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1.
PLoS One ; 15(6): e0234114, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32516325

RESUMO

We previously reported that a single 5 ns high intensity electric pulse (NEP) caused an E-field-dependent decrease in peak inward voltage-gated Na+ current (INa) in isolated bovine adrenal chromaffin cells. This study explored the effects of a pair of 5 ns pulses on INa recorded in the same cell type, and how varying the E-field amplitude and interval between the pulses altered its response. Regardless of the E-field strength (5 to 10 MV/m), twin NEPs having interpulse intervals ≥ than 5 s caused the inhibition of TTX-sensitive INa to approximately double relative to that produced by a single pulse. However, reducing the interval from 1 s to 10 ms between twin NEPs at E-fields of 5 and 8 MV/m but not 10 MV/m decreased the magnitude of the additive inhibitory effect by the second pulse in a pair on INa. The enhanced inhibitory effects of twin vs single NEPs on INa were not due to a shift in the voltage-dependence of steady-state activation and inactivation but were associated with a reduction in maximal Na+ conductance. Paradoxically, reducing the interval between twin NEPs at 5 or 8 MV/m but not 10 MV/m led to a progressive interval-dependent recovery of INa, which after 9 min exceeded the level of INa reached following the application of a single NEP. Disrupting lipid rafts by depleting membrane cholesterol with methyl-ß-cyclodextrin enhanced the inhibitory effects of twin NEPs on INa and ablated the progressive recovery of this current at short twin pulse intervals, suggesting a complete dissociation of the inhibitory effects of twin NEPs on this current from their ability to stimulate its recovery. Our results suggest that in contrast to a single NEP, twin NEPs may influence membrane lipid rafts in a manner that enhances the trafficking of newly synthesized and/or recycling of endocytosed voltage-gated Na+ channels, thereby pointing to novel means to regulate ion channels in excitable cells.


Assuntos
Células Cromafins/fisiologia , Eletricidade , Glândulas Suprarrenais/citologia , Animais , Bovinos , Células Cultivadas , Células Cromafins/citologia , Potenciais da Membrana/efeitos dos fármacos , Técnicas de Patch-Clamp , Canais de Sódio Disparados por Voltagem/metabolismo , beta-Ciclodextrinas/farmacologia
2.
Proc Natl Acad Sci U S A ; 117(25): 14187-14193, 2020 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-32513729

RESUMO

NaChBac, the first bacterial voltage-gated Na+ (Nav) channel to be characterized, has been the prokaryotic prototype for studying the structure-function relationship of Nav channels. Discovered nearly two decades ago, the structure of NaChBac has not been determined. Here we present the single particle electron cryomicroscopy (cryo-EM) analysis of NaChBac in both detergent micelles and nanodiscs. Under both conditions, the conformation of NaChBac is nearly identical to that of the potentially inactivated NavAb. Determining the structure of NaChBac in nanodiscs enabled us to examine gating modifier toxins (GMTs) of Nav channels in lipid bilayers. To study GMTs in mammalian Nav channels, we generated a chimera in which the extracellular fragment of the S3 and S4 segments in the second voltage-sensing domain from Nav1.7 replaced the corresponding sequence in NaChBac. Cryo-EM structures of the nanodisc-embedded chimera alone and in complex with HuwenToxin IV (HWTX-IV) were determined to 3.5 and 3.2 Å resolutions, respectively. Compared to the structure of HWTX-IV-bound human Nav1.7, which was obtained at an overall resolution of 3.2 Å, the local resolution of the toxin has been improved from ∼6 to ∼4 Å. This resolution enabled visualization of toxin docking. NaChBac can thus serve as a convenient surrogate for structural studies of the interactions between GMTs and Nav channels in a membrane environment.


