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1.
Comput Biol Chem ; 83: 107132, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31563636

RESUMO

Here, we focused on exploring the selectivity mechanism against Nav1.7 over Nav1.4 due to different binding modes of two selected inhibitors. By the superposition of Nav1.7 and Nav1.4 proteins, we selected the most homologous chain of Nav1.7 with Nav1.4, defining the active site of Nav1.4-VSD4 based on the aryl sulfonamide binding site of Nav1.7-VSD4. Comparison of the conformations exhibited by Tyr1386 (Nav1.4) and Tyr1537 (Nav1.7) suggested that the steric hindrance caused by Tyr1386 owned primary influence on inhibition selectivity, which was further verified through molecular docking and MD simulation of two representative inhibitors. Our finding would be helpful for discovery of selective Nav1.7 inhibitors over Nav1.4.


Assuntos
Modelos Moleculares , Canal de Sódio Disparado por Voltagem NAV1.4/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.7/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Sulfonamidas/farmacologia , Sítios de Ligação/efeitos dos fármacos , Relação Dose-Resposta a Droga , Humanos , Ligantes , Estrutura Molecular , Bibliotecas de Moléculas Pequenas/química , Software , Relação Estrutura-Atividade , Sulfonamidas/química , Termodinâmica
2.
Dokl Biochem Biophys ; 484(1): 9-12, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31012002

RESUMO

An effective bacterial system for the production of ß-toxin Ts1, the main component of the Brazilian scorpion Tityus serrulatus venom, was developed. Recombinant toxin and its 15N-labeled analogue were obtained via direct expression of synthetic gene in Escherichia coli with subsequent folding from the inclusion bodies. According to NMR spectroscopy data, the recombinant toxin is structured in an aqueous solution and contains a significant fraction of ß-structure. The formation of a stable disulfide-bond isomer of Ts1, having a disordered structure, has also been observed during folding. Recombinant Ts1 blocks Na+ current through NaV1.5 channels without affecting the processes of activation and inactivation. At the same time, the effect upon NaV1.4 channels is associated with a shift of the activation curve towards more negative membrane potentials.


Assuntos
Venenos de Escorpião , Bloqueadores dos Canais de Sódio , Animais , Humanos , Proteínas Musculares/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.4/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.5/metabolismo , Ressonância Magnética Nuclear Biomolecular , Estrutura Secundária de Proteína , Ratos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/farmacologia , Venenos de Escorpião/biossíntese , Venenos de Escorpião/química , Venenos de Escorpião/isolamento & purificação , Venenos de Escorpião/farmacologia , Bloqueadores dos Canais de Sódio/química , Bloqueadores dos Canais de Sódio/isolamento & purificação , Bloqueadores dos Canais de Sódio/farmacologia , Canais de Sódio/metabolismo , Relação Estrutura-Atividade , Xenopus laevis
3.
Nat Commun ; 10(1): 1514, 2019 04 03.
Artigo em Inglês | MEDLINE | ID: mdl-30944319

RESUMO

Skeletal muscle voltage-gated Na+ channel (NaV1.4) activity is subject to calmodulin (CaM) mediated Ca2+-dependent inactivation; no such inactivation is observed in the cardiac Na+ channel (NaV1.5). Taken together, the crystal structures of the NaV1.4 C-terminal domain relevant complexes and thermodynamic binding data presented here provide a rationale for this isoform difference. A Ca2+-dependent CaM N-lobe binding site previously identified in NaV1.5 is not present in NaV1.4 allowing the N-lobe to signal other regions of the NaV1.4 channel. Consistent with this mechanism, removing this binding site in NaV1.5 unveils robust Ca2+-dependent inactivation in the previously insensitive isoform. These findings suggest that Ca2+-dependent inactivation is effected by CaM's N-lobe binding outside the NaV C-terminal while CaM's C-lobe remains bound to the NaV C-terminal. As the N-lobe binding motif of NaV1.5 is a mutational hotspot for inherited arrhythmias, the contributions of mutation-induced changes in CDI to arrhythmia generation is an intriguing possibility.


Assuntos
Cálcio/metabolismo , Calmodulina/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.4/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.5/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Arritmias Cardíacas/genética , Arritmias Cardíacas/metabolismo , Sítios de Ligação , Cálcio/química , Calmodulina/química , Calmodulina/genética , Humanos , Modelos Moleculares , Músculo Esquelético/metabolismo , Mutação , Canal de Sódio Disparado por Voltagem NAV1.4/química , Canal de Sódio Disparado por Voltagem NAV1.4/genética , Canal de Sódio Disparado por Voltagem NAV1.5/química , Canal de Sódio Disparado por Voltagem NAV1.5/genética , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Domínios e Motivos de Interação entre Proteínas , Isoformas de Proteínas
4.
Neurology ; 92(13): e1405-e1415, 2019 03 26.
Artigo em Inglês | MEDLINE | ID: mdl-30824560

