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1.
Sci Rep ; 14(1): 6761, 2024 03 21.
Artigo em Inglês | MEDLINE | ID: mdl-38514708

RESUMO

Voltage-gated sodium channels (NaV) are pivotal proteins responsible for initiating and transmitting action potentials. Emerging evidence suggests that proteolytic cleavage of sodium channels by calpains is pivotal in diverse physiological scenarios, including ischemia, brain injury, and neuropathic pain associated with diabetes. Despite this significance, the precise mechanism by which calpains recognize sodium channels, especially given the multiple calpain isoforms expressed in neurons, remains elusive. In this work, we show the interaction of Calpain-10 with NaV's C-terminus through a yeast 2-hybrid assay screening of a mouse brain cDNA library and in vitro by GST-pulldown. Later, we also obtained a structural and dynamic hypothesis of this interaction by modeling, docking, and molecular dynamics simulation. These results indicate that Calpain-10 interacts differentially with the C-terminus of NaV1.2 and NaV1.6. Calpain-10 interacts with NaV1.2 through domains III and T in a stable manner. In contrast, its interaction with NaV1.6 involves domains II and III, which could promote proteolysis through the Cys-catalytic site and C2 motifs.


Assuntos
Calpaína , Canais de Sódio Disparados por Voltagem , Animais , Camundongos , Potenciais de Ação , Calpaína/metabolismo , Neurônios/metabolismo , Isoformas de Proteínas/metabolismo , Canais de Sódio Disparados por Voltagem/metabolismo
2.
PLoS One ; 19(3): e0298820, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38452156

RESUMO

BACKGROUND: 14-3-3 proteins are ubiquitous proteins that play a role in cardiac physiology (e.g., metabolism, development, and cell cycle). Furthermore, 14-3-3 proteins were proposed to regulate the electrical function of the heart by interacting with several cardiac ion channels, including the voltage-gated sodium channel Nav1.5. Given the many cardiac arrhythmias associated with Nav1.5 dysfunction, understanding its regulation by the protein partners is crucial. AIMS: In this study, we aimed to investigate the role of 14-3-3 proteins in the regulation of the human cardiac sodium channel Nav1.5. METHODS AND RESULTS: Amongst the seven 14-3-3 isoforms, only 14-3-3η (encoded by YWHAH gene) weakly co-immunoprecipitated with Nav1.5 when heterologously co-expressed in tsA201 cells. Total and cell surface expression of Nav1.5 was however not modified by 14-3-3η overexpression or inhibition with difopein, and 14-3-3η did not affect physical interaction between Nav1.5 α-α subunits. The current-voltage relationship and the amplitude of Nav1.5-mediated sodium peak current density were also not changed. CONCLUSIONS: Our findings illustrate that the direct implication of 14-3-3 proteins in regulating Nav1.5 is not evident in a transformed human kidney cell line tsA201.


Assuntos
Proteínas 14-3-3 , Canais de Sódio Disparados por Voltagem , Humanos , Proteínas 14-3-3/genética , Proteínas 14-3-3/metabolismo , Canais de Sódio Disparados por Voltagem/metabolismo , Miócitos Cardíacos/metabolismo , Linhagem Celular , Arritmias Cardíacas , Canal de Sódio Disparado por Voltagem NAV1.5/genética , Canal de Sódio Disparado por Voltagem NAV1.5/metabolismo
3.
Nat Commun ; 15(1): 2306, 2024 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-38485923

RESUMO

The poison dart toxin batrachotoxin is exceptional for its high potency and toxicity, and for its multifaceted modification of the function of voltage-gated sodium channels. By using cryogenic electron microscopy, we identify two homologous, but nonidentical receptor sites that simultaneously bind two molecules of toxin, one at the interface between Domains I and IV, and the other at the interface between Domains III and IV of the cardiac sodium channel. Together, these two bound toxin molecules stabilize α/π helical conformation in the S6 segments that gate the pore, and one of the bound BTX-B molecules interacts with the crucial Lys1421 residue that is essential for sodium conductance and selectivity via an apparent water-bridged hydrogen bond. Overall, our structure provides insight into batrachotoxin's potency, efficacy, and multifaceted functional effects on voltage-gated sodium channels via a dual receptor site mechanism.


Assuntos
Venenos , Canais de Sódio Disparados por Voltagem , Batraquiotoxinas/metabolismo , Sítios de Ligação , Conformação Molecular , Canais de Sódio Disparados por Voltagem/metabolismo
4.
PLoS Comput Biol ; 20(3): e1011846, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38489374