Assuntos
Proteínas de Bactérias/química , Microscopia Crioeletrônica/métodos , Nanoestruturas/química , Canais de Sódio Disparados por Voltagem/química , Canais de Sódio Disparados por Voltagem/metabolismo , Animais , Proteínas de Bactérias/genética , Humanos , Bicamadas Lipídicas/química , Modelos Moleculares , Conformação Proteica , Canais de Sódio , Venenos de Aranha/química , Canais de Sódio Disparados por Voltagem/genética
3.
PLoS Comput Biol ; 16(6): e1007766, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32542050

RESUMO

The delivery of kilohertz frequency alternating current (KHFAC) generates rapid, controlled, and reversible conduction block in motor, sensory, and autonomic nerves, but causes transient activation of action potentials at the onset of the blocking current. We implemented a novel engineering optimization approach to design blocking waveforms that eliminated the onset response by moving voltage-gated Na+ channels (VGSCs) to closed-state inactivation (CSI) without first opening. We used computational models and particle swarm optimization (PSO) to design a charge-balanced 10 kHz biphasic current waveform that produced conduction block without onset firing in peripheral axons at specific locations and with specific diameters. The results indicate that it is possible to achieve onset-free KHFAC nerve block by causing CSI of VGSCs. Our novel approach for designing blocking waveforms and the resulting waveform may have utility in clinical applications of conduction block of peripheral nerve hyperactivity, for example in pain and spasticity.


Assuntos
Bloqueio Nervoso/métodos , Condução Nervosa , Canais de Sódio Disparados por Voltagem/fisiologia , Potenciais de Ação , Axônios/fisiologia , Simulação por Computador , Estimulação Elétrica , Eletrodos , Humanos , Íons , Cadeias de Markov , Modelos Neurológicos , Nervos Periféricos/fisiologia , Nós Neurofibrosos/patologia
4.
PLoS One ; 15(4): e0232451, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32353050

RESUMO

Numerous neuronal properties including the synaptic vesicle release process, neurotransmitter receptor complement, and postsynaptic ion channels are involved in transforming synaptic inputs into postsynaptic spiking. Temperature is a significant influencer of neuronal function and synaptic integration. Changing temperature can affect neuronal physiology in a diversity of ways depending on how it affects different members of the cell's ion channel complement. Temperature's effects on neuronal function are critical for pathological states such as fever, which can trigger seizure activity, but are also important in interpreting and comparing results of experiments conducted at room vs physiological temperature. The goal of this study was to examine the influence of temperature on synaptic properties and ion channel function in thalamocortical (TC) relay neurons in acute brain slices of the dorsal lateral geniculate nucleus, a key synaptic target of retinal ganglion cells in the thalamus. Warming the superfusate in patch clamp experiments with acutely-prepared brain slices led to an overall inhibition of synaptically-driven spiking behavior in TC neurons in response to a retinal ganglion cell spike train. Further study revealed that this was associated with an increase in presynaptic synaptic vesicle release probability and synaptic depression and altered passive and active membrane properties. Additionally, warming the superfusate triggered activation of an inwardly rectifying potassium current and altered the voltage-dependence of voltage-gated Na+ currents and T-type calcium currents. This study highlights the importance of careful temperature control in ex vivo physiological experiments and illustrates how numerous properties such as synaptic inputs, active conductances, and passive membrane properties converge to determine spike output.


Assuntos
Corpos Geniculados/fisiologia , Temperatura Alta/efeitos adversos , Optogenética/métodos , Terminações Pré-Sinápticas/fisiologia , Células Ganglionares da Retina/fisiologia , Potenciais de Ação/fisiologia , Animais , Canais de Cálcio Tipo T/metabolismo , Feminino , Corpos Geniculados/citologia , Masculino , Camundongos , Técnicas de Patch-Clamp , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Transmissão Sináptica/fisiologia , Canais de Sódio Disparados por Voltagem/metabolismo
5.
Parasitol Res ; 119(7): 2075-2083, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32458116