RESUMO

OBJECTIVE: To identify the genetic and physiologic basis for recessive myasthenic congenital myopathy in 2 families, suggestive of a channelopathy involving the sodium channel gene, SCN4A. METHODS: A combination of whole exome sequencing and targeted mutation analysis, followed by voltage-clamp studies of mutant sodium channels expressed in fibroblasts (HEK cells) and Xenopus oocytes. RESULTS: Missense mutations of the same residue in the skeletal muscle sodium channel, R1460 of NaV1.4, were identified in a family and a single patient of Finnish origin (p.R1460Q) and a proband in the United States (p.R1460W). Congenital hypotonia, breathing difficulties, bulbar weakness, and fatigability had recessive inheritance (homozygous p.R1460W or compound heterozygous p.R1460Q and p.R1059X), whereas carriers were either asymptomatic (p.R1460W) or had myotonia (p.R1460Q). Sodium currents conducted by mutant channels showed unusual mixed defects with both loss-of-function (reduced amplitude, hyperpolarized shift of inactivation) and gain-of-function (slower entry and faster recovery from inactivation) changes. CONCLUSIONS: Novel mutations in families with myasthenic congenital myopathy have been identified at p.R1460 of the sodium channel. Recessive inheritance, with experimentally established loss-of-function, is a consistent feature of sodium channel based myasthenia, whereas the mixed gain of function for p.R1460 may also cause susceptibility to myotonia.


Assuntos
Síndromes Miastênicas Congênitas/genética , Canal de Sódio Disparado por Voltagem NAV1.4/genética , Adulto , Animais , Eletromiografia , Feminino , Finlândia , Humanos , Laringismo/genética , Laringismo/fisiopatologia , Mutação com Perda de Função , Masculino , Hipotonia Muscular/genética , Hipotonia Muscular/fisiopatologia , Músculo Esquelético/patologia , Mutação de Sentido Incorreto , Síndromes Miastênicas Congênitas/metabolismo , Síndromes Miastênicas Congênitas/fisiopatologia , Miotonia/genética , Miotonia/fisiopatologia , Canal de Sódio Disparado por Voltagem NAV1.4/metabolismo , Oócitos , Técnicas de Patch-Clamp , Linhagem , Sequenciamento Completo do Exoma , Xenopus
5.
Biochem Biophys Res Commun ; 506(4): 826-832, 2018 12 02.
Artigo em Inglês | MEDLINE | ID: mdl-30389137

RESUMO

Voltage-gated sodium channels play important roles in human physiology. However, their complexity hinders the understanding of their physiology and pathology at atomic level. We took advantage of the structural reports of similar channels obtained by cryo-EM (EeNav1.4, and NavPaS), and constructed models of human Nav1.4 channels at closed and open states. The open-state model is very similar to the recently published cryo-EM structure of hNav1.4. The comparison of both models shows shifts of the voltage sensors (VS) of DIII and DIV. The activated position of VS-DII in the closed model was demonstrated by Ts1 docking, thereby confirming the requirement that VS-DI, VS-DII and VS-DIII must be activated for the channel to open. The interactions observed with VS-DIII suggest a stepwise, yet fast, transition from resting to activated state. These models provide structural insights on the closed-open transition of the channel.


Assuntos
Ativação do Canal Iônico , Modelos Biológicos , Músculo Esquelético/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.4/química , Canal de Sódio Disparado por Voltagem NAV1.4/metabolismo , Humanos , Simulação de Acoplamento Molecular
6.
Sci Rep ; 8(1): 11058, 2018 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-30038349

RESUMO

A patient with an early severe myotonia diagnosed for Myotonic Dystrophy type 2 (DM2) was found bearing the combined effects of DM2 mutation and Nav1.4 S906T substitution. To investigate the mechanism underlying his atypical phenotype,whole-cell patch-clamp in voltage- and current-clamp mode was performed in myoblasts and myotubes obtained from his muscle biopsy. Results characterizing the properties of the sodium current and of the action potentials have been compared to those obtained in muscle cells derived from his mother, also affected by DM2, but without the S906T polymorphism. A faster inactivation kinetics and a +5 mV shift in the availability curve were found in the sodium current recorded in patient's myoblasts compared to his mother. 27% of his myotubes displayed spontaneous activity. Patient's myotubes showing a stable resting membrane potential had a lower rheobase current respect to the mother's while the overshoot and the maximum slope of the depolarizing phase of action potential were higher. These findings suggest that SCN4A polymorphisms may be responsible for a higher excitability of DM2 patients sarcolemma, supporting the severe myotonic phenotype observed. We suggest SCN4A as a modifier factor and that its screening should be performed in DM2 patients with uncommon clinical features.


Assuntos
Distrofia Miotônica/genética , Distrofia Miotônica/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.4/metabolismo , Potenciais de Ação/fisiologia , Adulto , Canais de Cloreto/genética , Canais de Cloreto/metabolismo , Feminino , Humanos , Masculino , Potenciais da Membrana/genética , Potenciais da Membrana/fisiologia , Pessoa de Meia-Idade , Fibras Musculares Esqueléticas/citologia , Fibras Musculares Esqueléticas/metabolismo , Mutação/genética , Mioblastos/citologia , Mioblastos/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.4/genética , Sarcolema/genética , Sarcolema/metabolismo
7.
Sci Rep ; 8(1): 9714, 2018 06 26.
Artigo em Inglês | MEDLINE | ID: mdl-29946067

