RESUMO
Recurrent episodes of weakness in periodic paralysis are caused by intermittent loss of muscle fibre excitability, as a consequence of sustained depolarization of the resting potential. Repolarization is favoured by increasing the fibre permeability to potassium. Based on this principle, we tested the efficacy of retigabine, a potassium channel opener, to suppress the loss of force induced by a low-K+ challenge in hypokalaemic periodic paralysis (HypoPP). Retigabine can prevent the episodic loss of force in HypoPP. Knock-in mutant mouse models of HypoPP (Cacna1s p.R528H and Scn4a p.R669H) were used to determine whether pre-treatment with retigabine prevented the loss of force, or post-treatment hastened recovery of force for a low-K+ challenge in an ex vivo contraction assay. Retigabine completely prevents the loss of force induced by a 2 mM K+ challenge (protection) in our mouse models of HypoPP, with 50% inhibitory concentrations of 0.8 ± 0.13 µM and 2.2 ± 0.42 µM for NaV1.4-R669H and CaV1.1-R528H, respectively. In comparison, the effective concentration for the KATP channel opener pinacidil was 10-fold higher. Application of retigabine also reversed the loss of force (rescue) for HypoPP muscle maintained in 2 mM K+. Our findings show that retigabine, a selective agonist of the KV7 family of potassium channels, is effective for the prevention of low-K+ induced attacks of weakness and to enhance recovery from an ongoing loss of force in mouse models of type 1 (Cacna1s) and type 2 (Scn4a) HypoPP. Substantial protection from the loss of force occurred in the low micromolar range, well within the therapeutic window for retigabine.
Assuntos
Paralisia Periódica Hipopotassêmica , Camundongos , Animais , Músculo Esquelético , Carbamatos/farmacologia , Carbamatos/uso terapêutico , Fenilenodiaminas/farmacologia , Fenilenodiaminas/uso terapêuticoRESUMO
Hypokalemic periodic paralysis (HypoPP) is a channelopathy of skeletal muscle caused by missense mutations in the voltage sensor domains (usually at an arginine of the S4 segment) of the CaV1.1 calcium channel or of the NaV1.4 sodium channel. The primary clinical manifestation is recurrent attacks of weakness, resulting from impaired excitability of anomalously depolarized fibers containing leaky mutant channels. Although the ictal loss of fiber excitability is sufficient to explain the acute episodes of weakness, a deleterious change in voltage sensor function for CaV1.1 mutant channels may also compromise excitation-contraction coupling (EC-coupling). We used the low-affinity Ca2+ indicator Oregon Green 488 BAPTA-5N (OGB-5N) to assess voltage-dependent Ca2+-release as a measure of EC-coupling for our knock-in mutant mouse models of HypoPP. The peak ΔF/F0 in fibers isolated from CaV1.1-R528H mice was about two-thirds of the amplitude observed in WT mice; whereas in HypoPP fibers from NaV1.4-R669H mice the ΔF/F0 was indistinguishable from WT. No difference in the voltage dependence of ΔF/F0 from WT was observed for fibers from either HypoPP mouse model. Because late-onset permanent muscle weakness is more severe for CaV1.1-associated HypoPP than for NaV1.4, we propose that the reduced Ca2+-release for CaV1.1-R528H mutant channels may increase the susceptibility to fixed myopathic weakness. In contrast, the episodes of transient weakness are similar for CaV1.1- and NaV1.4-associated HypoPP, consistent with the notion that acute attacks of weakness are primarily caused by leaky channels and are not a consequence of reduced Ca2+-release.