RESUMO

Aedes aegypti represents one of the main vectors of at least five relevant arthropod-borne viral infections in humans (i.e., Rift Valley fever, Dengue fever, Zika, chikungunya, and yellow fever) worldwide. Ae. aegypti control strategies are mostly based on using chemical insecticides (i.e., organophosphates, pyrethroids, carbamates, and organochlorines) and reducing larval sources. Furthermore, monitoring the growth activity and mapping the geographical distribution of insecticide resistance are mandatory, as recommended by the WHO. Accordingly, we conducted a study on the possible mechanism by which Ae. aegypti develops resistance to several frequently used chemical insecticides (i.e., λ-cyhalothrin, bendiocarb, cyfluthrin, deltamethrin, malathion, and permethrin) in the city of Makassar, Sulawesi, Indonesia. The results showed the progression of resistance toward the examined insecticides in Ae. aegypti populations in Makassar. The mortality rate of Ae. aegypti was less than 90%, with the highest resistance recorded against 0.75% permethrin. The molecular evaluation of the voltage-gated sodium channel gene (VGSC) showed a significant correlation of the V1016G gene mutation in the tested 0.75% permethrin-resistant Ae. aegypti phenotypes. Nevertheless, the F1534C point mutation in the VGSC gene of Ae. aegypti did not show a significant correlation with the phenotype exhibiting insecticide resistance to 0.75% permethrin. These results indicate that Ae. aegypti mosquitoes in Makassar City have developed resistance against the frequently used insecticide permethrin, which might spread to less-populated regions of Sulawesi. Therefore, we call for further entomological monitoring of insecticide resistance not only on Sulawesi but also on other closely located islands of the Indonesian archipelago to delay the spread of Ae. aegypti insecticide resistance.


Assuntos
Aedes/genética , Resistência a Inseticidas/genética , Inseticidas , Mosquitos Vetores/genética , Mutação , Canais de Sódio Disparados por Voltagem/genética , Animais , Indonésia
6.
Neuron ; 106(5): 806-815.e6, 2020 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-32209430

RESUMO

During development of the peripheral nervous system (PNS), Schwann-cell-secreted gliomedin induces the clustering of Na+ channels at the edges of each myelin segment to form nodes of Ranvier. Here we show that bone morphogenetic protein-1 (BMP1)/Tolloid (TLD)-like proteinases confine Na+ channel clustering to these sites by negatively regulating the activity of gliomedin. Eliminating the Bmp1/TLD cleavage site in gliomedin or treating myelinating cultures with a Bmp1/TLD inhibitor results in the formation of numerous ectopic Na+ channel clusters along axons that are devoid of myelin segments. Furthermore, genetic deletion of Bmp1 and Tll1 genes in mice using a Schwann-cell-specific Cre causes ectopic clustering of nodal proteins, premature formation of heminodes around early ensheathing Schwann cells, and altered nerve conduction during development. Our results demonstrate that by inactivating gliomedin, Bmp1/TLD functions as an additional regulatory mechanism to ensure the correct spatial and temporal assembly of PNS nodes of Ranvier.


Assuntos
Proteína Morfogenética Óssea 1/genética , Moléculas de Adesão Celular Neuronais/metabolismo , Bainha de Mielina/metabolismo , Nós Neurofibrosos/metabolismo , Metaloproteases Semelhantes a Toloide/genética , Canais de Sódio Disparados por Voltagem/metabolismo , Animais , Proteína Morfogenética Óssea 1/metabolismo , Camundongos , Camundongos Knockout , Condução Nervosa , Sistema Nervoso Periférico , Transporte Proteico , Células de Schwann/metabolismo , Metaloproteases Semelhantes a Toloide/metabolismo
7.
Lancet Child Adolesc Health ; 4(7): 536-547, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32142633

RESUMO

Voltage-gated sodium channels are essential for excitability of skeletal muscle fibres and neurons. An increasing number of disabling or fatal paediatric neurological disorders linked to mutations of voltage-gated sodium channel genes are recognised. Muscle phenotypes include episodic paralysis, myotonia, neonatal hypotonia, respiratory compromise, laryngospasm or stridor, congenital myasthenia, and myopathy. Evidence suggests a possible link between sodium channel dysfunction and sudden infant death. Increasingly recognised phenotypes of brain sodium channelopathies include several epilepsy disorders and complex encephalopathies. Together, these early-onset muscle and brain phenotypes have a substantial morbidity and a considerable mortality. Important advances in understanding the pathophysiological mechanisms underlying these channelopathies have helped to identify effective targeted therapies. The availability of effective treatments underlines the importance of increasing clinical awareness and the need to achieve a precise genetic diagnosis. In this Review, we describe the expanded range of phenotypes of muscle and brain sodium channelopathies and the underlying knowledge regarding mechanisms of sodium channel dysfunction. We also outline a diagnostic approach and review the available treatment options.


