Mouse Models of Periodic Paralysis in Andersen Tawil Syndrome and Functional Studies of Novel Nav1.4 mutations in Myotonia & Myopathy

The precise control of skeletal muscle excitation, contraction, and relaxation relies upon a highly choreographed sequence of ionic currents in the sarcolemmal membrane. Mutations of ion channels can lead to deleterious changes of ionic currents that impair muscle fiber excitability and thereby cause either weakness or involuntary after-contractions. The work presented here aims to define the functional consequences for newly identified disease mutations in a sodium channel and to establish the mechanism by which the partial loss of a potassium conductance creates susceptibility to episodic attacks of weakness. The majority of this work focuses on Andersen-Tawil syndrome, a form of periodic paralysis caused by loss-of-function mutations of the inward rectifying potassium channel Kir2.1. While a genetic basis was established for the disease, it was unknown how this defect creates susceptibility to episodes weakness, or whether as in other forms of periodic paralysis, the attacks are exacerbated by low or high potassium. To investigate this, we generated two different mouse models of ATS, one genetic and the other pharmacologic, that enabled us to explore the mechanistic relationship between the loss of a potassium conductance and the functional deficits in muscle excitation and contraction. We found that loss of muscle force was consistently induced by a high-K challenge, whereas only in the setting of a severely reduced the inward rectifier current (e.g. 6% remaining) did the muscle also exhibit weakness in a low-K challenge. Simulations of a model fiber provided insights on this divergent K-dependence for eliciting weakness and also identified approaches for manipulating conductances, transporters, or pumps to reduce the likelihood of an episode of paralysis. We tested a variety of agents and found that KATP channel openers may provide a means for pharmacologic intervention. The remainder of the work characterizes two newly identified mutations in the voltage-gated sodium channel (NaV1.4). The first is a novel loss-of-function mutation found in recessive congenital myopathy plus fluctuating weakness. The second study identified gain-of-function changes for a NaV1.4 mutation associated with an unusually early onset of myotonia with congenital anomalies, and defined a new syndrome, myotonic myopathy with secondary joint and bone anomalies

A Mixed Periodic Paralysis & Myotonia Mutant, P1158S, Imparts pH-Sensitivity in Skeletal Muscle Voltage-gated Sodium Channels

Skeletal muscle channelopathies, many of which are inherited as autosomal dominant mutations, include myotonia and periodic paralysis. Myotonia is defined by a delayed relaxation after muscular contraction, whereas periodic paralysis is defined by episodic attacks of weakness. One sub-type of periodic paralysis, known as hypokalemic periodic paralysis (hypoPP), is associated with low potassium levels. Interestingly, the P1158S missense mutant, located in the third domain S4-S5 linker of the “skeletal muscle”, Nav1.4, has been implicated in causing both myotonia and hypoPP. A common trigger for these conditions is physical activity. We previously reported that Nav1.4 is relatively insensitive to changes in extracellular pH compared to Nav1.2 and Nav1.5. Given that intense exercise is often accompanied by blood acidosis, we decided to test whether changes in pH would push gating in P1158S towards either phenotype. Our results suggest that, unlike in WT-Nav1.4, low pH depolarizes the voltage-dependence of activation and steady-state fast inactivation, decreases current density, and increases late currents in P1185S. Thus, P1185S turns the normally pH-insensitive Nav1.4 into a proton-sensitive channel. Using action potential modeling we predict a pH-to-phenotype correlation in patients with P1158S. We conclude that activities which alter blood pH may trigger the noted phenotypes in P1158S patients

Genetic neurological channelopathies: molecular genetics and clinical phenotypes

Evidence accumulated over recent years has shown that genetic neurological channelopathies can cause many different neurological diseases. Presentations relating to the brain, spinal cord, peripheral nerve or muscle mean that channelopathies can impact on almost any area of neurological practice. Typically, neurological channelopathies are inherited in an autosomal dominant fashion and cause paroxysmal disturbances of neurological function, although the impairment of function can become fixed with time. These disorders are individually rare, but an accurate diagnosis is important as it has genetic counselling and often treatment implications. Furthermore, the study of less common ion channel mutation-related diseases has increased our understanding of pathomechanisms that is relevant to common neurological diseases such as migraine and epilepsy. Here, we review the molecular genetic and clinical features of inherited neurological channelopathies