Assuntos
Canais de Cálcio Tipo L , Paralisia Periódica Hipopotassêmica , Canal de Sódio Disparado por Voltagem NAV1.4 , Animais , Canais de Cálcio Tipo L/genética , Modelos Animais de Doenças , Acoplamento Excitação-Contração , Paralisia Periódica Hipopotassêmica/genética , Camundongos , Músculo Esquelético/metabolismo , Mutação de Sentido Incorreto , Canal de Sódio Disparado por Voltagem NAV1.4/genéticaRESUMO
A de novo mutation in the KCND2 gene, which encodes the Kv4.2 K+ channel, was identified in twin boys with intractable, infant-onset epilepsy and autism. Kv4.2 channels undergo closed-state inactivation (CSI), a mechanism by which channels inactivate without opening during subthreshold depolarizations. CSI dynamically modulates neuronal excitability and action potential back propagation in response to excitatory synaptic input, controlling Ca2+ influx into dendrites and regulating spike timing-dependent plasticity. Here, we show that the V404M mutation specifically affects the mechanism of CSI, enhancing the inactivation of channels that have not opened while dramatically impairing the inactivation of channels that have opened. The mutation gives rise to these opposing effects by increasing the stability of the inactivated state and in parallel, profoundly slowing the closure of open channels, which according to our data, is required for CSI. The larger volume of methionine compared with valine is a major factor underlying altered inactivation gating. Our results suggest that V404M increases the strength of the physical interaction between the pore gate and the voltage sensor regardless of whether the gate is open or closed. Furthermore, in contrast to previous proposals, our data strongly suggest that physical coupling between the voltage sensor and the pore gate is maintained in the inactivated state. The state-dependent effects of V404M on CSI are expected to disturb the regulation of neuronal excitability and the induction of spike timing-dependent plasticity. Our results strongly support a role for altered CSI gating in the etiology of epilepsy and autism in the affected twins.
Assuntos
Transtorno Autístico/genética , Epilepsia/genética , Canais de Potássio Shal/genética , Animais , Transtorno Autístico/metabolismo , Epilepsia/metabolismo , Feminino , Humanos , Proteínas Interatuantes com Canais de Kv/genética , Proteínas Interatuantes com Canais de Kv/metabolismo , Potenciais da Membrana/fisiologia , Mutação , Oócitos/fisiologia , Técnicas de Patch-Clamp/métodos , Polimorfismo Genético , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Canais de Potássio Shal/metabolismo , Transfecção , Xenopus laevisRESUMO
The nondystrophic myotonias are rare muscle hyperexcitability disorders caused by gain-of-function mutations in the SCN4A gene or loss-of-function mutations in the CLCN1 gene. Clinically, they are characterized by myotonia, defined as delayed muscle relaxation after voluntary contraction, which leads to symptoms of muscle stiffness, pain, fatigue, and weakness. Diagnosis is based on history and examination findings, the presence of electrical myotonia on electromyography, and genetic confirmation. In the absence of genetic confirmation, the diagnosis is supported by detailed electrophysiological testing, exclusion of other related disorders, and analysis of a variant of uncertain significance if present. Symptomatic treatment with a sodium channel blocker, such as mexiletine, is usually the first step in management, as well as educating patients about potential anesthetic complications.
Assuntos
Fadiga/fisiopatologia , Debilidade Muscular/fisiopatologia , Músculo Esquelético/fisiopatologia , Mialgia/fisiopatologia , Transtornos Miotônicos/fisiopatologia , Acetazolamida/uso terapêutico , Idade de Início , Inibidores da Anidrase Carbônica/uso terapêutico , Canais de Cloreto/genética , Eletrodiagnóstico , Eletromiografia , Testes Genéticos , Humanos , Lamotrigina/uso terapêutico , Mexiletina/uso terapêutico , Miotonia Congênita/tratamento farmacológico , Miotonia Congênita/genética , Miotonia Congênita/fisiopatologia , Transtornos Miotônicos/genética , Canal de Sódio Disparado por Voltagem NAV1.4/genética , Guias de Prática Clínica como Assunto , Ranolazina/uso terapêutico , Bloqueadores dos Canais de Sódio/uso terapêutico , Bloqueadores do Canal de Sódio Disparado por Voltagem/uso terapêuticoRESUMO
Periodic paralyses (PPs) are rare neuromuscular disorders caused by mutations in skeletal muscle sodium, calcium, and potassium channel genes. PPs include hypokalemic paralysis, hyperkalemic paralysis, and Andersen-Tawil syndrome. Common features of PP include autosomal dominant inheritance, onset typically in the first or second decades, episodic attacks of flaccid weakness, which are often triggered by diet or rest after exercise. Diagnosis is based on the characteristic clinic presentation then confirmed by genetic testing. In the absence of an identified genetic mutation, documented low or high potassium levels during attacks or a decrement on long exercise testing support diagnosis. The treatment approach should include both management of acute attacks and prevention of attacks. Treatments include behavioral interventions directed at avoidance of triggers, modification of potassium levels, diuretics, and carbonic anhydrase inhibitors. Muscle Nerve 57: 522-530, 2018.