Assuntos
Encefalopatias/diagnóstico , Encefalopatias/genética , Canalopatias/diagnóstico , Canalopatias/genética , Doenças Musculares/diagnóstico , Doenças Musculares/genética , Canais de Sódio Disparados por Voltagem/genética , Transtorno do Espectro Autista/genética , Encefalopatias/terapia , Canalopatias/terapia , Morte Súbita/etiologia , Testes Genéticos , Humanos , Deficiência Intelectual/genética , Doenças Musculares/terapia , Prognóstico
8.
PLoS Negl Trop Dis ; 14(3): e0007753, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32176693

RESUMO

BACKGROUND: Resistance to pyrethroid insecticides in Aedes aegypti has become widespread after almost two decades of the frequent use of these pesticides to reduce arbovirus transmission. Despite this resistance, pyrethroids continue to be used because they are relatively inexpensive and have low human toxicity. Resistance management has been proposed as a way to retain the use of pyrethroids in natural populations. A key component of resistance management is the assumption that negative fitness is associated with resistance alleles such that resistance alleles will decline in frequency when the insecticides are removed. At least three studies in Ae. aegypti have demonstrated a decrease in pyrethroid resistance once the insecticide has been removed. METHODS/PRINCIPAL FINDINGS: The present study aims to evaluate variation in the loss of pyrethroid resistance among newly established laboratory populations of Ae. aegypti from Mexico. Eight field collections were maintained for up to eight generations, and we recorded changes in the frequencies of the mutations at the V1,016I locus and at the F1,534C locus in the voltage-gated sodium channel gene (VGSC). I1,016 and C1,534 confer resistance. We also examined resistance ratios (RR) with type 1 and 2 pyrethroids. CONCLUSIONS/SIGNIFICANCE: We demonstrate that, in general, the frequency of the Ae. aegypti pyrethroid-resistance alleles I1,016 and C1,534 decline when they are freed from pyrethroid pressure in the laboratory. However, the pattern of decline is strain dependent. In agreement with earlier studies, the RR was positively correlated with the frequencies of the resistance allele I1,016 and showed significant protection against permethrin, and deltamethrin, whereas F1,534C showed protection against permethrin but not against deltamethrin.


Assuntos
Aedes/efeitos dos fármacos , Aedes/crescimento & desenvolvimento , Resistência a Inseticidas , Inseticidas/farmacologia , Mutação , Piretrinas/farmacologia , Canais de Sódio Disparados por Voltagem/genética , Animais , Feminino , Frequência do Gene , Aptidão Genética , México , Seleção Genética , Canais de Sódio Disparados por Voltagem/metabolismo
9.
Proc Natl Acad Sci U S A ; 117(7): 3575-3582, 2020 02 18.
Artigo em Inglês | MEDLINE | ID: mdl-32024761

RESUMO

Excitability-a threshold-governed transient in transmembrane voltage-is a fundamental physiological process that controls the function of the heart, endocrine, muscles, and neuronal tissues. The 1950s Hodgkin and Huxley explicit formulation provides a mathematical framework for understanding excitability, as the consequence of the properties of voltage-gated sodium and potassium channels. The Hodgkin-Huxley model is more sensitive to parametric variations of protein densities and kinetics than biological systems whose excitability is apparently more robust. It is generally assumed that the model's sensitivity reflects missing functional relations between its parameters or other components present in biological systems. Here we experimentally assembled excitable membranes using the dynamic clamp and voltage-gated potassium ionic channels (Kv1.3) expressed in Xenopus oocytes. We take advantage of a theoretically derived phase diagram, where the phenomenon of excitability is reduced to two dimensions defined as combinations of the Hodgkin-Huxley model parameters, to examine functional relations in the parameter space. Moreover, we demonstrate activity dependence and hysteretic dynamics over the phase diagram due to the impacts of complex slow inactivation kinetics. The results suggest that maintenance of excitability amid parametric variation is a low-dimensional, physiologically tenable control process. In the context of model construction, the results point to a potentially significant gap between high-dimensional models that capture the full measure of complexity displayed by ion channel function and the lower dimensionality that captures physiological function.


