Do age-associated changes of voltage-gated sodium channel isoforms expressed in the mammalian heart predispose the elderly to atrial fibrillation?

Atrial fibrillation(AF)is the most common cardiac arrhythmia worldwide.The prevalence of the disease increases with age,strongly implying an age-related process underlying the pathology.At a time when people are living longer than ever before,an exponential increase in disease prevalence is predicted worldwide.Hence unraveling the underlying mechanics of the disease is paramount for the development of innovative treatment and prevention strategies.The role of voltage-gated sodium channels is fundamental in cardiac electrophysiology and may provide novel insights into the arrhythmogenesis of AF.Na_v1.5 is the predominant cardiac isoform,responsible for the action potential upstroke.Recent studies have demonstrated that Na_v1.8(an isoform predominantly expressed within the peripheral nervous system)is responsible for cellular arrhythmogenesis through the enhancement of pro-arrhythmogenic currents.Animal studies have shown a decline in Na_v1.5 leading to a diminished action potential upstroke during phase 0.Furthermore,the study of human tissue demonstrates an inverse expression of sodium channel isoforms;reduction of Na_v1.5 and increase of Na_v1.8 in both heart failure and ventricular hypertrophy.This strongly suggests that the expression of voltage-gated sodium channels play a crucial role in the development of arrhythmias in the diseased heart.Targeting aberrant sodium currents has led to novel therapeutic approaches in tackling AF and continues to be an area of emerging research.This review will explore how voltage-gated sodium channels may predispose the elderly heart to AF through the examination of laboratory and clinical based evidence.

Voltage-gated Sodium Channels and Blockers:An Overview and Where Will They Go?

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.Sofar,the cryoelectron microscopic structure of the human Nav 1.2,Nav 1.4,and Nav 1.7 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.

Molecular Docking Studies on Anticonvulsant Enaminones Inhibiting Voltage-Gated Sodium Channels

Epilepsy is described as the most common chronic brain disorder. A typical symptom of epilepsy results in uncontrolled convulsions caused by temporary excessive neuronal discharges. Although several new anticon-vulsants have been introduced, some types of seizures have still not been adequately controlled with these new and current therapies. There is an urgent need to develop new anticonvulsant drugs to control the many different types of seizures. Many studies have shown that the epilepsies involve more than one mechanism and therefore may be responsible for the various types of observed seizures. Recently reported studies have shown that a group of newly synthesized 6 Hz active anticonvulsant fluorinated N-benzamide enaminones exhibited selective inhibitions of voltage-gated sodium (Nav) channels. Nav channels are responsible for the initial inward currents during the depolarization phases of the action potential in excitable cells. The activation and opening of Nav channels result in the initial phases of action potentials. We hypothesize that there is an essential pharmacophore model for the interactions between these enaminones and the active sites of Nav channels. The research reported here is focused on molecular docking studies of the interactions that occur between the fluorinated N-benzamide enaminones and the Nav channels. These studies may open an avenue for designing anticonvulsant drugs by inhibiting Nav channels.

Research Progress on the Influence of Structural Changes inμ-Conotoxins on Sodium Channel Receptors

The voltage-gated sodium channel(Na v)is widely present in mammals and can generate cell action potentials,which are related to many diseases.Theμ-Conotoxins(μ-CTx)isolated from the venom of cone snails can specifically block the voltage-gated sodium channel;it can be widely used as a necessary probe to distinguish the Na v channel subtypes.In this study,the effects of eightμ-CTx on different Na v channel isoforms were reviewed,and sequence alignment and protein homologous modeling were used to predict their biological activities,and the structure-activity relationship betweenμ-CTx and mutagenesis strategies.

河豚毒素对BV-2细胞氧化应激损伤的诱导作用

目的探究河豚毒素(TTX)暴露对小鼠小胶质细胞BV-2的氧化应激损伤的诱导作用。方法选择BV-2细胞进行TTX体外暴露。首先采用不同浓度(0、0.01、0.1、1、10、100μmol·L^(-1))TTX对BV-2细胞进行单独暴露,观察细胞形态和活力变化,利用CCK8法确定TTX联合暴露浓度。使用藜芦定(VTD)和毒毛旋花苷G(O)的混合物与TTX对BV-2细胞进行联合暴露,根据细胞形态和细胞活力验证BV-2细胞膜上离子通道类型。通过测定TTX单独暴露后乳酸脱氢酶(LDH)释放量、钙离子荧光量、活性氧(ROS)含量和细胞凋亡率水平,分析TTX暴露对BV-2细胞的影响。结果100μmol·L^(-1)TTX可改变BV-2细胞形态,抑制细胞活力。联合暴露情况下,TTX能够拮抗VTD和O混合物造成的细胞膜内外钠离子浓度差异,延缓细胞损伤状态。与对照组比较,100μmol·L^(-1)TTX可促使细胞膜通透性改变,释放4.01 U/g LDH。在100μmol·L^(-1)TTX暴露下分别升高细胞内钙离子和ROS水平,细胞相对荧光强度提升至84.78%和63.48%;100μmol·L^(-1)TTX暴露下,BV-2细胞凋亡率增加2.9倍。结论TTX暴露可以抑制BV-2细胞增殖能力,改变细胞膜的通透性,促使ROS在细胞内蓄积,诱导细胞氧化应激损伤,造成细胞凋亡。