Ion Channel Gene Mutations Causing Skeletal Muscle Disorders: Pathomechanisms and Opportunities for Therapy

Skeletal muscle ion channelopathies (SMICs) are a large heterogeneous group of rare genetic disorders caused by mutations in genes encoding ion channel subunits in the skeletal muscle mainly characterized by myotonia or periodic paralysis, potentially resulting in long-term disabilities. However, with the development of new molecular technologies, new genes and new phenotypes, including progressive myopathies, have been recently discovered, markedly increasing the complexity in the field. In this regard, new advances in SMICs show a less conventional role of ion channels in muscle cell division, proliferation, differentiation, and survival. Hence, SMICs represent an expanding and exciting field. Here, we review current knowledge of SMICs, with a description of their clinical phenotypes, cellular and molecular pathomechanisms, and available treatments

Inhibition of voltage-dependent sodium currents by cannabidiol

Voltage-gated sodium channels initiate action potentials in excitable tissues. Altering these channels’ function can lead to many pathophysiological conditions. The family of voltage-gated sodium channel genes encodes 10 proteins (including Nav2.1) distributed throughout the central and peripheral nervous systems, cardiac and skeletal muscles. The SCN4A gene encodes the Nav1.4 channel, which is primarily responsible for depolarization of the skeletal muscle fibers. Many mutations in SCN4A are found and associated with the myotonic syndromes and periodic paralyses. These conditions are both considered gain-of-function and can be severely life-limiting with respect to performing everyday tasks. From a broader standpoint, hyperexcitability presents as a significant problem in other tissues besides skeletal muscles. Gain-of-function in sodium channels has been linked to a wide-range of pathophysiological conditions such as inherited erythromelalgia, epilepsy, and arrhythmias. Treating these types of pathologies requires an in-depth understanding of their underlying mechanisms. One way to gain this understanding is to investigate physiological triggers. There is also a dire need for novel ways of reducing the hyperexcitability associated with mutant sodium channels. One promising compound is the non-psychotropic component of the Cannabis sativa plant, cannabidiol. This compound has recently been shown to modulate some of the neuronal sodium channels. Although cannabidiol has shown efficacy in clinical trials, the underlying mechanism of action remains unknown. Sodium channels could be among the molecular targets for cannabidiol.In my doctoral research: 1) I studied how a single missense mutation, P1158S, in Nav1.4 causes various degrees of gain-of-function (myotonia and periodic paralysis) by using pH changes to probe P1158S gating modifications; 2) I studied the inhibitory effects of cannabidiol on voltage-dependent sodium currents; 3) I investigated the mechanism through which cannabidiol imparts inhibition. Overall, these data reveal novel insights into sodium channel hyperexcitability and pharmacologically targeting of this hyperexcitability using cannabidiol