Assuntos
Modelos Biológicos , Xenopus/metabolismo , Animais , Cinética , Potenciais da Membrana , Oócitos/química , Oócitos/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana/química , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Canais de Sódio Disparados por Voltagem/química , Canais de Sódio Disparados por Voltagem/metabolismo
10.
Eur J Paediatr Neurol ; 24: 129-133, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31928904

RESUMO

The four voltage-gated sodium channels SCN1/2/3/8A have been associated with heterogeneous types of developmental disorders, each presenting with disease specific temporal and cell type specific gene expression. Using single-cell RNA sequencing transcriptomic data from humans and mice, we observe that SCN1A is predominantly expressed in inhibitory neurons. In contrast, SCN2/3/8A are profoundly expressed in excitatory neurons with SCN2/3A starting prenatally, followed by SCN1/8A neonatally. In contrast to previous observations from low resolution RNA screens, we observe that all four genes are expressed in both excitatory and inhibitory neurons, however, exhibit differential expression strength. These findings provide molecular evidence, at single-cell resolution, to support the hypothesis that the excitatory/inhibitory (E/I) neuronal expression ratios of sodium channels are important regulatory mechanisms underlying brain homeostasis and neurological diseases. Modulating the E/I expression balance within cell types of sodium channels could serve as a potential strategy to develop targeted treatment for NaV-associated neuronal developmental disorders.


Assuntos
Encéfalo/metabolismo , Deficiências do Desenvolvimento/metabolismo , Neurônios/metabolismo , Canais de Sódio Disparados por Voltagem/metabolismo , Animais , Deficiências do Desenvolvimento/genética , Humanos , Camundongos , Canais de Sódio Disparados por Voltagem/genética
11.
Proc Natl Acad Sci U S A ; 117(4): 1988-1993, 2020 01 28.
Artigo em Inglês | MEDLINE | ID: mdl-31924746

RESUMO

Voltage-gated ion channels endow membranes with excitability and the means to propagate action potentials that form the basis of all neuronal signaling. We determined the structure of a voltage-gated sodium channel, two-pore channel 3 (TPC3), which generates ultralong action potentials. TPC3 is distinguished by activation only at extreme membrane depolarization (V50 ∼ +75 mV), in contrast to other TPCs and NaV channels that activate between -20 and 0 mV. We present electrophysiological evidence that TPC3 voltage activation depends only on voltage sensing domain 2 (VSD2) and that each of the three gating arginines in VSD2 reduces the activation threshold. The structure presents a chemical basis for sodium selectivity, and a constricted gate suggests a closed pore consistent with extreme voltage dependence. The structure, confirmed by our electrophysiology, illustrates the configuration of a bona fide resting state voltage sensor, observed without the need for any inhibitory ligand, and independent of any chemical or mutagenic alteration.


Assuntos
Ativação do Canal Iônico , Sódio/metabolismo , Canais de Sódio Disparados por Voltagem/química , Proteínas de Peixe-Zebra/química , Potenciais de Ação , Microscopia Crioeletrônica , Células HEK293 , Humanos , Modelos Moleculares , Conformação Proteica
12.
Eur J Paediatr Neurol ; 24: 123-128, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31889633

RESUMO

Voltage-gated sodium channels (VGSCs) play a crucial role in generation of action potentials. Pathogenic variants in the five human brain expressed VGSC genes, SCN1A, SCN2A, SCN3A, SCN8A and SCN1B have been associated with a spectrum of epilepsy phenotypes and neurodevelopmental disorders. In the last decade, next generation sequencing techniques have revolutionized the way we diagnose these channelopathies, which is paving the way towards precision medicine. Knowing the functional effect (Loss-of-function versus Gain-of-function) of a variant is not only important for understanding the underlying pathophysiology, but it is particularly crucial to orient therapeutic decisions. Here we provide a review of the literature dealing with treatment options in epilepsy-related sodium channelopathies, including the current and emerging medications.