Expression and Role of Voltage-Gated Sodium Channels in Human Dorsal Root Ganglion Neurons with Special Focus on Nav1.7,Species Differences, and Regulation by Paclitaxel

Voltage-gated sodium channels （Navs） play an important role in human pain sensation. However, the expression and role of Nav subtypes in native human sensory neurons are unclear. To address this issue, we obtained human dorsal root ganglion （hDRG） tissues from healthy donors. PCR analysis of seven DRG-expressed Nav subtypes revealed that the hDRG has higher expression of Navl.7 （,-~ 50% of total Nav expression） and lower expres- sion of Navl.8 （~ 12%）, whereas the mouse DRG has higher expression of Nav 1.8 （- 45%） and lower expression of Navl.7 （- 18%）. To mimic Nav regulation in chronic pain, we treated hDRG neurons in primary cultures with paclitaxel （0.1-1 μmol/L） for 24 h. Paclitaxel increased the Navl.7 but not Navl.8 expression and also increased the transient Na＋ currents and action potential firing frequency in small-diameter （〈50 ~tm） hDRG neurons. Thus, the hDRG provides a translational model in which to study ＂human pain in a dish＂ and test new pain therapeutics.

Exploring the obscure profiles of pharmacological binding sites on voltage-gated sodium channels by BmK neurotoxins

Diverse subtypes of voltage-gated sodium channels(VGSCs)have been found throughout tissues of the brain,muscles and the heart.Neurotoxins extracted from the venom of the Asian scorpion Buthus martensi Karsch(BmK)act as sodium channel-specific modulators and have therefore been widely used to study VGSCs.α-type neurotoxins,named BmK I,BmKαIV and BmK abT,bind to receptor site-3 on VGSCs and can strongly prolong the inactivation phase of VGSCs.In contrast,β-type neurotoxins,named BmK AS,BmK AS-1,BmK IT and BmK IT2,occupy receptor site-4 on VGSCs and can suppress peak currents and hyperpolarize the activation kinetics of sodium channels.Accumulating evidence from binding assays of scorpion neurotoxins on VGSCs,however,indicate that pharmacological sensitivity of VGSC subtypes to different modulators is much more complex than that suggested by the simpleα-type and β-type neurotoxin distinction.Exploring the mechanisms of possible dynamic interactions between site 3-/4-specific modulators and region-and/or speciesspecific subtypes of VGSCs would therefore greatly expand our understanding of the physiological and pharmacological properties of diverse VGSCs.In this review,we discuss the pharmacological and structural diversity of VGSCs as revealed by studies exploring the binding properties and cross-competitive binding of site 3-or site 4-specific modulators in VGSC subtypes in synaptosomes from distinct tissues of diverse species.

Theoretical and simulation studies on voltage-gated sodium channels

Voltage-gated sodium （Nav） channels are indispensable membrane elements for the generation and propagation of electric signals in excitable cells. The successes in the crystallographic studies on prokaryotic Nay chan- nels in recent years greatly promote the mechanistic investigation of these proteins and their eukaryotic counterparts. In this paper, we mainly review the pro- gress in computational studies, especially the simula- tion studies, on these proteins in the past years.

Distribution and Functional Characteristics of Voltage-Gated Sodium Channels in Immature Cochlear Hair Cells

Voltage-gated sodium channels(VGSCs)are transiently expressed in cochlear hair cells before hearing onset and play an indispensable role in shaping spontaneous activity.In this study,we showed that Na^+currents shaped the spontaneous action potentials in developing mouse inner hair cells(IHCs)by decreasing the time required for the membrane potential to reach the action-potential threshold.In immature IHCs,we identified 9 known VGSC subtypes(Navl.la-l.9ot),among which Navl.7a was the most highly expressed subtype and the main contributor to Na+currents in developing hair cells.Electrophysiological recordings of two cochlea-specific Navi.7 variants(CbmNavl.7a and CbmNavl.7b)revealed a novel loss-of-function mutation(C934R)at the extracellular linker between segments 5 and 6 of domain II.In addition,post-transcriptional modification events,such as alternative splicing and RNA editing,amended the gating properties and kinetic features of CbmNavl.7a(C934).These results provide molecular and functional characteristics of VGSCs in mammalian IHCs and their contributions to spontaneous physiological activity during cochlear maturation.