Assuntos
Síndrome de Andersen/diagnóstico , Paralisias Periódicas Familiares/diagnóstico , Acetazolamida/uso terapêutico , Síndrome de Andersen/terapia , Antiarrítmicos/uso terapêutico , Terapia Comportamental , Inibidores da Anidrase Carbônica/uso terapêutico , Diuréticos/uso terapêutico , Diurético Poupador de Potássio/uso terapêutico , Humanos , Hidroclorotiazida/uso terapêutico , Paralisia Periódica Hipopotassêmica/diagnóstico , Paralisia Periódica Hipopotassêmica/terapia , Paralisias Periódicas Familiares/terapia , Paralisia Periódica Hiperpotassêmica/diagnóstico , Paralisia Periódica Hiperpotassêmica/terapia , Potássio/uso terapêuticoRESUMO
The NaV1.4 sodium channel is highly expressed in skeletal muscle, where it carries almost all of the inward Na+ current that generates the action potential, but is not present at significant levels in other tissues. Consequently, mutations of SCN4A encoding NaV1.4 produce pure skeletal muscle phenotypes that now include six allelic disorders: sodium channel myotonia, paramyotonia congenita, hyperkalemic periodic paralysis, hypokalemic periodic paralysis, congenital myasthenia, and congenital myopathy with hypotonia. Mutation-specific alternations of NaV1.4 function explain the mechanistic basis for the diverse phenotypes and identify opportunities for strategic intervention to modify the burden of disease.
Assuntos
Canalopatias/etiologia , Doenças Musculares/etiologia , Mutação , Canal de Sódio Disparado por Voltagem NAV1.4/genética , Animais , Humanos , Miotonia/etiologia , Miotonia Congênita/etiologia , Paralisia Periódica Hiperpotassêmica/etiologiaRESUMO
Over 60 mutations of SCN4A encoding the NaV1.4 sodium channel of skeletal muscle have been identified in patients with myotonia, periodic paralysis, myasthenia, or congenital myopathy. Most mutations are missense with gain-of-function defects that cause susceptibility to myotonia or periodic paralysis. Loss-of-function from enhanced inactivation or null alleles is rare and has been associated with myasthenia and congenital myopathy, while a mix of loss and gain of function changes has an uncertain relation to hypokalaemic periodic paralysis. To better define the functional consequences for a loss-of-function, we generated NaV1.4 null mice by deletion of exon 12. Heterozygous null mice have latent myasthenia and a right shift of the force-stimulus relation, without evidence of periodic paralysis. Sodium current density was half that of wild-type muscle and no compensation by retained expression of the foetal NaV1.5 isoform was detected. Mice null for NaV1.4 did not survive beyond the second postnatal day. This mouse model shows remarkable preservation of muscle function and viability for haploinsufficiency of NaV1.4, as has been reported in humans, with a propensity for pseudo-myasthenia caused by a marginal Na(+) current density to support sustained high-frequency action potentials in muscle.