RESUMO

In a variety of neurons, action potentials (APs) initiate at the proximal axon, within a region called the axon initial segment (AIS), which has a high density of voltage-gated sodium channels (NaVs) on its membrane. In pyramidal neurons, the proximal AIS has been reported to exhibit a higher proportion of NaVs with gating properties that are "right-shifted" to more depolarized voltages, compared to the distal AIS. Further, recent experiments have revealed that as neurons develop, the spatial distribution of NaV subtypes along the AIS can change substantially, suggesting that neurons tune their excitability by modifying said distribution. When neurons are stimulated axonally, computational modelling has shown that this spatial separation of gating properties in the AIS enhances the backpropagation of APs into the dendrites. In contrast, in the more natural scenario of somatic stimulation, our simulations show that the same distribution can impede backpropagation, suggesting that the choice of orthodromic versus antidromic stimulation can bias or even invert experimental findings regarding the role of NaV subtypes in the AIS. We implemented a range of hypothetical NaV distributions in the AIS of three multicompartmental pyramidal cell models and investigated the precise kinetic mechanisms underlying such effects, as the spatial distribution of NaV subtypes is varied. With axonal stimulation, proximal NaV availability dominates, such that concentrating right-shifted NaVs in the proximal AIS promotes backpropagation. However, with somatic stimulation, the models are insensitive to availability kinetics. Instead, the higher activation threshold of right-shifted NaVs in the AIS impedes backpropagation. Therefore, recently observed developmental changes to the spatial separation and relative proportions of NaV1.2 and NaV1.6 in the AIS differentially impact activation and availability. The observed effects on backpropagation, and potentially learning via its putative role in synaptic plasticity (e.g. through spike-timing-dependent plasticity), are opposite for orthodromic versus antidromic stimulation, which should inform hypotheses about the impact of the developmentally regulated subcellular localization of these NaV subtypes.


Assuntos
Segmento Inicial do Axônio , Canais de Sódio Disparados por Voltagem , Segmento Inicial do Axônio/fisiologia , Canal de Sódio Disparado por Voltagem NAV1.6/ultraestrutura , Axônios/fisiologia , Neurônios/fisiologia , Potenciais de Ação/fisiologia
5.
Elife ; 122024 Mar 11.
Artigo em Inglês | MEDLINE | ID: mdl-38465747

RESUMO

Voltage-gated sodium channels (Naáµ¥) are membrane proteins which open to facilitate the inward flux of sodium ions into excitable cells. In response to stimuli, Naáµ¥ channels transition from the resting, closed state to an open, conductive state, before rapidly inactivating. Dysregulation of this functional cycle due to mutations causes diseases including epilepsy, pain conditions, and cardiac disorders, making Naáµ¥ channels a significant pharmacological target. Phosphoinositides are important lipid cofactors for ion channel function. The phosphoinositide PI(4,5)P2 decreases Naáµ¥1.4 activity by increasing the difficulty of channel opening, accelerating fast inactivation and slowing recovery from fast inactivation. Using multiscale molecular dynamics simulations, we show that PI(4,5)P2 binds stably to inactivated Naáµ¥ at a conserved site within the DIV S4-S5 linker, which couples the voltage-sensing domain (VSD) to the pore. As the Naáµ¥ C-terminal domain is proposed to also bind here during recovery from inactivation, we hypothesize that PI(4,5)P2 prolongs inactivation by competitively binding to this site. In atomistic simulations, PI(4,5)P2 reduces the mobility of both the DIV S4-S5 linker and the DIII-IV linker, responsible for fast inactivation, slowing the conformational changes required for the channel to recover to the resting state. We further show that in a resting state Naáµ¥ model, phosphoinositides bind to VSD gating charges, which may anchor them and impede VSD activation. Our results provide a mechanism by which phosphoinositides alter the voltage dependence of activation and the rate of recovery from inactivation, an important step for the development of novel therapies to treat Naáµ¥-related diseases.


Assuntos
Ativação do Canal Iônico , Canais de Sódio Disparados por Voltagem , Ativação do Canal Iônico/fisiologia , Domínios Proteicos , Canais Iônicos , Sítios de Ligação
6.
Parasit Vectors ; 17(1): 91, 2024 Feb 27.
Artigo em Inglês | MEDLINE | ID: mdl-38414050