Research progress on the correlation between ion channel and excitability of striatum neurons in Parkinson's disease

Parkinson's disease(PD)is a neurodegenerative disorder due to gradual loss of dopaminergic neurons in the substantia nigra in the midbrain,however the pathogenesis is unclear.There is a correlation between the excitability of striatal neurons and PD.Ion channels are important to maintain membrane potential and regulate excitability of neurons,while ionic mechanisms for modulation of neurons excitability are not fully understood.This article reviews the relationship between ion channels and excitability of striatal neurons in PD and ion channel changes in the pathogenesis of PD.In order to find new targets to treatment PD by intervening ion channels.

The Effect of Peony and Licorice Decoction on the Voltage-Gated Sodium Channel Subtype 1.4 Based on Standard Decoction

Objective:The objective of this study is to investigate the inhibitory effect of peony and licorice decoction and its compatibility components on the Nav1.4 voltage-gated sodium channels(VGSCs).Materials and Methods:Writhing test was carried out with ICR mice.Paeonia lactiflora and Glycyrrhiza uralensis group were administrated 0.2 ml of solution of freeze-dried powder dissolved in normal saline with the concentration of 2.94 mg/ml,1.47 mg/ml,and 0.74 mg/ml using intragastric administration,respectively.Peony and licorice decoction groups were administrated 0.2 ml of solution of freeze-dried powder dissolved in normal saline with the concentration of 5.89 mg/ml,2.94 mg/ml,and 1.47 mg/ml using intragastric administration,respectively.For electrophysiology studies,each freeze-dried powder was dissolved in DMSO to make 10 mg/ml and 50 mg/ml stock solutions.The electrophysiological recordings were obtained under visual control of a microscope.For UPLC analysis,the freeze-dried powder was dissolved in methanol and then determines the contents of the nine marker compounds.Results:The effect of G.uralensis on incubation period and writhing frequency was significantly better than that of peony and licorice decoction group and P.lactiflora group.The inhibition rate of 50 mg/ml water extracts of the three samples was significantly higher than that of the 10 mg/ml group.Moreover,the water extract of G.uralensis at 50 mg/ml had the strongest inhibitory effect on I_(Nav) 1.4 of the three.Conclusion:The possible mechanism of peony and licorice decoction in relieving spasm and pain is most likely by inhibiting Voltage-Gated Sodium Channel Subtype 1.4.

Therapeutic efficacy of voltage‑gated sodium channel inhibitors in epilepsy

Epilepsy is a neurological disease characterized by excessive and abnormal hyper-synchrony of electrical discharges of the brain and a predisposition to generate epileptic seizures resulting in a broad spectrum of neurobiological insults,imposing psychological,cognitive,social and also economic burdens to the sufferer.Voltage-gated sodium channels(VGSCs)are essential for the generation and propagation of action potentials throughout the central nervous system.Dysfunction of these channels has been implicated in the pathogenesis of epilepsy.VGSC inhibitors have been demonstrated to act as anticonvulsants to suppress the abnormal neuronal firing underlying epileptic seizures,and are used for the management and treatment of both genetic-idiopathic and acquired epilepsies.We discuss the forms of idiopathic and acquired epilepsies caused by VGSC mutations and the therapeutic efficacy of VGSC blockers in idiopathic,acquired and pharmacoresistant forms of epilepsy in this review.We conclude that there is a need for better alternative therapies that can be used alone or in combination with VGSC inhibitors in the management of epilepsies.The current anti-seizure medications(ASMs)especially for pharmacoresistant epilepsies and some other types of epilepsy have not yielded expected therapeutic efficacy partly because they do not show subtype-selectivity in blocking sodium channels while also bringing side effects.Therefore,there is a need to develop novel drug cocktails with enhanced selectivity for specific VGSC isoforms,to achieve better treatment of pharmacoresistant epilepsies and other types of epileptic seizures.