Assuntos
Canalopatias/tratamento farmacológico , Canalopatias/genética , Epilepsia/tratamento farmacológico , Epilepsia/genética , Canais de Sódio Disparados por Voltagem/genética , Humanos
13.
Pest Manag Sci ; 76(6): 2095-2104, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31944525

RESUMO

BACKGROUND: Apolygus lucorum (Meyer-Dür) is a serious worldwide agricultural pest, especially for Bt cotton in China. Pyrethroids, neonicotinoids and organophosphates are the most effective insecticides to control piercing and sucking insects, including A. lucorum. The voltage-gated sodium channel (Nav ) is major target site of pyrethroids. Extensive alternative splicing and RNA editing, two major post-transcriptional mechanisms, contribute to generate different functional sodium channel variants. In our research, we characterized the sodium channel variants of A. lucorum. RESULTS: In this study, we isolated numerous sodium channel variants that cover the entire coding region of the VGSC gene from A. lucorum. All clones could be grouped into 47 splice types based on the presence of nine alternative exons (exons j, n, o, a, p, b, s, q and t). Exons j, b and t were located independently, while exons n, o, a and p were located adjacently, as were exons s and q. We also found 35 nucleotide changes in different positions in individual variants, of which 18 nucleotide changes were A-to-I RNA editing, 11 nucleotide changes were likely due to U-to-C or C-to-U editing, and the others were likely natural sequence polymorphisms in the population. Furthermore, we expressed all of the variants in Xenopus oocytes. Eighteen of them were expressed in oocytes and sensitive to tetrodotoxin. CONCLUSION: Our results provide a functional basis for understanding how A. lucorum sodium channel variants work in regulating channel expression, pharmacology and gating properties for agricultural insects. Apolygus lucorum is widely distributed in cotton production. Our results suggest how AlNav (the sodium channel of A.lucorum) variants work in regulating channel expression, pharmacology and sodium channel gating for agricultural insects in the future. © 2020 Society of Chemical Industry.


Assuntos
Heterópteros , Animais , China , Piretrinas , Sódio , Canais de Sódio Disparados por Voltagem
14.
Chemistry ; 26(9): 2025-2033, 2020 Feb 11.
Artigo em Inglês | MEDLINE | ID: mdl-31769085

RESUMO

A novel series of C12-keto-type saxitoxin (STX) derivatives bearing an unusual nonhydrated form of the ketone at C12 has been synthesized, and their NaV -inhibitory activity has been evaluated in a cell-based assay as well as whole-cell patch-clamp recording. Among these compounds, 11-benzylidene STX (3 a) showed potent inhibitory activity against neuroblastoma Neuro 2A in both cell-based and electrophysiological analyses, with EC50 and IC50 values of 8.5 and 30.7 nm, respectively. Interestingly, the compound showed potent inhibitory activity against tetrodotoxin-resistant subtype of NaV 1.5, with an IC50 value of 94.1 nm. Derivatives 3 a-d and 3 f showed low recovery rates from NaV 1.2 subtype (ca 45-79 %) compared to natural dcSTX (2), strongly suggesting an irreversible mode of interaction. We propose an interaction model for the C12-keto derivatives with NaV in which the enone moiety in the STX derivatives 3 works as Michael acceptor for the carboxylate of Asp1717 .


Assuntos
Saxitoxina/química , Bloqueadores dos Canais de Sódio/síntese química , Canais de Sódio Disparados por Voltagem/metabolismo , Potenciais de Ação/efeitos dos fármacos , Sequência de Aminoácidos , Sítios de Ligação , Linhagem Celular Tumoral , Humanos , Concentração Inibidora 50 , Simulação de Acoplamento Molecular , Técnicas de Patch-Clamp , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Teoria Quântica , Saxitoxina/metabolismo , Saxitoxina/farmacologia , Bloqueadores dos Canais de Sódio/metabolismo , Bloqueadores dos Canais de Sódio/farmacologia , Tetrodotoxina/química , Tetrodotoxina/metabolismo , Canais de Sódio Disparados por Voltagem/química , Canais de Sódio Disparados por Voltagem/genética
15.
Insect Mol Biol ; 29(1): 9-18, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31206812