RESUMO

BACKGROUND: Over the past two decades, dengue fever (DF) has emerged as a significant arboviral disease in Yunnan province, China, particularly in the China-Myanmar border area. Aedes aegypti, an invasive mosquito species, plays a crucial role in transmitting the dengue virus to the local population. Insecticide-based vector control has been the primary tool employed to combat DF, but the current susceptibility status of Ae. aegypti to commonly used insecticides is unknown. Assessment of Ae. aegypti resistance to pyrethroid insecticides and an understanding of the underlying mechanisms of this resistance in the China-Myanmar border region is of significant strategic importance for effectively controlling the DF epidemic in the area. METHODS: Aedes aegypti larvae collected from Ruili and Gengma counties in Yunnan Province were reared to adults in the laboratory and tested for susceptibility to three pyrethroid insecticides (3.20% permethrin, 0.08% lambda-cyhalothrin and 0.20% deltamethrin) by the standard WHO susceptibility bioassay. Genotyping of mutations in the knockdown gene (kdr), namely S989P, V1016G and F1534C, that are responsible for resistance to pyrethroid insecticides was performed using allele-specific PCR methods. A possible association between the observed resistant phenotype and mutations in the voltage-gated sodium channel gene (VGSC) was also studied. RESULTS: Aedes aegypti mosquitoes collected from the two counties and reared in the laboratory were resistant to all of the pyrethroids tested, with the exception of Ae. aegypti from Gengma County, which showed sensitivity to 0.20% deltamethrin. The mortality rate of Ae. aegypti from Ruili county exposed to 3.20% permethrin did not differ significantly from that of Ae. aegypti from Gengma County (χ2 = 0.311, P = 0.577). By contrast, the mortality rate of Ae. aegypti from Ruili County exposed to 0.08% lambda-cyhalothrin and 0.20% deltamethrin, respectively, was significantly different from that of Ae. aegypti from Gengma. There was no significant difference in the observed KDT50 of Ae. aegypti from the two counties to various insecticides. Four mutation types and 12 genotypes were detected at three kdr mutation sites. Based on results from all tested Ae. aegypti, the V1016G mutation was the most prevalent kdr mutation (100% prevalence), followed by the S989P mutation (81.6%) and the F1534C mutation (78.9%). The constituent ratio of VGSC gene mutation types was significantly different in Ae. aegypti mosquitoes from Ruili and those Gengma. The triple mutant S989P + V1016G + F1534C was observed in 274 Ae. aegypti mosquitoes (60.8%), with the most common genotype being SP + GG + FC (31.4%). The prevalence of the F1534C mutation was significantly higher in resistant Ae. aegypti from Ruili (odds ratio [OR] 7.43; 95% confidence interval [CI] 1.71-32.29; P = 0.01) and Gengma (OR 9.29; 95% CI 3.38-25.50; P = 0.00) counties than in susceptible Ae. aegypti when exposed to 3.20% permethrin and 0.08% lambda-cyhalothrin, respectively. No significant association was observed in the triple mutation genotypes with the Ae. aegypti population exposed to 3.20% permethrin and 0.20% deltamethrin resistance (P > 0.05), except for Ae. aegypti from Gengma County when exposed to 0.08% lambda-cyhalothrin (OR 2.86; 95% CI 1.20-6.81; P = 0.02). CONCLUSIONS: Aedes aegypti from Ruili and Gengma counties have developed resistance to various pyrethroid insecticides. The occurrence of multiple mutant sites in VGSC strongly correlated with the high levels of resistance to pyrethroids in the Ae. aegypti populations, highlighting the need for alternative strategies to manage the spread of resistance. A region-specific control strategy for dengue vectors needs to be implemented in the future based on the status of insecticide resistance and kdr mutations.


Assuntos
Aedes , Dengue , Inseticidas , Nitrilas , Piretrinas , Canais de Sódio Disparados por Voltagem , Animais , Inseticidas/farmacologia , Aedes/genética , Permetrina , Mianmar , China/epidemiologia , Piretrinas/farmacologia , Mutação , Resistência a Inseticidas/genética , Canais de Sódio Disparados por Voltagem/genética , Dengue/epidemiologia , Dengue/prevenção & controle , Surtos de Doenças , Mosquitos Vetores/genética
7.
Int J Mol Sci ; 25(3)2024 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-38338757

RESUMO

Tetrodotoxin (TTX) is a neurotoxic molecule used by many animals for defense and/or predation, as well as an important biomedical tool. Its ubiquity as a defensive agent has led to repeated independent evolution of tetrodotoxin resistance in animals. TTX binds to voltage-gated sodium channels (VGSC) consisting of α and ß subunits. Virtually all studies investigating the mechanisms behind TTX resistance have focused on the α subunit of voltage-gated sodium channels, where tetrodotoxin binds. However, the possibility of ß subunits also contributing to tetrodotoxin resistance was never explored, though these subunits act in concert. In this study, we present preliminary evidence suggesting a potential role of ß subunits in the evolution of TTX resistance. We gathered mRNA sequences for all ß subunit types found in vertebrates across 12 species (three TTX-resistant and nine TTX-sensitive) and tested for signatures of positive selection with a maximum likelihood approach. Our results revealed several sites experiencing positive selection in TTX-resistant taxa, though none were exclusive to those species in subunit ß1, which forms a complex with the main physiological target of TTX (VGSC Nav1.4). While experimental data validating these findings would be necessary, this work suggests that deeper investigation into ß subunits as potential players in tetrodotoxin resistance may be worthwhile.