Mechanisms of Phenotypic Variability in Myotonia Congenita

The severity of Myotonia Congenita varies not only across individuals with different CLCN1 genotypes, but also within a pedigree, and can even fluctuate over time within a single individual in response to environmental circumstances. The functional consequences of eight naturally occurring sequence variants in the skeletal muscle chloride channel gene, CLCN1, were examined by whole cell patch-clamp of HEK293T cells expressing the gene product, ClC-1, in order to investigate potential differences in their mechanisms of pathogenicity. G276D and G523D caused complete loss of function, while S289G produced altered kinetics and a marked depolarizing shift of voltage dependence. H369P, A566T and M646T all tested normal in the HEK293T assay despite strong clinical support for pathogenicity. Their mechanism of pathogenicity may rely on muscle-specific processes that are not faithfully recapitulated in HEK293T cells. W118G and P744T were selected as examples of variants for which pathogenicity is unclear from the clinical evidence. The former is present in controls, but over-represented in the Myotonia Congenita population. The latter is present in an individual who also harbours a large deletion in CLCN1. Both variants tested normal in the HEK293T assay. A potent trigger for worsening of myotonia in some female patients is pregnancy. In order to clarify the role of sex hormones in non-genomic modulation of skeletal muscle excitability, the effects of progesterone and oestrogen on endogenous chloride currents through the wildtype ClC-1 of mouse skeletal muscle were tested by whole cell patch clamp. Progesterone and oestrogen rapidly reduced the chloride conductance and shifted its voltage dependence, thus a non-genomic mechanism exists in skeletal muscle linking sex hormones to ClC-1. However the effect was only significant at 500 times the highest physiological concentration encountered in pregnancy. The macroscopic chloride conductance of a membrane expressing wildtype ClC-1 was simulated in Matlab. The simulation improves on published models by recapitulating both time-dependence and voltage-dependence of the channel through a method based on independent representations of the fast and the slow gates. The applicability of the model for the purposes of exploring the effects of specific mutations was assessed by attempting to simulate the currents through S289G channels; the effects of S289G could be mimicked by slowing and inverting the kinetics of the fast gate and shifting the fast gate opening probability to more depolarized potentials. The mechanism of low chloride conductance myotonia and electrical factors likely to impact on its severity are discussed in the context of experiments conducted in a model of myotonic muscle. Slowing of ClC-1 kinetics alone did not produce myotonia, but could lower the threshold for myotonia caused by shifts in voltage dependence. Muscle fibre diameter is an important factor in the propensity to myotonia, which can be driven by asynchrony between surface and t-tubular action potentials in large muscle fibres. Increasing muscle fibre diameter could underly the age-dependence of symptom onset in Myotonia Congenita, and differences in diameter could contribute to phenotypic variability, including male-female differences

Sarcolemmal-restricted localization of functional ClC-1 channels in mouse skeletal muscle