人脑胶质瘤组织高表达nNav1.5促进肿瘤细胞的迁移和侵袭

目的：观察电压-门控钠离子通道（voltage-gated sodium channel,VGSC）亚型nNav1.5在人脑胶质瘤组织中的表达,并探讨其对脑胶质瘤U251细胞迁移及侵袭的影响。方法：收集中国医科大学附属第一医院神经外科于2011年10月至2012年10月手术切除并经病理证实的脑胶质瘤组织标本68例,应用免疫组织化学S-P法检测脑胶质瘤组织中nNav1.5的表达。设计并化学合成nNav1.5基因特异性小干扰RNA（nNav1.5-siRNA）,用脂质体介导转染胶质瘤U251细胞,应用Real-time PCR和Western blotting法分别检测U251细胞中nNav1.5 mRNA和蛋白的表达水平,并采用细胞划痕实验和Transwell侵袭实验检测U251细胞迁移和侵袭能力的变化。结果：nNav1.5在人脑胶质瘤组织中表达的阳性率显著高于正常组织（72.6%vs23.0%,P〈0.01）,并且其在高级别胶质瘤（WHOⅢ~Ⅳ级）组织中的阳性率明显高于低级别胶质瘤（WHOⅠ~Ⅱ级）组织（85.8%vs 52.9%,P〈0.01）。nNav1.5-siRNA转染可显著抑制U251细胞中nNav1.5 mRNA和蛋白的表达（P〈0.01）;转染后U251细胞的迁移距离明显小于未转染细胞[（0.019±0.015）vs（0.223±0.031）mm,P〈0.01],且其侵袭指数明显低于未转染细胞[（2.99±0.15）%vs（6.77±0.26）%,P〈0.01]。结论：nNav1.5在人脑胶质瘤组织中高表达,干扰nNav1.5表达可显著抑制胶质瘤细胞的迁移和侵袭能力,nNav1.5是胶质瘤恶性侵袭的调控因子并有望成为胶质瘤的新标志物和治疗靶点。

电压门控钠离子通道:律动生命乐章的主音符

生命个体传递神经冲动时扩布的电位变化过程以动作电位发放形式为特质表征.电压门控钠离子通道(voltage-gated sodium channels,VGSCs)是形成动作电位的核心蛋白构件,在细胞的电兴奋产生和律动中起主角作用.VGSCs决定神经元细胞的兴奋性以及从突触输入到轴突输出的信号传导过程.VGSCs也是众多外源性天然或人工合成化学产物作用的靶器.大多数临床抗癫药和局麻药、杀虫剂、天然生物神经毒素等均以阻断或调制靶通道对离子的通透性,以及通道的门控动力学性质的方式异化通道结构与功能的平衡,导致临床病理事件的产生或生理机能的缓解与恢复.从内源和外源两个方向略览VGSCs,即赋予生命乐章主音符涵义的生理与病理功能地位及其靶向药理学与毒理学身价.

Modeling protein-protein interactions in axon initial segment to understand their potential impact on action potential initiation

The axon initial segment(AIS)region is crucial for action potential initiation due to the presence of high-density AIS protein voltage-gated sodium channels(Nav).Nav channels comprise several serine residues responsible for the recruitment of Nav channels into the structure of AIS through interactions with ankyrin-G(AnkG).In this study,a series of computational experiments are performed to understand the role of AIS proteins casein kinase 2 and AnkG on Nav channel recruitment into the AIS.The computational simulation results using Virtual cell software indicate that Nav channels with all serine sites available for phosphorylation bind to AnkG with strong affinity.At the low initial concentration of AnkG and casein kinase 2,the concentration of Nav channels reduces significantly,suggesting the importance of casein kinase 2 and AnkG in the recruitment of Nav channels.

Highlights for the 6th International Ion Channel Conference:ion channel structure,function,disease and therapeutics

To foster communication and interactions amongst international scholars and scientists in the field of ion channel research, the 6 th International Ion Channel Conference(IICC-2017) was held between June 23–27, 2017 in the eastern coastal city of Qingdao, China. The meeting consisted of 450 attendees and 130 speakers and poster presenters. The program consisted of research progress, new findings and ongoing studies that were focused on(1) Ion channel structure and function;(2) Ion channel physiology and human diseases;(3) Ion channels as targets for drug discovery;(4) Technological advances in ion channel research. An insightful overview was presented on the structure and function of the mechanotransduction channel Drosophila NOMPC(No mechanoreceptor potential C), a member of the transient receptor potential(TRP) channel family. Recent studies on Transmembrane protein 16 or Anoctamin-1(TMEM16A, a member of the calcium-activated chloride channel [CaCC] family) were summarized as well. In addition, topics for ion channel regulation, homeostatic feedback and brain disorders were thoroughly discussed. The presentations at the IICC-2017 offer new insights into our understanding of ion channel structures and functions, and ion channels as targets for drug discovery.

