects in cell-surface channels or nutrient transporters. In this study, we describe a family with paroxysmal exertion-induced dyskinesia (PED) over 3 generations. Their PED was accompanied by epilepsy, mild developmental delay, reduced CSF glucose levels, hemolytic anemia with echinocytosis, and altered erythrocyte ion concentrations. Using a candidate gene approach, we identified a causative deletion of 4 highly conserved amino acids (Q282_S285del) in the pore region of the glucose transporter 1 (GLUT1). Functional studies in Xenopus oocytes and human erythrocytes revealed that this mutation decreased glucose transport and caused a cation leak that alters intracellular concentrations of sodium, potassium, and calcium. We screened 4 additional families, in which PED is combined with epilepsy, developmental delay, or migraine, but not with hemolysis or echinocytosis, and identified 2 additional GLUT1 mutations (A275T, G314S) that decreased glucose transport but did not affect cation permeability. Combining these data with brain imaging studies, we propose that the dyskinesias result from an exertion-induced energy deficit that may cause episodic dysfunction of the basal ganglia, and that the hemolysis with echinocytosis may result from alterations in intracellular electrolytes caused by a cation leak through mutant GLUT1.
PMCID: PMC2350432  PMID: 18451999
14.  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.
PMCID: PMC1180551  PMID: 16075040

Results 1-14 (14)