Assuntos
Canais de Sódio Disparados por Voltagem , Animais , Tetrodotoxina/farmacologia , Funções Verossimilhança , Canais de Sódio Disparados por Voltagem/genética , Bloqueadores dos Canais de Sódio/farmacologia
8.
Proc Natl Acad Sci U S A ; 121(9): e2322899121, 2024 Feb 27.
Artigo em Inglês | MEDLINE | ID: mdl-38381792

RESUMO

Voltage-gated sodium channels (Nav) undergo conformational shifts in response to membrane potential changes, a mechanism known as the electromechanical coupling. To delineate the structure-function relationship of human Nav channels, we have performed systematic structural analysis using human Nav1.7 as a prototype. Guided by the structural differences between wild-type (WT) Nav1.7 and an eleven mutation-containing variant, designated Nav1.7-M11, we generated three additional intermediate mutants and solved their structures at overall resolutions of 2.9-3.4 Å. The mutant with nine-point mutations in the pore domain (PD), named Nav1.7-M9, has a reduced cavity volume and a sealed gate, with all voltage-sensing domains (VSDs) remaining up. Structural comparison of WT and Nav1.7-M9 pinpoints two residues that may be critical to the tightening of the PD. However, the variant containing these two mutations, Nav1.7-M2, or even in combination with two additional mutations in the VSDs, named Nav1.7-M4, failed to tighten the PD. Our structural analysis reveals a tendency of PD contraction correlated with the right shift of the static inactivation I-V curves. We predict that the channel in the resting state should have a "tight" PD with down VSDs.


Assuntos
Canais de Sódio Disparados por Voltagem , Humanos , Canais de Sódio Disparados por Voltagem/genética , Potenciais da Membrana , Mutação , Relação Estrutura-Atividade
9.
ACS Chem Neurosci ; 15(6): 1169-1184, 2024 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-38359277

RESUMO

Voltage-gated sodium channel (NaV) inhibitors are used to treat neurological disorders of hyperexcitability such as epilepsy. These drugs act by attenuating neuronal action potential firing to reduce excitability in the brain. However, all currently available NaV-targeting antiseizure medications nonselectively inhibit the brain channels NaV1.1, NaV1.2, and NaV1.6, which potentially limits the efficacy and therapeutic safety margins of these drugs. Here, we report on XPC-7724 and XPC-5462, which represent a new class of small molecule NaV-targeting compounds. These compounds specifically target inhibition of the NaV1.6 and NaV1.2 channels, which are abundantly expressed in excitatory pyramidal neurons. They have a > 100-fold molecular selectivity against NaV1.1 channels, which are predominantly expressed in inhibitory neurons. Sparing NaV1.1 preserves the inhibitory activity in the brain. These compounds bind to and stabilize the inactivated state of the channels thereby reducing the activity of excitatory neurons. They have higher potency, with longer residency times and slower off-rates, than the clinically used antiseizure medications carbamazepine and phenytoin. The neuronal selectivity of these compounds is demonstrated in brain slices by inhibition of firing in cortical excitatory pyramidal neurons, without impacting fast spiking inhibitory interneurons. XPC-5462 also suppresses epileptiform activity in an ex vivo brain slice seizure model, whereas XPC-7224 does not, suggesting a possible requirement of Nav1.2 inhibition in 0-Mg2+- or 4-AP-induced brain slice seizure models. The profiles of these compounds will facilitate pharmacological dissection of the physiological roles of NaV1.2 and NaV1.6 in neurons and help define the role of specific channels in disease states. This unique selectivity profile provides a new approach to potentially treat disorders of neuronal hyperexcitability by selectively downregulating excitatory circuits.


Assuntos
Epilepsia , Canais de Sódio Disparados por Voltagem , Humanos , Neurônios/metabolismo , Canais de Sódio Disparados por Voltagem/metabolismo , Epilepsia/metabolismo , Encéfalo/metabolismo , Convulsões/tratamento farmacológico , Convulsões/metabolismo , Potenciais de Ação/fisiologia
10.
Mol Pharmacol ; 105(3): 233-249, 2024 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-38195157

RESUMO

Discovery and development of new molecules directed against validated pain targets is required to advance the treatment of pain disorders. Voltage-gated sodium channels (NaVs) are responsible for action potential initiation and transmission of pain signals. NaV1.8 is specifically expressed in peripheral nociceptors and has been genetically and pharmacologically validated as a human pain target. Selective inhibition of NaV1.8 can ameliorate pain while minimizing effects on other NaV isoforms essential for cardiac, respiratory, and central nervous system physiology. Here we present the pharmacology, interaction site, and mechanism of action of LTGO-33, a novel NaV1.8 small molecule inhibitor. LTGO-33 inhibited NaV1.8 in the nM potency range and exhibited over 600-fold selectivity against human NaV1.1-NaV1.7 and NaV1.9. Unlike prior reported NaV1.8 inhibitors that preferentially interacted with an inactivated state via the pore region, LTGO-33 was state-independent with similar potencies against closed and inactivated channels. LTGO-33 displayed species specificity for primate NaV1.8 over dog and rodent NaV1.8 and inhibited action potential firing in human dorsal root ganglia neurons. Using chimeras combined with mutagenesis, the extracellular cleft of the second voltage-sensing domain was identified as the key site required for channel inhibition. Biophysical mechanism of action studies demonstrated that LTGO-33 inhibition was relieved by membrane depolarization, suggesting the molecule stabilized the deactivated state to prevent channel opening. LTGO-33 equally inhibited wild-type and multiple NaV1.8 variants associated with human pain disorders. These collective results illustrate LTGO-33 inhibition via both a novel interaction site and mechanism of action previously undescribed in NaV1.8 small molecule pharmacologic space. SIGNIFICANCE STATEMENT: NaV1.8 sodium channels primarily expressed in peripheral pain-sensing neurons represent a validated target for the development of novel analgesics. Here we present the selective small molecule NaV1.8 inhibitor LTGO-33 that interdicts a distinct site in a voltage-sensor domain to inhibit channel opening. These results inform the development of new analgesics for pain disorders.