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

CANALI-LIGANDO E VOLTAGGIO- DIPENDENTI IN CELLULE SATELLITI DURANTE IL PROCESSO DELLA MIOGENESI IN VITRO

2001/2002Cell implantation has been proposed as a possible strategy for muscle regeneration and a good therapy for muscle diseases (Partridge et al, 1978). Primary cells and in particular muscle satellite cells are among the best candidates for transplantation. The satellite cells are quiescent precursor cells located beneath the basai lamina of mature muscle fibres; they can proliferate generating myoblasts that fuse into multinucleated fibres for muscle growth or in response to :muscle injury (for review Seale & Rudnicki, 2000). During differentiation, myoblasts express specific proteins as myosin, tropomyosin and different ionic channels. Voltagedependent sodium channels and acetylcholine receptors channels (AChRs) are among the frrst channels to be expressed during in uitro myogenesis: the former are already expressed in rat quiescent satellite cells (Bader et al., 1988), whereas functional AChRs appear only in proliferating myoblasts (Cossu et aJ., 1987). At this developmental stage sodium channels are responsible of action potential generation whereas AChRs are found to be involved in myoblasts fusion (Krause et aZ., 1995). Severallines of evidence demonstrated that there are ionic channels involved in neuromuscular diseases, for example Myotonic Dystrophy (MD). MD is the most common inherited neuromuscular disease in adults, with a global incidence of l in 8000 individuals. MD is an autosomal dominant, multisystemic disorder characterised primarily by myotonia and progressive muscle weakness. The genetic defect is a trinucleotide (CTG) repeat expansion within the 3'-untranslated region of a protein kinase gene (DMPK) located on chromosome 19ql3.3 (for review Tapscott & Thornton, 2001). Severallines of evidence demonstrate that altered modulation of Na+ channels may play an important role in the pathogenesis of MD. (Chahine et al., 1997; Mounsey et al., 2000). The aim of this work was to analyse the properties of voltage-dependent sod.ium channels and AChR during in uitro myogenesis of murine satellite cells. The properties of these channels have been investigated in human satellite cells coming from healthy and MD donors. The last part of the work was dealing with the same property on healthy donor of different age. Materials And Methods Cell culture. Cultures of i28 cells were established from mouse satellite cells (Irintchev et al, 1997) The cells have been expanded in HAM'S F-10 med.ium containing 20% fetal calf serum (FCS), L-glutamine (2mM), penicillin (l 00 units/ml) and streptomycin (100 uM/ml). To induce cell d.ifferentiation and myotube fusion, the medium was replaced, l day after plating, with DMEM supplemented with 2% horse serum and L-glutamine, penicillin, and streptomycin. Human myoblasts were grown in HAM'S F-10 medium supplemented with 50 ug/ml gentamycine and 20% FCS. For myoblast d.ifferentiation, growth medium was replaced by DMEM containing 10 uM/ml insulin and 100 ug/ml transferrin. Cell nuclea were counted using DAPI staining; fusion index was expressed (in percentage) as the ratio between the number of nuclei in multinucleated myotubes and total number of nuclea, for each considered field in the Petri dish. Human satellite cells have been coded as follows: DSQ satellite cells coming from 29 week-old DM foetus, BQ29 satellite cells coming from 29 week-old foetus, 30/00 coming from 2 year-old donor and STR13 satellite cells coming from 21 year-old don or, Electrophysiological recording. Patch-clamp recordings were performed, at room temperature, in the whole-cell and cell-attached con:figurations using borosilicate glass pipettes. ACh currents: ACh-induced total currents were measured in the whole-cell con:figuration in the voltage-clamp mode. Bath solution (NES) contained (mM): NaCl 140, KCl 2.8, CaCh 2, MgCh 2, glucose 10, HEPES-NaOH 10, pH 7,3. Pipettes (3-5 MQ) were filled with the following solution (mM): KC1120, CaCh l, MgCh 2, EGTA 11, HEPES-KOH 10, pH 7.3. ACh (10 J.LM) was applied by a gravity-driven perfusion system. In the cell-attached con:figuration, pipettes ( -8 MQ) were filled with NES containing ACh 100 nM. Na+ current: total Na+ currents were measured in the whole-cell con:figuration in the voltage-clamp mode. The extracellular solution was (mM): 40 TEA, 2.8 KCI, 100 NaCl, 2 CaCh, 2 MgCh, 10 HEPES, 10 glucose, pH=7.3. The pipette solution was (mM): 120 CsCl, 11 EGTA, l CaCh, 2 MgCh, 10 HEPES, pH=7,3. Currents were recorded using on Axopatch 200 amplifi.er; signals were fùtered at 2kHz with a Bessel fùter (-3bB) and transferred to hard disk using the Digi.Data 1200 interface. Total and single-channel currents were analyzed using the pClamp 6 software (Axon Instruments). Results And Conclusion ACHR AND VOLTAGE-DEPENDENT SODIUM CHANNELS PROPERTIES IN MURINE SATELLITE CELLS Murine satellite cells started to express functional ACbRs wben they were stili undifferentiated. ACb-induced mean total current increased during differentiation; after 2 days in differentiation medium ali the cells were responsive to ACb application. The kinetics and the biopbysical properties of single ACb cbannels reveal that these cells express the foetal type of ACbRs, as observed in immature skeletal muscle and denervated adult muscles. Sodium currents in differentiated myoblasts were analysed plating the cell at low density in order to reduce the myoblasts fusion. In this condition only 20% of the total nuclea belonged to multinucleated myotubes, wbereas most of the cells were mononucleated. Tetrodotoxin (TTX)-insensitive and TTX-sensitive sodium current bave been found in mononucleated differentiated myoblasts. No significant differences bave been found in the kinetic properties of sodium currents in the presence or absence of TTX. Murine satellite cells maintained in cell culture fused in electrically excitable multinucleated myotubes whic, after 5-6 days in differentiation medium, contracted spontaneously. Spontaneous action potentials recorded in current-clamp configuration were blocked by TTX. Preliminary results sbow that a-bungarotoxin (a specific blocker for ACbR) could modulate the action potential frequency. ACHR AND VOLTAGE-DEPENDENT SODIUM CHANNELS PROPERTIES IN HUMAN SATELLITE CELLS For the frrst time the properties of AChRs in MD human satellite cells have been investigated. AChRs expressed during in vitro myogenesis by human healthy and MD satellite cells differed in single channel conductance which was significantly lower in pathological condition. Different subunits arrangement mechanism could be suggested to explain such differend single channel conductance: Kullberg et al ( 1990) showed that when AChR with only a.J3 and y subunits were expressed in Xenopus oocytes, the channels had a lower conductance than native receptors. We can conclude that a defect in AChR conductance, during in vitro differentiation of MD satellite cells, could be at least partially responsible of the reduced fusion already observed in MD myoblasts (Furling et al., 2001). ITX-sensitive and TTX-insensitive sodium channels were both present and the total current density was lower in DM cells. As previously observed (Rudel et al., 1989), sodium channels expressed in MD cells were activated at more positive membrane potentials. A difference in the kinetics of activation could be explained by phosphorilation processes induced by kinase like DMPK. Finally regarding the different age of the donors of satellite muscle cells, we have up to now analysed the byphysical properties of AChR. Single channels properties are not different and resemble those of the fetal type of AChR. The amplitude of total current was also not related of the age of donor increasing in the same way during the processes of myogenesis. Further experiments are needed to demonstrate if AChR induced current could be, at least in part, responsible for the different differentiation capability already observed.XV Ciclo1972Versione digitalizzata della tesi di dottorato cartacea. Nell'originale cartaceo errata numerazione delle pagin