RESUMO

Dominantly inherited channelopathies of the skeletal muscle voltage-gated sodium channel NaV1.4 include hypokalaemic and hyperkalaemic periodic paralysis (hypoPP and hyperPP) and myotonia. HyperPP and myotonia are caused by NaV1.4 channel overactivity and overlap clinically. Instead, hypoPP is caused by gating pore currents through the voltage sensing domains (VSDs) of NaV1.4 and seldom co-exists clinically with myotonia. Recessive loss-of-function NaV1.4 mutations have been described in congenital myopathy and myasthenic syndromes. We report two families with the NaV1.4 mutation p.R1451L, located in VSD-IV. Heterozygous carriers in both families manifest with myotonia and/or hyperPP. In contrast, a homozygous case presents with both hypoPP and myotonia, but unlike carriers of recessive NaV1.4 mutations does not manifest symptoms of myopathy or myasthenia. Functional analysis revealed reduced current density and enhanced closed state inactivation of the mutant channel, but no evidence for gating pore currents. The rate of recovery from inactivation was hastened, explaining the myotonia in p.R1451L carriers and the absence of myasthenic presentations in the homozygous proband. Our data suggest that recessive loss-of-function NaV1.4 variants can present with hypoPP without congenital myopathy or myasthenia and that myotonia can present even in carriers of homozygous NaV1.4 loss-of-function mutations.


Assuntos
Paralisia Periódica Hipopotassêmica/genética , Paralisia Periódica Hipopotassêmica/metabolismo , Miotonia/genética , Miotonia/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.4/metabolismo , Adulto , Animais , Eletrofisiologia , Células HEK293 , Heterozigoto , Sequenciamento de Nucleotídeos em Larga Escala , Homozigoto , Humanos , Masculino , Mutação/genética , Canal de Sódio Disparado por Voltagem NAV1.4/genética , Linhagem , Estrutura Secundária de Proteína , Adulto Jovem
8.
Biomol NMR Assign ; 12(2): 283-289, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-29728980

RESUMO

Human voltage-gated sodium (NaV) channels are critical for initiating and propagating action potentials in excitable cells. Nine isoforms have different roles but similar topologies, with a pore-forming α-subunit and auxiliary transmembrane ß-subunits. NaV pathologies lead to debilitating conditions including epilepsy, chronic pain, cardiac arrhythmias, and skeletal muscle paralysis. The ubiquitous calcium sensor calmodulin (CaM) binds to an IQ motif in the C-terminal tail of the α-subunit of all NaV isoforms, and contributes to calcium-dependent pore-gating in some channels. Previous structural studies of calcium-free (apo) CaM bound to the IQ motifs of NaV1.2, NaV1.5, and NaV1.6 showed that CaM binding was mediated by the C-domain of CaM (CaMC), while the N-domain (CaMN) made no detectable contacts. To determine whether this domain-specific recognition mechanism is conserved in other NaV isoforms, we used solution NMR spectroscopy to assign the backbone resonances of complexes of apo CaM bound to peptides of IQ motifs of NaV1.1, NaV1.4, and NaV1.7. Analysis of chemical shift differences showed that peptide binding only perturbed resonances in CaMC; resonances of CaMN were identical to free CaM. Thus, CaMC residues contribute to the interface with the IQ motif, while CaMN is available to interact elsewhere on the channel.


Assuntos
Apoproteínas/química , Apoproteínas/metabolismo , Calmodulina/química , Calmodulina/metabolismo , Ressonância Magnética Nuclear Biomolecular , Canais de Sódio Disparados por Voltagem/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Humanos , Canal de Sódio Disparado por Voltagem NAV1.1/química , Canal de Sódio Disparado por Voltagem NAV1.1/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.4/química , Canal de Sódio Disparado por Voltagem NAV1.4/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.7/química , Canal de Sódio Disparado por Voltagem NAV1.7/metabolismo , Canais de Sódio Disparados por Voltagem/química
9.
Nature ; 557(7706): 590-594, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29769724

RESUMO

Potassium-sensitive hypokalaemic and normokalaemic periodic paralysis are inherited skeletal muscle diseases characterized by episodes of flaccid muscle weakness1,2. They are caused by single mutations in positively charged residues ('gating charges') in the S4 transmembrane segment of the voltage sensor of the voltage-gated sodium channel Nav1.4 or the calcium channel Cav1.11,2. Mutations of the outermost gating charges (R1 and R2) cause hypokalaemic periodic paralysis1,2 by creating a pathogenic gating pore in the voltage sensor through which cations leak in the resting state3,4. Mutations of the third gating charge (R3) cause normokalaemic periodic paralysis 5 owing to cation leak in both activated and inactivated states 6 . Here we present high-resolution structures of the model bacterial sodium channel NavAb with the analogous gating-charge mutations7,8, which have similar functional effects as in the human channels. The R2G and R3G mutations have no effect on the backbone structures of the voltage sensor, but they create an aqueous cavity near the hydrophobic constriction site that controls gating charge movement through the voltage sensor. The R3G mutation extends the extracellular aqueous cleft through the entire length of the activated voltage sensor, creating an aqueous path through the membrane. Conversely, molecular modelling shows that the R2G mutation creates a continuous aqueous path through the membrane only in the resting state. Crystal structures of NavAb(R2G) in complex with guanidinium define a potential drug target site. Molecular dynamics simulations illustrate the mechanism of Na+ permeation through the mutant gating pore in concert with conformational fluctuations of the gating charge R4. Our results reveal pathogenic mechanisms of periodic paralysis at the atomic level and suggest designs of drugs that may prevent ionic leak and provide symptomatic relief from hypokalaemic and normokalaemic periodic paralysis.