Assuntos
Debilidade Muscular/genética , Canal de Sódio Disparado por Voltagem NAV1.4/genética , Potenciais de Ação/genética , Potenciais de Ação/fisiologia , Animais , Modelos Animais de Doenças , Heterozigoto , Potenciais da Membrana/genética , Potenciais da Membrana/fisiologia , Camundongos , Camundongos Knockout , Músculo Esquelético/fisiologia , LinhagemRESUMO
Excitation-contraction (EC) coupling comprises events in muscle that convert electrical signals to Ca(2+) transients, which then trigger contraction of the sarcomere. Defects in these processes cause a spectrum of muscle diseases. We report that STAC3, a skeletal muscle-specific protein that localizes to T tubules, is essential for coupling membrane depolarization to Ca(2+) release from the sarcoplasmic reticulum (SR). Consequently, homozygous deletion of src homology 3 and cysteine rich domain 3 (Stac3) in mice results in complete paralysis and perinatal lethality with a range of musculoskeletal defects that reflect a blockade of EC coupling. Muscle contractility and Ca(2+) release from the SR of cultured myotubes from Stac3 mutant mice could be restored by application of 4-chloro-m-cresol, a ryanodine receptor agonist, indicating that the sarcomeres, SR Ca(2+) store, and ryanodine receptors are functional in Stac3 mutant skeletal muscle. These findings reveal a previously uncharacterized, but required, component of the EC coupling machinery of skeletal muscle and introduce a candidate for consideration in myopathic disorders.
Assuntos
Cálcio/metabolismo , Contração Muscular/fisiologia , Músculo Esquelético/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Potenciais de Ação/fisiologia , Proteínas Adaptadoras de Transdução de Sinal , Animais , Northern Blotting , Western Blotting , Primers do DNA/genética , Eletroporação , Genótipo , Hibridização In Situ , Camundongos , Camundongos Knockout , Microscopia Eletrônica , Músculo Esquelético/fisiologia , Músculo Esquelético/ultraestrutura , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/fisiologia , Reação em Cadeia da Polimerase em Tempo Real , beta-GalactosidaseAssuntos
Canalopatias , Morte Súbita do Lactente , Humanos , Lactente , Músculo Esquelético , Fatores de RiscoRESUMO
Transient attacks of weakness in hypokalaemic periodic paralysis are caused by reduced fibre excitability from paradoxical depolarization of the resting potential in low potassium. Mutations of calcium channel and sodium channel genes have been identified as the underlying molecular defects that cause instability of the resting potential. Despite these scientific advances, therapeutic options remain limited. In a mouse model of hypokalaemic periodic paralysis from a sodium channel mutation (NaV1.4-R669H), we recently showed that inhibition of chloride influx with bumetanide reduced the susceptibility to attacks of weakness, in vitro. The R528H mutation in the calcium channel gene (CACNA1S encoding CaV1.1) is the most common cause of hypokalaemic periodic paralysis. We developed a CaV1.1-R528H knock-in mouse model of hypokalaemic periodic paralysis and show herein that bumetanide protects against both muscle weakness from low K+ challenge in vitro and loss of muscle excitability in vivo from a glucose plus insulin infusion. This work demonstrates the critical role of the chloride gradient in modulating the susceptibility to ictal weakness and establishes bumetanide as a potential therapy for hypokalaemic periodic paralysis arising from either NaV1.4 or CaV1.1 mutations.
Assuntos
Bumetanida/uso terapêutico , Canais de Cálcio Tipo L/genética , Paralisia Periódica Hipopotassêmica/tratamento farmacológico , Paralisia Periódica Hipopotassêmica/genética , Mutação/genética , Inibidores de Simportadores de Cloreto de Sódio e Potássio/uso terapêutico , Acetazolamida/farmacologia , Animais , Arginina/genética , Inibidores da Anidrase Carbônica/farmacologia , Modelos Animais de Doenças , Potencial Evocado Motor/efeitos dos fármacos , Potencial Evocado Motor/genética , Furosemida/farmacologia , Glucose/metabolismo , Histidina/genética , Paralisia Periódica Hipopotassêmica/patologia , Paralisia Periódica Hipopotassêmica/fisiopatologia , Técnicas In Vitro , Contração Isométrica/efeitos dos fármacos , Contração Isométrica/genética , Masculino , Camundongos , Camundongos Transgênicos , Músculo Esquelético/fisiopatologiaRESUMO
Periodic paralysis is a rare, dominantly inherited disorder of skeletal muscle in which episodic attacks of weakness are caused by a transient impairment of fiber excitability. Attacks of weakness are often elicited by characteristic environmental triggers, which were the basis for clinically delineating subtypes of periodic paralysis and are an important distinction for optimal disease management. All forms of familial periodic paralysis are caused by mutations of ion channels, often selectively expressed in skeletal muscle, that destabilize the resting potential. The missense mutations usually alter channel function through gain-of-function changes rather than producing a complete loss-of-function null. The knowledge of which channel gene harbors a variant, whether that variant is expected to (or known to) alter function, and how altered function impairs fiber excitability aides in the interpretation of patient signs and symptoms, the interpretation of gene test results, and how to optimize therapeutic intervention for symptom management and improve quality of life.