Alternative splicing in sodium channels: biophysical and functional effects in NaV1.1, NaV1.2 & NaV1.7

Alternative splicing in voltage-gated sodium channels can affect pathophysiological conditions, including epilepsy and pain. A conserved alternative splicing event in sodium channel genes, including SCN1A, SCN2A and SCN9A, gives rise to the neonatal (5N) and adult (5A) isoforms. Differences in the ratio of 5A/5N in Nav1.1 (encoded by SCN1A) in patients may lead to different predisposition to epilepsy or response to antiepileptic drugs (AED). Previous HEK293T whole-cell voltage-clamp recordings showed that Nav1.1-5N channels recover more quickly from fast inactivation than 5A. However it was unknown whether this effect is conserved in Nav1.2 (encoded by SCN2A) and Nav1.7 (SCN9A) channels, or what the functional consequences of this splicing event are for neurons. This project used whole-cell voltage-clamp recordings on heterologously expressed neonatal and adult channels to compare the biophysical properties of the splice isoforms for all three channel types and their modulation by AEDs. It also used current-clamp and dynamic-clamp recordings on transfected hippocampal cultured neurons to assess the effect of splicing on neuronal properties during epileptiform activity. Biophysical analysis in HEK293T cells revealed that splicing profoundly regulates fast inactivation and channel availability during fast, repetitive stimulation, with neonatal channels showing higher availability compared to adult channels and this difference was conserved among Nav1.1, Nav1.2 and Nav1.7. The change in inactivation imposed by splicing can be modeled as a modification of the stability of the inactivation statein resting channels. This change can be eradicated by administration of the AEDs phenytoin and carbamazepine. Current-clamp recordings in transfected neurons showed that the alternatively spliced variantmodifies the rising phase of action potentials for Nav1.1 and Nav1.2 at high firing frequencies, implying a consistent splice-dependent modulation of channel availability. For Nav1.1 in interneurons, this translated to higher firing frequency for the neonatal isoform, which also conferred a higher maximal firing rate during epileptiform events imposed under dynamic-clamp recordings