Assuntos
Canais de Sódio Disparados por Voltagem , Humanos , Animais , Cães , Dor/tratamento farmacológico , Analgésicos/farmacologia , Neurônios , Potenciais de Ação , Gânglios Espinais , Canal de Sódio Disparado por Voltagem NAV1.7 , Bloqueadores dos Canais de Sódio/farmacologia
11.
Toxins (Basel) ; 16(1)2024 01 18.
Artigo em Inglês | MEDLINE | ID: mdl-38251271

RESUMO

µ-Conotoxins are small, potent pore-blocker inhibitors of voltage-gated sodium (NaV) channels, which have been identified as pharmacological probes and putative leads for analgesic development. A limiting factor in their therapeutic development has been their promiscuity for different NaV channel subtypes, which can lead to undesirable side-effects. This review will focus on four areas of µ-conotoxin research: (1) mapping the interactions of µ-conotoxins with different NaV channel subtypes, (2) µ-conotoxin structure-activity relationship studies, (3) observed species selectivity of µ-conotoxins and (4) the effects of µ-conotoxin disulfide connectivity on activity. Our aim is to provide a clear overview of the current status of µ-conotoxin research.


Assuntos
Conotoxinas , Canais de Sódio Disparados por Voltagem , Conotoxinas/farmacologia , Dissulfetos , Relação Estrutura-Atividade
12.
Curr Opin Pharmacol ; 75: 102433, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38277942

RESUMO

Neuronal electrochemical signals involve the flux of sodium ions through voltage-gated sodium channels (NaV) located in the neurolemma. Of the nine sodium channel subtypes, NaV-1.7, 1.8, and 1.9 are predominantly located on nociceptors, making them prime targets to control pain. This review highlights some of the latest discoveries targeting NaV channel activity, including: (1) charged local anaesthetic derivatives; (2) NaV channel toxins and associated small peptide blockers; (3) regulation of NaV channel accessory proteins; and (4) genetic manipulation of NaV channel function. While the translation of preclinical findings to a viable treatment in humans has remained a challenge, a greater understanding of NaV channel physiology could lead to the development of a new stream of therapies aimed at alleviating chronic pain.


Assuntos
Dor , Canais de Sódio Disparados por Voltagem , Humanos , Dor/tratamento farmacológico , Dor/metabolismo , Canais de Sódio Disparados por Voltagem/metabolismo , Analgésicos/farmacologia , Analgésicos/uso terapêutico , Bloqueadores dos Canais de Sódio/farmacologia , Bloqueadores dos Canais de Sódio/uso terapêutico
13.
Bull Entomol Res ; 114(1): 49-56, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38180110

RESUMO

Aphis spiraecola Patch is one of the most economically important tree fruit pests worldwide. The pyrethroid insecticide lambda-cyhalothrin is commonly used to control A. spiraecola. In this 2-year study, we quantified the resistance level of A. spiraecola to lambda-cyhalothrin in different regions of the Shaanxi province, China. The results showed that A. spiraecola had reached extremely high resistance levels with a 174-fold resistance ratio (RR) found in the Xunyi region. In addition, we compared the enzymatic activity and expression level of P450 genes among eight A. spiraecola populations. The P450 activity of A. spiraecola was significantly increased in five regions (Xunyi, Liquan, Fengxiang, Luochuan, and Xinping) compared to susceptible strain (SS). The expression levels of CYP6CY7, CYP6CY14, CYP6CY22, P4504C1-like, P4506a13, CYP4CZ1, CYP380C47, and CYP4CJ2 genes were significantly increased under lambda-cyhalothrin treatment and in the resistant field populations. A L1014F mutation in the sodium channel gene was found and the mutation rate was positively correlated with the LC50 of lambda-cyhalothrin. In conclusion, the levels of lambda-cyhalothrin resistance of A. spiraecola field populations were associated with P450s and L1014F mutations. Our combined findings provide evidence on the resistance mechanism of A. spiraecola to lambda-cyhalothrin and give a theoretical basis for rational and effective control of this pest species.