Assuntos
Paralisias Periódicas Familiares , Humanos , Paralisias Periódicas Familiares/genética , Paralisias Periódicas Familiares/diagnóstico , Paralisias Periódicas Familiares/terapia , Mutação/genética , Canais Iônicos/genética , Músculo Esquelético/fisiopatologiaRESUMO
Mutations in tripartite motif protein 32 (TRIM32) are responsible for several hereditary disorders that include limb girdle muscular dystrophy type 2H (LGMD2H), sarcotubular myopathy (STM) and Bardet Biedl syndrome. Most LGMD2H mutations in TRIM32 are clustered in the NHL ß-propeller domain at the C-terminus and are predicted to interfere with homodimerization. To get insight into TRIM32's role in the pathogenesis of LGMD2H and to create an accurate model of disease, we have generated a knock-in mouse (T32KI) carrying the c.1465G > A (p.D489N) mutation in murine Trim32 corresponding to the human LGMD2H/STM pathogenic mutation c.1459G > A (p.D487N). Our data indicate that T32KI mice have both a myopathic and a neurogenic phenotype, very similar to the one described in the Trim32-null mice that we created previously. Analysis of Trim32 gene expression in T32KI mice revealed normal mRNA levels, but a severe reduction in mutant TRIM32 (D489N) at the protein level. Our results suggest that the D489N pathogenic mutation destabilizes the protein, leading to its degradation, and results in the same mild myopathic and neurogenic phenotype as that found in Trim32-null mice. Thus, one potential mechanism of LGMD2H might be destabilization of mutated TRIM32 protein leading to a null phenotype.
Assuntos
Distrofia Muscular do Cíngulo dos Membros/genética , Mutação de Sentido Incorreto , Fenótipo , Ubiquitina-Proteína Ligases/genética , Animais , Regulação da Expressão Gênica , Técnicas de Introdução de Genes , Marcação de Genes , Camundongos , Camundongos Transgênicos , Distrofia Muscular do Cíngulo dos Membros/metabolismo , Distrofia Muscular do Cíngulo dos Membros/patologia , Miosinas/metabolismo , Isoformas de Proteínas/metabolismo , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Hypokalemic periodic paralysis (hypoKPP) is characterized by episodic flaccid paralysis of muscle and acute hypokalemia during attacks. Familial forms of hypoKPP are predominantly caused by mutations of either voltage-gated Ca(2+) or Na(+) channels. The pathogenic gene mutation in non-familial hypoKPP, consisting mainly of thyrotoxic periodic paralysis (TPP) and sporadic periodic paralysis (SPP), is largely unknown. Recently, mutations in KCNJ18, which encodes a skeletal muscle-specific inwardly rectifying K(+) channel Kir2.6, were reported in some TPP patients. Whether mutations of Kir2.6 occur in other patients with non-familial hypoKPP and how mutations of the channel predispose patients to paralysis are unknown. Here, we report one conserved heterozygous mutation in KCNJ18 in two TPP patients and two separate heterozygous mutations in two SPP patients. These mutations result in V168M, R43C, and A200P amino acid substitution of Kir2.6, respectively. Compared with the wild type channel, whole-cell currents of R43C and V168M mutants were reduced by â¼78 and 43%, respectively. No current was detected for the A200P mutant. Single channel conductance and open probability were reduced for R43C and V168M, respectively. Biotinylation assays showed reduced cell surface abundance for R43C and A200P. All three mutants exerted dominant negative inhibition on wild type Kir2.6 as well as wild type Kir2.1, another Kir channel expressed in the skeletal muscle. Thus, mutations of Kir2.6 are associated with SPP as well as TPP. We suggest that decreased outward K(+) current from hypofunction of Kir2.6 predisposes the sarcolemma to hypokalemia-induced paradoxical depolarization during attacks, which in turn leads to Na(+) channel inactivation and inexcitability of muscles.