Assuntos
Ativação do Canal Iônico , Canal de Sódio Disparado por Voltagem NAV1.4/química , Canal de Sódio Disparado por Voltagem NAV1.4/metabolismo , Paralisias Periódicas Familiares/metabolismo , Sítios de Ligação , Condutividade Elétrica , Guanidina/metabolismo , Humanos , Paralisia Periódica Hipopotassêmica/genética , Paralisia Periódica Hipopotassêmica/metabolismo , Ativação do Canal Iônico/genética , Simulação de Dinâmica Molecular , Mutação , Canal de Sódio Disparado por Voltagem NAV1.4/genética , Paralisias Periódicas Familiares/genética , Sódio/metabolismo , Termodinâmica
10.
Proc Natl Acad Sci U S A ; 115(17): 4495-4500, 2018 04 24.
Artigo em Inglês | MEDLINE | ID: mdl-29636418

RESUMO

Gating pore currents through the voltage-sensing domains (VSDs) of the skeletal muscle voltage-gated sodium channel NaV1.4 underlie hypokalemic periodic paralysis (HypoPP) type 2. Gating modifier toxins target ion channels by modifying the function of the VSDs. We tested the hypothesis that these toxins could function as blockers of the pathogenic gating pore currents. We report that a crab spider toxin Hm-3 from Heriaeus melloteei can inhibit gating pore currents due to mutations affecting the second arginine residue in the S4 helix of VSD-I that we have found in patients with HypoPP and describe here. NMR studies show that Hm-3 partitions into micelles through a hydrophobic cluster formed by aromatic residues and reveal complex formation with VSD-I through electrostatic and hydrophobic interactions with the S3b helix and the S3-S4 extracellular loop. Our data identify VSD-I as a specific binding site for neurotoxins on sodium channels. Gating modifier toxins may constitute useful hits for the treatment of HypoPP.


Assuntos
Mutação de Sentido Incorreto , Canal de Sódio Disparado por Voltagem NAV1.4/metabolismo , Neurotoxinas/toxicidade , Paralisia Periódica Hiperpotassêmica/metabolismo , Estrutura Secundária de Proteína , Venenos de Aranha/toxicidade , Substituição de Aminoácidos , Animais , Feminino , Células HEK293 , Humanos , Ativação do Canal Iônico , Canal de Sódio Disparado por Voltagem NAV1.4/química , Canal de Sódio Disparado por Voltagem NAV1.4/genética , Paralisia Periódica Hiperpotassêmica/genética , Paralisia Periódica Hiperpotassêmica/patologia , Xenopus laevis
11.
PLoS One ; 13(3): e0194265, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29534110

RESUMO

Spatial heterogeneity in the strength or agents of selection can lead to geographic variation in ecologically important phenotypes. Many dendrobatid frogs sequester alkaloid toxins from their diets and often exhibit fixed mutations at NaV1.4, a voltage-gated sodium ion channel associated with alkaloid toxin resistance. Yet previous studies have noted an absence of resistance mutations in individuals from several species known to sequester alkaloid toxins, suggesting possible intraspecific variation for alkaloid resistance in these species. Toxicity and alkaloid profiles vary substantially between populations in several poison frog species (genus Dendrobates) and are correlated with variation in a suite of related traits such as aposematic coloration. If resistance mutations are costly, due to alterations of channel gating properties, we expect that low toxicity populations will have reduced frequencies and potentially even the loss of resistance alleles. Here, we examine whether intraspecific variation in toxicity in three dendrobatid frogs is associated with intraspecific variation in alleles conferring toxin resistance. Specifically, we examine two species that display marked variation in toxicity throughout their native ranges (Dendrobates pumilio and D. granuliferus) and one species with reduced toxicity in its introduced range (D. auratus). However, we find no evidence for population-level variation in alkaloid resistance at NaV1.4. In fact, contrary to previous studies, we found that alkaloid resistance alleles were not absent in any populations of these species. All three species exhibit fixed alkaloid resistance mutations throughout their ranges, suggesting that these mutations are maintained even when alkaloid sequestration is substantially reduced.