Scorpion toxin BmKI directly activates Nav1.8 in primary sensory neurons to induce neuronal hyperexcitability in rats

Voltage-gated sodium channels （VGSCs） in primary sensory neurons play a key role in transmitting pain signals to the central nervous system. BmK I, a site-3 sodium channel-specific toxin from scorpion Buthus martensi Karsch, induces pain behaviors in rats. How- ever, the subtypes of VGSCs targeted by BmK I were not entirely clear. We therefore investigated the effects of BmK I on the current amplitude, gating and kinetic properties of Nav1.8, which is associated with neuronal hyperexcitability in DRG neurons. It was found that BmK I dose-dependently increased Nav1.8 current in small- sized （〈25 μm） acutely dissociated DRG neurons, which correlated with its inhibition on both fast and slow in- activation. Moreover, voltage-dependent activation and steady-state inactivation curves of Nay1.8 were shifted in a hyperpolarized direction. Thus, BmK I reduced the threshold of neuronal excitability and increased action potential firing in DRG neurons. In conclusion, our data clearly demonstrated that BmK I modulated Nav1.8 re- markably, suggesting BmK I as a valuable probe for studying Nay1.8. And Navl.8 is an important target re- lated to BmK I-evoked pain.

Negative cross-resistance of a pyrethroid-resistant Drosophila mutant to Phryma leptostachya-derived haedoxan A

Voltage-gated sodium channels are the primary target of pyrethroid insecticides.Mutations in sodium channel confer knockdown resistance(kdr)to pyrethroids in various arthropod pests.Haedoxan A(HA)is the major insecticidal component from Phryma leptostachya.It has been shown that HA alters electrical responses at the Drosophila neuromuscular junction and modifies the gating properties of cockroach sodium channels expressed in Xenopus oocytes.However,whether sodium channel mutations that confer pyrethroid resistance also affect the action of HA is unknown.In this study,we conducted bioassays using HA and permethrin in two Drosophila melanogaster strains:w^(1118),an insecticide-susceptible strain,and para^(tsl),a pyrethroid-resistant strain due to a I265N mutation in the sodium channel,and identified a new case of negative cross-resistance(NCR)between permethrin and HA.Both para^(tsl) larvae and adults were more resistant to permethrin,as expected.However,both para^(tsl) larvae and adults were more sensitive to HA compared to w^(1118).We confirmed that the I265N mutation reduced the sensitivity to permethrin of a Drosophila sodium channel variant,DmNa_(v)22,expressed in Xenopus oocytes.Interestingly,the I265N mutation also abolished the effect of HA on sodium channels.Further characterization showed that I265 on the sodium channels is critical for the action of both pyrethroids and HA on sodium channels,pointing to an overlapping mode of action between pyrethroids and HA on the sodium channel.Overall,our results suggest an I265N-independnt mechanism(s)in para^(tsl) flies that is responsible for the NCR between permethrin and HA at the whole insect level.

SCN9A Epileptic Encephalopathy Mutations Display a Gain-offunction Phenotype and Distinct Sensitivity to Oxcarbazepine

Genetic mutants of voltage-gated sodium channels(VGSCs)are considered to be responsible for the increasing number of epilepsy syndromes.Previous research has indicated that mutations of one of the VGSC genes,SCN9A(Navl.7),result in febrile seizures and Dravet syndrome in humans.Despite these recent efforts,the electrophysiological basis of SCN9A mutations remains unclear.Here,we performed a genetic screen of patients with febrile seizures and identified a novel missense mutation of SCN9A(W1150R).Electrophysiological characterization of different SCN9A mutants in HEK293T cells,the previously-reported N641Y and K655R variants,as well as the newly-found W1150R variant,revealed that the current density of the W1150R and N641Y variants was significantly larger than that of the wild-type(WT)channel.The time constants of recovery from fast inactivation of the N641Y and K655R variants were markedly lower than in the WT channel.The W1150R variant caused a negative shift of the G-V curve in the voltage dependence of steady-state activation.All mutants displayed persistent currents larger than the WT channel.In addition,we found that oxcarbazepine(OXC),one of the antiepileptic drugs targeting VGSCs,caused a significant shift to more negative potential for the activation and inactivation in WT and mutant channels.OXC-induced inhibition of currents was weaker in the W1150R variant than in the WT.Furthermore,with administering OXC the time constant of the N641Y variant was longer than those of the other two SCN9A mutants.In all,our results indicated that the point mutation W1150R resulted in a novel gain-of-function variant.These findings indicated that SCN9A mutants contribute to an increase in seizure,and show distinct sensitivity to OXC.