RESUMO

The voltage-sensitive sodium channel (VSSC) is essential for the generation and propagation of action potentials. VSSC kinetics can be modified by producing different splice variants. The functionality of VSSC depends on features such as the voltage sensors, the selectivity filter and the inactivation loop. Mutations in Vssc conferring resistance to pyrethroid insecticides are known as knockdown resistance (kdr). We analysed the conservation of VSSC in both a broad scope and a narrow scope by three approaches: (1) we compared conservation of sequences and of differential exon use across orders of the Insecta; (2) we determined which kdr mutations were possible with a single nucleotide mutation in nine populations of Aedes aegypti; and (3) we examined the individual VSSC variation that exists within a population of Drosophila melanogaster. There is an increasing amount of transcript diversity possible from Diplura towards Diptera. The residues of the voltage sensors, selectivity filter and inactivation loop are highly conserved. The majority of exon sequences were >88.6% similar. Strain-specific differences in codon constraints exist for kdr mutations in nine strains of A. aegypti. Three Vssc mutations were found in one population of D. melanogaster. This study shows that, overall, Vssc is highly conserved across Insecta and within a population of an insect, but that important differences do exist.


Assuntos
Insetos/genética , Resistência a Inseticidas/genética , Canais de Sódio Disparados por Voltagem/genética , Aedes/genética , Sequência de Aminoácidos , Animais , Drosophila melanogaster/genética , Éxons , Proteínas de Insetos/genética , Mutação , Piretrinas/farmacologia , Análise de Sequência de DNA
16.
Chem Biol Interact ; 315: 108890, 2020 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-31678597

RESUMO

The monoterpenoid terpinen-4-ol (4TERP) is known to inhibit cell excitability, has low toxicity and important pharmacological activities, which are likely related to neural excitability, such as anti-inflammatory, antiepileptic and antinociceptive effects. However, the pharmacological characteristics and mechanisms underlying the effects of 4TERP on blockade of neural action potential are not completely elucidated. Since Na+ current (INa) through voltage-dependent Na+ channels (NaV) is a major mechanism for excitability, the present study investigated the pharmacological characteristics and mechanisms of the action of 4TERP on INa through NaV. For this aim, dissociated small neurons of dorsal root ganglia of adult rats were used for whole cell patch-clamp recordings. 4TERP concentration-dependently inhibits INa (IC50 0.8 ±â€¯0.3 mM; pharmacological efficacy 42.89 ±â€¯5.54%). 4TERP interfered with INa through a mechanism with various components, which includes predominantly channel pore block and sensitivity to frequency of use. In presence of 4TERP (3 mM), decreasing stimulation from 5 Hz to very low frequency (75 s of quiescence previously to stimulation) induced INa decrease to 65.17 ±â€¯5.86% of control. 4TERP also altered (left shift) voltage sensitivity of the steady state activation of NaV. Data are discussed aiming to interpret the importance of blockade of INa through NaV as participant of 4TERP-induced inhibition of membrane excitability.


Assuntos
Gânglios Espinais/efeitos dos fármacos , Monoterpenos/farmacologia , Neurônios/efeitos dos fármacos , Terpenos/farmacologia , Bloqueadores do Canal de Sódio Disparado por Voltagem/farmacologia , Canais de Sódio Disparados por Voltagem/metabolismo , Potenciais de Ação/efeitos dos fármacos , Animais , Feminino , Gânglios Espinais/metabolismo , Masculino , Neurônios/metabolismo , Técnicas de Patch-Clamp/métodos , Ratos , Ratos Wistar
17.
Cell ; 180(1): 122-134.e10, 2020 01 09.
Artigo em Inglês | MEDLINE | ID: mdl-31866066

RESUMO

Voltage-gated sodium channel Nav1.5 generates cardiac action potentials and initiates the heartbeat. Here, we report structures of NaV1.5 at 3.2-3.5 Å resolution. NaV1.5 is distinguished from other sodium channels by a unique glycosyl moiety and loss of disulfide-bonding capability at the NaVß subunit-interaction sites. The antiarrhythmic drug flecainide specifically targets the central cavity of the pore. The voltage sensors are partially activated, and the fast-inactivation gate is partially closed. Activation of the voltage sensor of Domain III allows binding of the isoleucine-phenylalanine-methionine (IFM) motif to the inactivation-gate receptor. Asp and Ala, in the selectivity motif DEKA, line the walls of the ion-selectivity filter, whereas Glu and Lys are in positions to accept and release Na+ ions via a charge-delocalization network. Arrhythmia mutation sites undergo large translocations during gating, providing a potential mechanism for pathogenic effects. Our results provide detailed insights into Nav1.5 structure, pharmacology, activation, inactivation, ion selectivity, and arrhythmias.