Assuntos
Alcaloides/toxicidade , Anuros/fisiologia , Evolução Biológica , Resistência a Medicamentos/genética , Canal de Sódio Disparado por Voltagem NAV1.4/genética , Venenos/toxicidade , Alelos , Animais , Comportamento Animal/fisiologia , Variação Biológica da População/genética , Comportamento Alimentar/fisiologia , Cromatografia Gasosa-Espectrometria de Massas , Geografia , Haplótipos , Fígado/anatomia & histologia , Camundongos , Mutação , Canal de Sódio Disparado por Voltagem NAV1.4/metabolismo , Fenótipo , Testes de Toxicidade/métodos
12.
Curr Med Chem ; 25(42): 5822-5834, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29589531

RESUMO

Tocainide is an antiarrhythmic agent belonging to class IB that was primarily used for suppression of symptomatic ventricular arrhythmias. Tocainide was also reported to relieve pain such as tic douloureux, trigemina neuralgia in humans and tinnitus. Significant antinociception, as assayed on the hot-plate test, was observed after intraperitoneal injection of tocainide, too. By the mid-1980s tocainide was emerging as a more consistently effective treatment for myotonic disorders. Numerous reports of serious adverse reactions led to the use of tocainide being discontinued, even though research on tocainide and its analogues, endowed with a better pharmacological profile, is still in progress for their potential usefulness in the treatment of myotonias. This review is focused on the description of the different synthetic routes to racemic and optically active tocainide developed in the last decades, as well as analytical studies regarding enantioseparation methods. Finally, some analogues of tocainide reported in the literature, most of which with pharmacological studies, have been mentioned.


Assuntos
Antiarrítmicos/síntese química , Tocainide/análogos & derivados , Antiarrítmicos/farmacocinética , Antiarrítmicos/uso terapêutico , Arritmias Cardíacas/tratamento farmacológico , Meia-Vida , Humanos , Canal de Sódio Disparado por Voltagem NAV1.4/química , Canal de Sódio Disparado por Voltagem NAV1.4/metabolismo , Relação Quantitativa Estrutura-Atividade , Tocainide/farmacocinética , Tocainide/uso terapêutico , Bloqueadores do Canal de Sódio Disparado por Voltagem/síntese química , Bloqueadores do Canal de Sódio Disparado por Voltagem/farmacocinética , Bloqueadores do Canal de Sódio Disparado por Voltagem/uso terapêutico
13.
Sci Rep ; 8(1): 2041, 2018 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-29391559

RESUMO

Mutations in NaV1.4, the skeletal muscle voltage-gated Na+ channel, underlie several skeletal muscle channelopathies. We report here the functional characterization of two substitutions targeting the R1451 residue and resulting in 3 distinct clinical phenotypes. The R1451L is a novel pathogenic substitution found in two unrelated individuals. The first individual was diagnosed with non-dystrophic myotonia, whereas the second suffered from an unusual phenotype combining hyperkalemic and hypokalemic episodes of periodic paralysis (PP). The R1451C substitution was found in one individual with a single attack of hypoPP induced by glucocorticoids. To elucidate the biophysical mechanism underlying the phenotypes, we used the patch-clamp technique to study tsA201 cells expressing WT or R1451C/L channels. Our results showed that both substitutions shifted the inactivation to hyperpolarized potentials, slowed the kinetics of inactivation, slowed the recovery from slow inactivation and reduced the current density. Cooling further enhanced these abnormalities. Homology modeling revealed a disruption of hydrogen bonds in the voltage sensor domain caused by R1451C/L. We concluded that the altered biophysical properties of R1451C/L well account for the PMC-hyperPP cluster and that additional factors likely play a critical role in the inter-individual differences of clinical expression resulting from R1451C/L.


Assuntos
Mutação de Sentido Incorreto , Transtornos Miotônicos/genética , Canal de Sódio Disparado por Voltagem NAV1.4/metabolismo , Células HEK293 , Humanos , Ativação do Canal Iônico , Canal de Sódio Disparado por Voltagem NAV1.4/química , Canal de Sódio Disparado por Voltagem NAV1.4/genética
14.
Proc Natl Acad Sci U S A ; 114(39): 10491-10496, 2017 09 26.
Artigo em Inglês | MEDLINE | ID: mdl-28874544

RESUMO

Poison-dart Phyllobates terribilis frogs sequester lethal amounts of steroidal alkaloid batrachotoxin (BTX) in their skin as a defense mechanism against predators. BTX targets voltage-gated Na+ channels and enables them to open persistently. How BTX autoresistance arises in such frogs remains a mystery. The BTX receptor has been delineated along the Na+ channel inner cavity, which is formed jointly by four S6 transmembrane segments from domains D1 to D4. Within the P. terribilis muscle Na+ channel, five amino acid (AA) substitutions have been identified at D1/S6 and D4/S6. We therefore investigated the role of these naturally occurring substitutions in BTX autoresistance by introducing them into rat Nav1.4 muscle Na+ channel, both individually and in combination. Our results showed that combination mutants containing an N1584T substitution all conferred a complete BTX-resistant phenotype when expressed in mammalian HEK293t cells. The single N1584T mutant also retained its functional integrity and became exceptionally resistant to 5 µM BTX, aside from a small residual BTX effect. Single and combination mutants with the other four S6 residues (S429A, I433V, A445D, and V1583I) all remained highly BTX sensitive. These findings, along with diverse BTX phenotypes of N1584K/A/D/T mutant channels, led us to conclude that the conserved N1584 residue is indispensable for BTX actions, probably functioning as an integral part of the BTX receptor. Thus, complete BTX autoresistance found in P. terribilis muscle Na+ channels could emerge primarily from a single AA substitution (asparagine→threonine) via a single nucleotide mutation (AAC→ACC).