Assuntos
Afídeos , Inseticidas , Piretrinas , Canais de Sódio Disparados por Voltagem , Animais , Afídeos/genética , Piretrinas/farmacologia , Nitrilas/farmacologia , Mutação , Canais de Sódio Disparados por Voltagem/genética , Expressão Gênica , Inseticidas/farmacologia , Resistência a Inseticidas/genética
14.
Pestic Biochem Physiol ; 198: 105710, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-38225068

RESUMO

Aedes aegypti, the primary vector responsible for transmitting dengue fever in southern Taiwan, has developed a relatively high resistance to synthetic pyrethroids. It has evolved four amino acid substitutions in the voltage-gated sodium channel (VGSC), namely S996P, V1023G, F1565C, and D1794Y. To unveil the distribution and correlation of VGSC mutations and pyrethroid resistance among different field populations, Ae. aegypti collected from various districts in Kaohsiung and Tainan Cities underwent tests for resistance development against different pyrethroids and frequency of S996P, V1023G, F1565C, and D1794Y substitutions. The adult knockdown assay revealed a relatively high knockdown resistance in the Ae. aegypti populations from Kaohsiung and Tainan against permethrin, cypermethrin, and fenvalerate (averaging >50-fold). Conversely, less resistance was observed against α-cypermethrin, deltamethrin, λ-cyhalothrin, cyfluthrin, and etofenprox (averaging <35-fold). Using Polymerase Chain Reaction/restriction fragment length polymorphism analysis, four mutant haplotypes were identified in these field populations. Notably, the SIAVFD and SIBVFD wild haplotypes were absent. Analysis utilizing IBM SPSS Statistics 20.0 and Spearman's rank correlation coefficient indicated that Haplotype C (PIAGFD), especially P allele, frequency displayed a significant positive correlation with five Type II pyrethroid resistance, while 1023G and 1023G/G exhibited a significant association with permethrin and fevalerate resistance. Conversely, Haplotype E (SIBVCD) negatively correlated with pyrethroid resistance, particularly fenvalerate resistance (-0.776). Haplotype C and E were the most prevalent and widely distributed among the investigated field populations. This prevalence of haplotype C is likely tied to the extensive and excessive use of Type II pyrethroids for dengue control over the past three decades. Given the significant positive correlation, the best-fit lines and R2 values were established to facilitate the swift prediction of knockdown resistance levels to various pyrethroids based on VGSC mutation frequency. This predictive approach aims to guide insecticide usage and the management of pyrethroid resistance in the field populations of Ae. aegypti in Taiwan.


Assuntos
Aedes , Inseticidas , Nitrilas , Piretrinas , Canais de Sódio Disparados por Voltagem , Animais , Permetrina , Aedes/genética , Aedes/metabolismo , Taxa de Mutação , Resistência a Inseticidas/genética , Piretrinas/farmacologia , Piretrinas/metabolismo , Inseticidas/farmacologia , Inseticidas/metabolismo , Mutação , Canais de Sódio Disparados por Voltagem/genética , Canais de Sódio Disparados por Voltagem/metabolismo , Mosquitos Vetores/genética
15.
Parasit Vectors ; 17(1): 34, 2024 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-38273349

RESUMO

BACKGROUND: Aedes aegypti is the main vector of arboviral diseases worldwide. The species invaded and became established in southern Iran in 2020. Insecticide-based interventions are primarily used for its control. With insecticide resistance widespread, knowledge of resistance mechanisms is vital for informed deployment of insecticidal interventions, but information from Iranian Ae. aegypti is lacking. METHODS: Fifty-six Ae. aegypti specimens were collected from the port city of Bandar Lengeh in Hormozgan Province in the South of Iran in 2020 and screened for kdr mutations. The most common kdr mutations in Latin America and Asia (V410L, S989P, V1016G/I and F1534C), especially when present in combinations, are highly predictive of DDT and pyrethroid resistance were detected. Phylogenetic analyses based on the diversity of S989P and V1016G/I mutations were undertaken to assess the phylogeography of these kdr mutations. RESULTS: Genotyping all four kdr positions of V410L, S989P, V1016G/I and F1534C revealed that only 16 out of the 56 (28.57%) specimens were homozygous wild type for all kdr mutation sites. Six haplotypes including VSVF (0.537), VSVC (0.107), LSVF (0.016), LSIF (0.071), VPGC (0.257) and LPGC (0.011) were detected in this study. For the first time, 11 specimens harbouring the V410L mutation, and 8 samples with V1016I mutation were found. V410L and V1016I were coincided in 8 specimens. Also, six specimens contained 1016G/I double mutation which was not reported before. CONCLUSIONS: The relatively high frequency of these kdr mutations in Iranian Ae. aegypti indicates a population exhibiting substantial resistance to pyrethroid insecticides, which are used widely in control operations and household formulations. The detection of the 410L/1016I kdr mutant haplotype in Iranian Ae. aegypti suggests possible convergence of invasive populations from West Africa or Latin America. However, as Iran has very limited maritime/air connections with those African countries, a Latin American origin for the invasive Ae. aegypti in Iran is more plausible.