Assuntos
Canal de Sódio Disparado por Voltagem NAV1.5/genética , Canal de Sódio Disparado por Voltagem NAV1.5/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.5/ultraestrutura , Animais , Linhagem Celular , Células HEK293 , Coração/fisiologia , Humanos , Ativação do Canal Iônico/fisiologia , Potenciais da Membrana/fisiologia , Técnicas de Patch-Clamp/métodos , Ratos , Sódio/metabolismo , Canais de Sódio/química , Relação Estrutura-Atividade , Canais de Sódio Disparados por Voltagem/metabolismo , Canais de Sódio Disparados por Voltagem/ultraestrutura
18.
Curr Med Sci ; 39(6): 863-873, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31845216

RESUMO

Voltage-gated sodium (Nav) channels are critical players in the generation and propagation of action potentials by triggering membrane depolarization. Mutations in Nav channels are associated with a variety of channelopathies, which makes them relevant targets for pharmaceutical intervention. So far, the cryoelectron microscopic structure of the human Nav1.2, Nav1.4, and Nav1.7 has been reported, which sheds light on the molecular basis of functional mechanism of Nav channels and provides a path toward structure-based drug discovery. In this review, we focus on the recent advances in the structure, molecular mechanism and modulation of Nav channels, and state updated sodium channel blockers for the treatment of pathophysiology disorders and briefly discuss where the blockers may be developed in the future.


Assuntos
Canalopatias/genética , Mutação , Bloqueadores dos Canais de Sódio/química , Canais de Sódio Disparados por Voltagem/metabolismo , Potenciais de Ação , Canalopatias/tratamento farmacológico , Microscopia Crioeletrônica , Desenho de Fármacos , Humanos , Modelos Moleculares , Conformação Proteica , Bloqueadores dos Canais de Sódio/farmacologia , Bloqueadores dos Canais de Sódio/uso terapêutico , Relação Estrutura-Atividade , Canais de Sódio Disparados por Voltagem/química , Canais de Sódio Disparados por Voltagem/genética
19.
PLoS Negl Trop Dis ; 13(11): e0007818, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31738756

RESUMO

In insects, the voltage-gated sodium channel (VGSC) is the primary target site of pyrethroid insecticides. Various amino acid substitutions in the VGSC protein, which are selected under insecticide pressure, are known to confer insecticide resistance. In the genome, the VGSC gene consists of more than 30 exons sparsely distributed across a large genomic region, which often exceeds 100 kbp. Due to this complex genomic structure, it is often challenging to genotype full coding nucleotide sequences (CDSs) of VGSC from individual genomic DNA (gDNA). In this study, we designed biotinylated oligonucleotide probes from CDSs of VGSC of Asian tiger mosquito, Aedes albopictus. The probe set effectively concentrated (>80,000-fold) all targeted regions of gene VGSC from pooled barcoded Illumina libraries each constructed from individual A. albopictus gDNAs. The probe set also captured all orthologous VGSC CDSs, except some tiny exons, from the gDNA of other Culicinae mosquitos, A. aegypti and Culex pipiens complex, with comparable efficiency as a result of the high nucleotide-level conservation of VGSC. To improve efficiency of the downstream bioinformatic process, we developed an automated pipeline-MoNaS (Mosquito Na+ channel mutation Search)-which calls amino acid substitutions in the VGSC from NGS reads and compares those to known resistance mutations. The proposed method and our bioinformatic tool should facilitate the discovery of novel amino acid variants conferring insecticide resistance on VGSC and population genetic studies on resistance alleles (with respect to the origin, selection, and migration etc.) in both clinically and agriculturally important insect pests.


Assuntos
Aedes/genética , Genômica , Genótipo , Resistência a Inseticidas/genética , Mutação , Canais de Sódio Disparados por Voltagem/genética , Alelos , Animais , Culex/genética , DNA/metabolismo , Éxons , Piretrinas/farmacologia , Sódio/metabolismo
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