Assuntos
Substituição de Aminoácidos/genética , Batraquiotoxinas/toxicidade , Resistência a Medicamentos/genética , Canal de Sódio Disparado por Voltagem NAV1.4/genética , Animais , Anuros/metabolismo , Células HEK293 , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Mutagênese Sítio-Dirigida , Canal de Sódio Disparado por Voltagem NAV1.4/metabolismo , Domínios Proteicos/genética , Domínios Proteicos/fisiologia , Ratos , Canais de Sódio/genética
15.
J Physiol ; 595(22): 6837-6850, 2017 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-28940424

RESUMO

KEY POINTS: Paramyotonia congenita is a hereditary channelopathy caused by missense mutations in the SCN4A gene, which encodes the α subunit of the human skeletal muscle voltage-gated sodium channel NaV1.4. Affected individuals suffered from myotonia and paralysis of muscles, which were aggravated by exposure to cold. We report a three-generation Chinese family with patients presenting paramyotonia congenita and identify a novel N1366S mutation of NaV1.4. Whole-cell electrophysiological recordings of the N1366S channel reveal a gain-of-function change of gating in response to cold. Modelling and molecular dynamic simulation data suggest that an arginine-to-serine substitution at position 1366 increases the distance from N1366 to R1454 and disrupts the hydrogen bond formed between them at low temperature. We demonstrate that N1366S is a disease-causing mutation and that the temperature-sensitive alteration of N1366S channel activity may be responsible for the pronounced paramyotonia congenita symptoms of these patients. ABSTRACT: Paramyotonia congenita is an autosomal dominant skeletal muscle channelopathy caused by missense mutations in SCN4A, the gene encoding the α subunit of the human skeletal muscle voltage-gated sodium channel NaV1.4. We report a three-generation family in which six members present clinical symptoms of paramyotonia congenita characterized by a marked worsening of myotonia by cold and by the presence of clear episodes of paralysis. We identified a novel mutation in SCN4A (Asn1366Ser, N1366S) in all patients in the family but not in healthy relatives or in 500 normal control subjects. Functional analysis of the channel protein expressed in HEK293 cells by whole-cell patch clamp recording revealed that the N1366S mutation led to significant alterations in the gating process of the NaV1.4 channel. The N1366S mutant displayed a cold-induced hyperpolarizing shift in the voltage dependence of activation and a depolarizing shift in fast inactivation, as well as a reduced rate of fast inactivation and accelerated recovery from fast inactivation. In addition, homology modelling and molecular dynamic simulation of N1366S and wild-type NaV1.4 channels indicated that the arginine-to-serine substitution disrupted the hydrogen bond formed between N1366 and R1454. Together, our results suggest that N1366S is a gain-of-function mutation of NaV1.4 at low temperature and the mutation may be responsible for the clinical symptoms of paramyotonia congenita in the affected family and constitute a basis for studies into its pathogenesis.


Assuntos
Mutação com Ganho de Função , Ativação do Canal Iônico , Transtornos Miotônicos/genética , Canal de Sódio Disparado por Voltagem NAV1.4/genética , Adulto , Idoso , Temperatura Baixa , Feminino , Células HEK293 , Humanos , Masculino , Pessoa de Meia-Idade , Simulação de Dinâmica Molecular , Transtornos Miotônicos/metabolismo , Transtornos Miotônicos/patologia , Canal de Sódio Disparado por Voltagem NAV1.4/metabolismo
16.
J Biol Chem ; 292(44): 18270-18280, 2017 11 03.
Artigo em Inglês | MEDLINE | ID: mdl-28924048

RESUMO

Scorpion toxins can kill other animals by inducing paralysis and arrhythmia, which limits the potential applications of these agents in the clinical management of diseases. Antitumor-analgesic peptide (AGAP), purified from Buthus martensii Karsch, has been proved to possess analgesic and antitumor activities. Trp38, a conserved aromatic residue of AGAP, might play an important role in mediating AGAP activities according to the sequence and homology-modeling analyses. Therefore, an AGAP mutant, W38G, was generated, and effects of both AGAP and the mutant W38G were examined by whole-cell patch clamp techniques on the sodium channels hNav1.4 and hNav1.5, which were closely associated with the biotoxicity of skeletal and cardiac muscles, respectively. The data showed that both W38G and AGAP inhibited the peak currents of hNav1.4 and hNav1.5; however, W38G induced a much weaker inhibition of both channels than AGAP. Accordingly, W38G exhibited much less toxic effect on both skeletal and cardiac muscles than AGAP in vivo The analgesic activity of W38G and AGAP were verified in vivo as well, and W38G retained analgesic activity similar to AGAP. Inhibition to both Nav1.7 and Nav1.8 was involved in the analgesic mechanism of AGAP and W38G. These findings indicated that Trp38 was a key amino acid involved in the biotoxicity of AGAP, and the AGAP mutant W38G might be a safer alternative for clinical application because it retains the analgesic efficacy with less toxicity to skeletal and cardiac muscles.