Assuntos
Paralisia Periódica Hipopotassêmica/genética , Mutação , Canais de Potássio Corretores do Fluxo de Internalização/genética , Adulto , Sequência de Aminoácidos , Sequência de Bases , Primers do DNA , Feminino , Humanos , Masculino , Dados de Sequência MolecularRESUMO
Hyperkalemic periodic paralysis (HyperKPP) produces myotonia and attacks of muscle weakness triggered by rest after exercise or by K+ ingestion. We introduced a missense substitution corresponding to a human familial HyperKPP mutation (Met1592Val) into the mouse gene encoding the skeletal muscle voltage-gated Na+ channel NaV1.4. Mice heterozygous for this mutation exhibited prominent myotonia at rest and muscle fiber-type switching to a more oxidative phenotype compared with controls. Isolated mutant extensor digitorum longus muscles were abnormally sensitive to the Na+/K+ pump inhibitor ouabain and exhibited age-dependent changes, including delayed relaxation and altered generation of tetanic force. Moreover, rapid and sustained weakness of isolated mutant muscles was induced when the extracellular K+ concentration was increased from 4 mM to 10 mM, a level observed in the muscle interstitium of humans during exercise. Mutant muscle recovered from stimulation-induced fatigue more slowly than did control muscle, and the extent of recovery was decreased in the presence of high extracellular K+ levels. These findings demonstrate that expression of the Met1592ValNa+ channel in mouse muscle is sufficient to produce important features of HyperKPP, including myotonia, K+-sensitive paralysis, and susceptibility to delayed weakness during recovery from fatigue.
Assuntos
Músculo Esquelético/metabolismo , Miotonia/metabolismo , Miotonia/patologia , Potássio/metabolismo , Canais de Sódio/metabolismo , Envelhecimento/fisiologia , Animais , Progressão da Doença , Eletrofisiologia , Regulação da Expressão Gênica , Humanos , Camundongos , Camundongos Transgênicos , Mutação/genética , Miotonia/genética , Oxirredução , Paralisia Periódica Hiperpotassêmica/genética , Paralisia Periódica Hiperpotassêmica/metabolismo , Paralisia Periódica Hiperpotassêmica/patologia , Fenótipo , RNA Mensageiro/genética , Sensibilidade e Especificidade , Canais de Sódio/genéticaRESUMO
Loss-of-function mutations of SCN1A encoding the pore-forming α subunit of the NaV1.1 neuronal sodium channel cause a severe developmental epileptic encephalopathy, Dravet syndrome (DS). In this issue of the JCI, Chen, Luo, Gao, et al. describe a phenocopy for DS in mice deficient for posttranslational conjugation with neural precursor cell expressed, developmentally downregulated 8 (NEDD8) (neddylation), selectively engineered in inhibitory interneurons. Pursuing the possibility that this phenotype is also caused by loss of NaV1.1, Chen, Luo, Gao, and colleagues show that interneuron excitability and GABA release are impaired, NaV1.1 degradation rate is increased with a commensurate decrease of NaV1.1 protein, and NaV1.1 is a substrate for neddylation. These findings establish neddylation as a mechanism for stabilizing NaV1.1 subunits and suggest another pathomechanism for epileptic sodium channelopathy.