Assuntos
Aedes , Inseticidas , Piretrinas , Canais de Sódio Disparados por Voltagem , Animais , Aedes/genética , Irã (Geográfico) , Genótipo , Filogenia , Inseticidas/farmacologia , Piretrinas/farmacologia , Mutação , Canais de Sódio Disparados por Voltagem/genética , Resistência a Inseticidas/genética , Mosquitos Vetores/genética
16.
Exp Brain Res ; 242(1): 205-224, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-37994916

RESUMO

Traumatic brain injury (TBI) leads to disturbed brain discharge rhythm, elevated excitability, anxiety-like behaviors, and decreased learning and memory capabilities. Cognitive dysfunctions severely affect the quality of life and prognosis of TBI patients, requiring effective rehabilitation treatment. Evidence indicates that moderate exercise after brain injury decreases TBI-induced cognitive decline. However, the underlying mechanism remains unelucidated. Our results demonstrate that TBI causes cognitive impairment behavior abnormalities and overexpression of Nav1.1, Nav1.3 and Nav1.6 proteins inside the hippocampus of mice models. Three weeks of voluntary running wheel (RW) exercise treatments before or/and post-injury effectively redressed the aberrant changes caused by TBI. Additionally, a 10% exercise-conditioned medium helped recover cell viability, neuronal sodium current and expressions of Nav1.1, Nav1.3 and Nav1.6 proteins across cultured neurons after injury. Therefore, the results validate the neuroprotection induced by voluntary RW exercise treatment before or/and post-TBI. The RW exercise-induced improvement in cognitive behaviors and neuronal excitability could be associated with correcting the Nav1.1, Nav1.3, and Nav1.6 expression levels. The current study proves that voluntary exercise is an effective treatment strategy against TBI. The study also highlights novel potential targets for rehabilitating TBI, including the Navs proteins.


Assuntos
Lesões Encefálicas Traumáticas , Lesões Encefálicas , Canais de Sódio Disparados por Voltagem , Humanos , Camundongos , Animais , Qualidade de Vida , Lesões Encefálicas Traumáticas/complicações , Lesões Encefálicas Traumáticas/terapia , Cognição
17.
Pest Manag Sci ; 80(2): 592-601, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-37740934

RESUMO

BACKGROUND: The larvae of Drosophila suzukii Matsumura feed directly inside the fruit, causing catastrophic damage to orchards. The misuse of pyrethroid insecticides during the control period has led to increasing resistance of D. suzukii to pyrethroids acting on the voltage-gated sodium channel (VGSC). RESULTS: In this study, the sodium channel of D. suzukii was cloned (DsNav 5 GenBank number: OQ871532). The results of multiple-sequence alignment showed that the homology of sodium channel between D. suzukii and Drosophila melanogaster was as high as 95.3%. Analysis of transcripts from 62 variants of D. suzukii VGSC revealed a total of six alternative splicing sites (exons u, j, a, b, e, and h) and 33 RNA editing. Exons j, a, b, e, and h are conserved in D. melanogaster and other insects, whereas exon u has never been reported before. The number of A-to-I was distinctly more than that of U-to-C for RNA editing. All D. suzukii VGSC variants were expressed in Xenopus oocytes, but only one (type 5) was able to produce robust currents and nine produce weak currents. DsNav 5 with TipE of D. melanogaster co-expresses current better than its own TipE. Subsequently, tetrodotoxin was verified to be a blocker of VGSC, and the gating properties of DsNav 5 were investigated. CONCLUSION: These findings proved that the VGSC of D. suzukii has not only the basic gating properties, but also the diversity of gating properties. This study also laid a foundation for the study of pyrethroid resistance mechanism of VGSC in D. suzukii. © 2023 Society of Chemical Industry.


Assuntos
Proteínas de Drosophila , Inseticidas , Piretrinas , Canais de Sódio Disparados por Voltagem , Animais , Drosophila melanogaster/genética , Drosophila/genética , Canais de Sódio Disparados por Voltagem/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Piretrinas/farmacologia , Inseticidas/farmacologia
18.
Circ Res ; 134(1): 46-59, 2024 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-38095085