Assuntos
Analgésicos não Entorpecentes/efeitos adversos , Antineoplásicos/efeitos adversos , Proteínas de Artrópodes/efeitos adversos , Mutação , Peptídeos/efeitos adversos , Venenos de Escorpião/efeitos adversos , Bloqueadores do Canal de Sódio Disparado por Voltagem/efeitos adversos , Substituição de Aminoácidos , Analgésicos não Entorpecentes/administração & dosagem , Analgésicos não Entorpecentes/farmacologia , Analgésicos não Entorpecentes/uso terapêutico , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/farmacologia , Proteínas de Artrópodes/uso terapêutico , Células CHO , Cricetulus , Relação Dose-Resposta a Droga , Feminino , Humanos , Masculino , Camundongos , Canal de Sódio Disparado por Voltagem NAV1.4/química , Canal de Sódio Disparado por Voltagem NAV1.4/genética , Canal de Sódio Disparado por Voltagem NAV1.4/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.5/química , Canal de Sódio Disparado por Voltagem NAV1.5/genética , Canal de Sódio Disparado por Voltagem NAV1.5/metabolismo , Peptídeos/genética , Peptídeos/farmacologia , Peptídeos/uso terapêutico , Distribuição Aleatória , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Venenos de Escorpião/genética , Venenos de Escorpião/farmacologia , Venenos de Escorpião/uso terapêutico , Escorpiões , Testes de Toxicidade Aguda , Testes de Toxicidade Subaguda , Bloqueadores do Canal de Sódio Disparado por Voltagem/administração & dosagem , Bloqueadores do Canal de Sódio Disparado por Voltagem/farmacologia , Bloqueadores do Canal de Sódio Disparado por Voltagem/uso terapêutico
17.
PLoS One ; 12(7): e0180154, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28683073

RESUMO

Ion channels regulate a variety of physiological processes and represent an important class of drug target. Among the many methods of studying ion channel function, patch clamp electrophysiology is considered the gold standard by providing the ultimate precision and flexibility. However, its utility in ion channel drug discovery is impeded by low throughput. Additionally, characterization of endogenous ion channels in primary cells remains technical challenging. In recent years, many automated patch clamp (APC) platforms have been developed to overcome these challenges, albeit with varying throughput, data quality and success rate. In this study, we utilized SyncroPatch 768PE, one of the latest generation APC platforms which conducts parallel recording from two-384 modules with giga-seal data quality, to push these 2 boundaries. By optimizing various cell patching parameters and a two-step voltage protocol, we developed a high throughput APC assay for the voltage-gated sodium channel Nav1.7. By testing a group of Nav1.7 reference compounds' IC50, this assay was proved to be highly consistent with manual patch clamp (R > 0.9). In a pilot screening of 10,000 compounds, the success rate, defined by > 500 MΩ seal resistance and >500 pA peak current, was 79%. The assay was robust with daily throughput ~ 6,000 data points and Z' factor 0.72. Using the same platform, we also successfully recorded endogenous voltage-gated potassium channel Kv1.3 in primary T cells. Together, our data suggest that SyncroPatch 768PE provides a powerful platform for ion channel research and drug discovery.


Assuntos
Ensaios de Triagem em Larga Escala/métodos , Potenciais da Membrana/fisiologia , Canal de Sódio Disparado por Voltagem NAV1.7/metabolismo , Técnicas de Patch-Clamp/métodos , Bloqueadores dos Canais de Potássio/farmacologia , Bloqueadores dos Canais de Sódio/farmacologia , Animais , Células CHO , Cricetulus , Avaliação Pré-Clínica de Medicamentos , Expressão Gênica , Ensaios de Triagem em Larga Escala/instrumentação , Canal de Potássio Kv1.3/deficiência , Canal de Potássio Kv1.3/genética , Canal de Sódio Disparado por Voltagem NAV1.1/genética , Canal de Sódio Disparado por Voltagem NAV1.1/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.2/genética , Canal de Sódio Disparado por Voltagem NAV1.2/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.3/genética , Canal de Sódio Disparado por Voltagem NAV1.3/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.4/genética , Canal de Sódio Disparado por Voltagem NAV1.4/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.5/genética , Canal de Sódio Disparado por Voltagem NAV1.5/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.6/genética , Canal de Sódio Disparado por Voltagem NAV1.6/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.7/genética , Técnicas de Patch-Clamp/instrumentação , Cultura Primária de Células , Ratos , Canais de Sódio/genética , Canais de Sódio/metabolismo , Linfócitos T/citologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismo , Transgenes
18.
ACS Chem Neurosci ; 8(8): 1747-1755, 2017 08 16.
Artigo em Inglês | MEDLINE | ID: mdl-28586206

RESUMO

Mutations of hNav1.7 that cause its activities to be enhanced contribute to severe neuropathic pain. Only a small number of hNav1.7 specific inhibitors have been identified, most of which interact with the voltage-sensing domain of the voltage-activated sodium ion channel. In our previous computational study, we demonstrated that a [Lys6]-C84 fullerene binds tightly (affinity of 46 nM) to NavAb, the voltage-gated sodium channel from the bacterium Arcobacter butzleri. Here, we extend this work and, using molecular dynamics simulations, demonstrate that the same [Lys6]-C84 fullerene binds strongly (2.7 nM) to the pore of a modeled human sodium ion channel hNav1.7. In contrast, the fullerene binds only weakly to a mutated model of hNav1.7 (I1399D) (14.5 mM) and a model of the skeletal muscle hNav1.4 (3.7 mM). Comparison of one representative sequence from each of the nine human sodium channel isoforms shows that only hNav1.7 possesses residues that are critical for binding the fullerene derivative and blocking the channel pore.