Assuntos
Canalopatias , Epilepsias Mioclônicas , Epilepsia , Animais , Canalopatias/genética , Epilepsias Mioclônicas/genética , Epilepsia/genética , Interneurônios , Camundongos , Canal de Sódio Disparado por Voltagem NAV1.1/genéticaRESUMO
Mutations in the voltage sensor domain (VSD) of CaV1.1, the α1S subunit of the L-type calcium channel in skeletal muscle, are an established cause of hypokalemic periodic paralysis (HypoPP). Of the 10 reported mutations, 9 are missense substitutions of outer arginine residues (R1 or R2) in the S4 transmembrane segments of the homologous domain II, III (DIII), or IV. The prevailing view is that R/X mutations create an anomalous ion conduction pathway in the VSD, and this so-called gating pore current is the basis for paradoxical depolarization of the resting potential and weakness in low potassium for HypoPP fibers. Gating pore currents have been observed for four of the five CaV1.1 HypoPP mutant channels studied to date, the one exception being the charge-conserving R897K in R1 of DIII. We tested whether gating pore currents are detectable for the other three HypoPP CaV1.1 mutations in DIII. For the less conserved R1 mutation, R897S, gating pore currents with exceptionally large amplitude were observed, correlating with the severe clinical phenotype of these patients. At the R2 residue, gating pore currents were detected for R900G but not R900S. These findings show that gating pore currents may occur with missense mutations at R1 or R2 in S4 of DIII and that the magnitude of this anomalous inward current is mutation specific.
Assuntos
Paralisia Periódica Hipopotassêmica , Canais de Cálcio Tipo L/genética , Humanos , Paralisia Periódica Hipopotassêmica/genética , Potenciais da Membrana , Músculo Esquelético , Mutação , Mutação de Sentido IncorretoRESUMO
Initiation of skeletal muscle contraction is triggered by rapid activation of RYR1 channels in response to sarcolemmal depolarization. RYR1 is intracellular and has no voltage-sensing structures, but it is coupled with the voltage-sensing apparatus of CaV1.1 channels to inherit voltage sensitivity. Using an opto-electrophysiological approach, we resolved the excitation-driven molecular events controlling both CaV1.1 and RYR1 activations, reported as fluorescence changes. We discovered that each of the four human CaV1.1 voltage-sensing domains (VSDs) exhibits unique biophysical properties: VSD-I time-dependent properties were similar to ionic current activation kinetics, suggesting a critical role of this voltage sensor in CaV1.1 activation; VSD-II, VSD-III, and VSD-IV displayed faster activation, compatible with kinetics of sarcoplasmic reticulum Ca2+ release. The prominent role of VSD-I in governing CaV1.1 activation was also confirmed using a naturally occurring, charge-neutralizing mutation in VSD-I (R174W). This mutation abolished CaV1.1 current at physiological membrane potentials by impairing VSD-I activation without affecting the other VSDs. Using a structurally relevant allosteric model of CaV activation, which accounted for both time- and voltage-dependent properties of CaV1.1, to predict VSD-pore coupling energies, we found that VSD-I contributed the most energy (~75 meV or â¼3 kT) toward the stabilization of the open states of the channel, with smaller (VSD-IV) or negligible (VSDs II and III) energetic contribution from the other voltage sensors (<25 meV or â¼1 kT). This study settles the longstanding question of how CaV1.1, a slowly activating channel, can trigger RYR1 rapid activation, and reveals a new mechanism for voltage-dependent activation in ion channels, whereby pore opening of human CaV1.1 channels is primarily driven by the activation of one voltage sensor, a mechanism distinct from that of all other voltage-gated channels.
Assuntos
Canais de Cálcio Tipo L , Contração Muscular , Canais de Cálcio Tipo L/metabolismo , Fenômenos Eletrofisiológicos , Humanos , Cinética , Potenciais da MembranaRESUMO
Mutations of voltage-gated ion channels cause several channelopathies of skeletal muscle, which present clinically with myotonia, periodic paralysis, or a combination of both. Expression studies have revealed both loss-of-function and gain-of-function defects for the currents passed by mutant channels. In many cases, these functional changes could be mechanistically linked to the defects of fibre excitability underlying myotonia or periodic paralysis. One remaining enigma was the basis for depolarization-induced weakness in hypokalaemic periodic paralysis (HypoPP) arising from mutations in either sodium or calcium channels. Curiously, 14 of 15 HypoPP mutations are at arginines in S4 voltage sensors, and recent observations show that these substitutions support an alternative pathway for ion conduction, the gating pore, that may be the source of the aberrant depolarization during an attack of paralysis.