RESUMO

BACKGROUND: Brugada syndrome is associated with loss-of-function SCN5A variants, yet these account for only ≈20% of cases. A recent genome-wide association study identified a novel locus within MAPRE2, which encodes EB2 (microtubule end-binding protein 2), implicating microtubule involvement in Brugada syndrome. METHODS: A mapre2 knockout zebrafish model was generated using CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeat-associated protein 9) and validated by Western blot. Larval hearts at 5 days post-fertilization were isolated for voltage mapping and immunocytochemistry. Adult fish hearts were used for ECG, patch clamping, and immunocytochemistry. Morpholinos were injected into embryos at 1-cell stage for knockdown experiments. A transgenic zebrafish line with cdh2 tandem fluorescent timer was used to study adherens junctions. Microtubule plus-end tracking and patch clamping were performed in human induced pluripotent stem cell derived cardiomyocytes (iPSC-CMs) with MAPRE2 knockdown and knockout, respectively. RESULTS: Voltage mapping of mapre2 knockout hearts showed a decrease in ventricular maximum upstroke velocity of the action potential and conduction velocity, suggesting loss of cardiac voltage-gated sodium channel function. ECG showed QRS prolongation in adult knockout fish, and patch clamping showed decreased sodium current density in knockout ventricular myocytes and arrhythmias in knockout iPSC-CMs. Confocal imaging showed disorganized adherens junctions and mislocalization of mature Ncad (N-cadherin) with mapre2 loss of function, associated with a decrease of detyrosinated tubulin. MAPRE2 knockdown in iPSC-CMs led to an increase in microtubule growth velocity and distance, indicating changes in microtubule dynamics. Finally, knockdown of ttl encoding tubulin tyrosine ligase in mapre2 knockout larvae rescued tubulin detyrosination and ventricular maximum upstroke velocity of the action potential. CONCLUSIONS: Genetic ablation of mapre2 led to a decrease in voltage-gated sodium channel function, a hallmark of Brugada syndrome, associated with disruption of adherens junctions, decrease of detyrosinated tubulin as a marker of microtubule stability, and changes in microtubule dynamics. Restoration of the detyrosinated tubulin fraction with ttl knockdown led to rescue of voltage-gated sodium channel-related functional parameters in mapre2 knockout hearts. Taken together, our study implicates microtubule dynamics in the modulation of ventricular conduction.


Assuntos
Síndrome de Brugada , Células-Tronco Pluripotentes Induzidas , Canais de Sódio Disparados por Voltagem , Animais , Humanos , Potenciais de Ação , Síndrome de Brugada/genética , Síndrome de Brugada/metabolismo , Estudo de Associação Genômica Ampla , Células-Tronco Pluripotentes Induzidas/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Microtúbulos/metabolismo , Miócitos Cardíacos/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.5/genética , Canal de Sódio Disparado por Voltagem NAV1.5/metabolismo , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismo , Canais de Sódio Disparados por Voltagem/metabolismo , Peixe-Zebra/genética , Peixe-Zebra/metabolismo
19.
Cell Signal ; 113: 110970, 2024 01.
Artigo em Inglês | MEDLINE | ID: mdl-37967692

RESUMO

Voltage-gated sodium channels (VGSC) are essential for triggering and relaying action potentials (AP), which perform critical functions in a variety of physiological processes, such as controlling muscle contractions and facilitating the release of neurotransmitters. In this study, we used a mouse C2C12 cell differentiation model to study the molecular expression and channel dynamics of VGSC and to investigate the exact role of VGSC in the development of muscle regeneration. Immunofluorescence, Real-time quantitative polymerase chain reaction, Western blot, and whole-cell patch clamp were employed for this purpose in mouse myoblasts. The findings revealed an increase in intracellular sodium concentration, NaV1.4 gene expression, and protein expression with the progress of differentiation (days 0, 1, 3, 5 and 7). Furthermore, VGSC dynamics exhibit the following characteristics: ① The increase of sodium current (INa); ② The decrease in the activation threshold and the voltage trigger maximum of INa; ③ A positive shift in the steady-state inactivation curve; ④ The recovery of INa during repolarization is delayed, the activity-dependent decay rate of INa was accelerated, and the proportionate amount of the fraction of activated channels was reduced. Based on these results, it is postulated that the activation threshold of AP could be decreased, and the refractory period could be extended with the extension of differentiation duration, which may contribute to muscle contraction. Taken together, VGSC provides a theoretical and empirical basis for exploring potential targets for neuromuscular diseases and other therapeutic muscle regeneration dysfunctions.


Assuntos
Canais de Sódio Disparados por Voltagem , Animais , Camundongos , Canais de Sódio Disparados por Voltagem/metabolismo , Potenciais de Ação , Diferenciação Celular , Sódio/metabolismo
20.
Channels (Austin) ; 18(1): 2287832, 2024 12.
Artigo em Inglês | MEDLINE | ID: mdl-38033122

RESUMO

Voltage-gated sodium (Nav) channels govern membrane excitability by initiating and propagating action potentials. Consistent with their physiological significance, dysfunction, or mutations in these channels are associated with various channelopathies. Nav channels are thereby major targets for various clinical and investigational drugs. In addition, a large number of natural toxins, both small molecules and peptides, can bind to Nav channels and modulate their functions. Technological breakthrough in cryo-electron microscopy (cryo-EM) has enabled the determination of high-resolution structures of eukaryotic and eventually human Nav channels, alone or in complex with auxiliary subunits, toxins, and drugs. These studies have not only advanced our comprehension of channel architecture and working mechanisms but also afforded unprecedented clarity to the molecular basis for the binding and mechanism of action (MOA) of prototypical drugs and toxins. In this review, we will provide an overview of the recent advances in structural pharmacology of Nav channels, encompassing the structural map for ligand binding on Nav channels. These findings have established a vital groundwork for future drug development.


Assuntos
Canais de Sódio Disparados por Voltagem , Humanos , Canais de Sódio Disparados por Voltagem/metabolismo , Microscopia Crioeletrônica , Potenciais de Ação , Peptídeos , Sódio/metabolismo
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