Assuntos
Fulerenos/farmacologia , Canal de Sódio Disparado por Voltagem NAV1.7/metabolismo , Bloqueadores dos Canais de Sódio/farmacologia , Sequência de Aminoácidos , Fulerenos/química , Humanos , Ligações de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Bicamadas Lipídicas/metabolismo , Simulação de Dinâmica Molecular , Mutação , Canal de Sódio Disparado por Voltagem NAV1.4/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.7/genética , Ligação Proteica , Alinhamento de Sequência , Bloqueadores dos Canais de Sódio/química
19.
Neurology ; 88(16): 1520-1527, 2017 Apr 18.
Artigo em Inglês | MEDLINE | ID: mdl-28330959

RESUMO

OBJECTIVE: To characterize the clinical phenotype in patients with p.A1156T sodium channel mutation. METHODS: Twenty-nine Finnish patients identified with the c.3466G>A p.A1156T mutation in the SCN4A gene were extensively examined. In a subsequent study, 63 patients with similar myalgic phenotype and with negative results in myotonic dystrophy type 2 genetic screening (DM2-neg group) and 93 patients diagnosed with fibromyalgia were screened for the mutation. Functional consequences of the p.A1156T mutation were studied in HEK293 cells with whole-cell patch clamp. RESULTS: The main clinical manifestation in p.A1156T patients was not myotonia or periodic paralysis but exercise- and cold-induced muscle cramps, muscle stiffness, and myalgia. EMG myotonic discharges were detected in most but not all. Electrophysiologic compound muscle action potentials exercise test showed variable results. The p.A1156T mutation was identified in one patient in the DM2-neg group but not in the fibromyalgia group, making a total of 30 patients so far identified. Functional studies of the p.A1156T mutation showed mild attenuation of channel fast inactivation. CONCLUSIONS: The unspecific symptoms of myalgia stiffness and exercise intolerance without clinical myotonia or periodic paralysis in p.A1156T patients make the diagnosis challenging. The symptoms of milder SCN4A mutations may be confused with other similar myalgic syndromes, including fibromyalgia and myotonic dystrophy type 2.


Assuntos
Mutação , Mialgia/genética , Mialgia/fisiopatologia , Canal de Sódio Disparado por Voltagem NAV1.4/genética , Canal de Sódio Disparado por Voltagem NAV1.4/metabolismo , Adolescente , Adulto , Criança , Pré-Escolar , Estudos de Coortes , Análise Mutacional de DNA , Grupo com Ancestrais do Continente Europeu/genética , Família , Feminino , Fibromialgia/genética , Fibromialgia/fisiopatologia , Finlândia , Células HEK293 , Humanos , Masculino , Pessoa de Meia-Idade , Distrofia Miotônica/genética , Distrofia Miotônica/fisiopatologia , Técnicas de Patch-Clamp , Fenótipo , Adulto Jovem
20.
J Gen Physiol ; 149(4): 465-481, 2017 Apr 03.
Artigo em Inglês | MEDLINE | ID: mdl-28258204

RESUMO

Local anesthetics, antiarrhythmics, and anticonvulsants include both charged and electroneutral compounds that block voltage-gated sodium channels. Prior studies have revealed a common drug-binding region within the pore, but details about the binding sites and mechanism of block remain unclear. Here, we use the x-ray structure of a prokaryotic sodium channel, NavMs, to model a eukaryotic channel and dock representative ligands. These include lidocaine, QX-314, cocaine, quinidine, lamotrigine, carbamazepine (CMZ), phenytoin, lacosamide, sipatrigine, and bisphenol A. Preliminary calculations demonstrated that a sodium ion near the selectivity filter attracts electroneutral CMZ but repels cationic lidocaine. Therefore, we further docked electroneutral and cationic drugs with and without a sodium ion, respectively. In our models, all the drugs interact with a phenylalanine in helix IVS6. Electroneutral drugs trap a sodium ion in the proximity of the selectivity filter, and this same site attracts the charged group of cationic ligands. At this position, even small drugs can block the permeation pathway by an electrostatic or steric mechanism. Our study proposes a common pharmacophore for these diverse drugs. It includes a cationic moiety and an aromatic moiety, which are usually linked by four bonds.


Assuntos
Anestésicos Locais/farmacologia , Antiarrítmicos/farmacologia , Anticonvulsivantes/farmacologia , Simulação de Acoplamento Molecular , Canal de Sódio Disparado por Voltagem NAV1.4/química , Bloqueadores dos Canais de Sódio/farmacologia , Acetamidas/farmacologia , Animais , Compostos Benzidrílicos/farmacologia , Sítios de Ligação , Carbamazepina/farmacologia , Cocaína/farmacologia , Humanos , Lacosamida , Lamotrigina , Lidocaína/análogos & derivados , Lidocaína/farmacologia , Canal de Sódio Disparado por Voltagem NAV1.4/metabolismo , Fenóis/farmacologia , Fenitoína/farmacologia , Piperazinas/farmacologia , Ligação Proteica , Pirimidinas/farmacologia , Quinidina/farmacologia , Triazinas/farmacologia
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