Deglycosylation altered the gating properties of rNav1.3:glycosylation/deglycosylation homeostasis probably complicates the functional regulation of voltage-gated sodium channel

Objective To examine the effect of deglycosylation on gating properties of rNav1.3. Methods rNav1.3 was expressed in Xenopus oocyte, with glycosylation inhibition by using tunicamycin. Two-electrode voltage clamp was employed to record the whole-cell sodium current and data were analyzed by Origin software. Those of glycosylated rNav1.3 were kept as control. Results Compared with glycosylated ones, the steady-state activation curve of deglycosylated rNav1.3 was positively shifted by about 10 mV, while inactivation curve was negatively shifted by about 8 mV. Conclusion Glycosylation altered the gating properties of rNav 1.3 and contributed to the functional diversity.

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.

Effects of Intrathecally Administerd NaV1.8 Antisense Oligonucleotide on the Expression of Sodium Channel mRNA in Dorsal Root Ganglion

Neuropathic pain has been hypothesized to be the result of aberrant expression and function of sodium channels at the site of injury. To investigate the effects of NaV1.8 antisense oligonucleotide on the expression of sodium channel mRNA in dorsal root ganglion （DRG） neurons in chronic neuropathic pain. 24 Sprague-Dawley rats weighing 200--260 g were anesthetized with the intraperitoneal injection of 300 mg· kg^-1 choral hydrate. The CCI model was made by loose ligation of sciatic nerve trunk by 4--0 chromic gut. The mechanical and thermal pain threshold were measured before operation and 1, 3, 5, 7, 9, 11, 13 days after operation. A PE-10 catheter was implanted in subarachnoid space at lumbar region. On the 7th postoperative day the animals were randomly divided into 4 groups. The drugs were injected intrathecally twice a day for 5 consecutive days in group 2--4. The animals were decapitated 14 days after the surgery. The L4--L6 DRG of the operated side was removed and crushed, and total RNA was extracted with Trizol reagent. The contralateral side was used as control. The change of NaV1.8 sodium channel transcripts was determined by RT-PCR. Pain threshold was significantly lowered after CCI as compared with that in control group and was elevated 3 days after antisense oligonucleotide injection. Sensory neuron specific TTX-R sodium channel NaV1.8 transcript was down-regulated after antisense oligonucleotide injection at the dosage of 45 μg as compared with that in CCI group （P〈0.01）, and it was even greater at the dosage of 90 μg. The intrathecally injected NaV1.8 antisense oligonucleotide can reduce the mechanical allodynia and thermal hyperalgesia partially by downregulating the SNS transcript expression.

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.

Transforming growth factor-beta 1 enhances discharge activity of cortical neurons

Transforming growth factor-beta 1(TGF-β1)has been extensively studied for its pleiotropic effects on central nervous system diseases.The neuroprotective or neurotoxic effects of TGF-β1 in specific brain areas may depend on the pathological process and cell types involved.Voltage-gated sodium channels(VGSCs)are essential ion channels for the generation of action potentials in neurons,and are involved in various neuroexcitation-related diseases.However,the effects of TGF-β1 on the functional properties of VGSCs and firing properties in cortical neurons remain unclear.In this study,we investigated the effects of TGF-β1 on VGSC function and firing properties in primary cortical neurons from mice.We found that TGF-β1 increased VGSC current density in a dose-and time-dependent manner,which was attributable to the upregulation of Nav1.3 expression.Increased VGSC current density and Nav1.3 expression were significantly abolished by preincubation with inhibitors of mitogen-activated protein kinase kinase(PD98059),p38 mitogen-activated protein kinase(SB203580),and Jun NH2-terminal kinase 1/2 inhibitor(SP600125).Interestingly,TGF-β1 significantly increased the firing threshold of action potentials but did not change their firing rate in cortical neurons.These findings suggest that TGF-β1 can increase Nav1.3 expression through activation of the ERK1/2-JNK-MAPK pathway,which leads to a decrease in the firing threshold of action potentials in cortical neurons under pathological conditions.Thus,this contributes to the occurrence and progression of neuroexcitatory-related diseases of the central nervous system.

Modulatory effect of auxiliary β_1 subunit on Nav1.3 voltage-gated sodium channel expressed in Xenopus oocyte

Voltage-gated sodium channels play an important role in the generation and propagation of action potentials in excitable cells. They are composed of a pore-forming α subunit and auxiliary β subunits. To date, nine subtypes of the α subunit, designated Nav1.1 to Nav1.9, have been shown to form functional sodium channels. In addition, four different mammalian subunits （β1-β4） isoforms have been cloned from the nervous system. The β subunits are structurally homologous and form single transmembrane glycoproteins with short intracellular loops and immunoglobulin-like extracellular segments. The association of the various α subtypes with different combinations of auxiliary β subunits creates the possibility of additional molecular and functional complexity for neuronal sodium channels.

电压门控钠通道在慢性疼痛药物发现中的研究进展

由于发病率高、药物效果有限或治疗药物受限等原因,慢性疼痛的治疗一直是世界范围内研究人员关注的难题。电压门控钠通道(VGSCs)阻滞剂有较为显著的镇痛作用,目前已知与慢性疼痛相关的钠通道亚型主要有Nav1.3、Nav1.7、Nav1.8、Nav1.9。2021年8—9月进行了该研究,全面概括了上述钠通道亚型与慢性疼痛的关系,归纳出潜在候选药物临床前研究方法,以及已被证实安全有效的选择性钠通道阻滞剂品种,为选择性钠通道阻滞剂的开发提供参考。

钠离子通道Na_v1.7在大鼠牙髓炎中的表达

目的通过检测大鼠实验性牙髓炎牙髓中电压门控钠离子通道Nav1.7的表达,探讨Nav1.7的表达与牙痛的关系。方法大鼠切牙髓腔暴露,置入脂多糖(LPS)诱导产生牙髓炎。大鼠分为正常对照组和封LPS 1、3、5 d组,HE染色观察各组牙髓组织病理学改变。通过免疫组化、ELISA和逆转录PCR(RT-PCR)方法检测大鼠正常牙髓和炎症牙髓中Nav1.7的表达。结果封LPS 1 d组,牙髓中未见明显炎症,而封LPS 3和5 d组牙髓中炎症反应程度逐渐上升。免疫组化结果显示,Nav1.7在所有牙髓中均有表达,封LPS 3和5 d组牙髓中Nav1.7表达量较正常对照组明显增加(P<0.05);ELISA结果与上述结果相似。RT-PCR结果显示,封LPS 3和5 d组牙髓中Nav1.7 mRNA较正常对照组有显著上调(P<0.05)。结论在牙髓炎中,Nav1.7可能以时序控制方式表达明显增多,且与牙髓炎炎症程度相关。鉴于Nav1.7在疼痛中发挥了重要作用,牙髓中Nav1.7的表达可能涉及牙痛的病理生理机制。

Growth Differentiation Factor-15 Produces Analgesia by Inhibiting Tetrodotoxin-Resistant Nav1.8 Sodium Channel Activity in Rat Primary Sensory Neurons

Growth differentiation factor 15(GDF-15)is a member of the transforming growth factor-βsuperfamily.It is widely distributed in the central and peripheral nervous systems.Whether and how GDF-15 modulates nociceptive signaling remains unclear.Behaviorally,we found that peripheral GDF-15 significantly elevated nociceptive response thresholds to mechanical and thermal stimuli in naïve and arthritic rats.Electrophysiologically,we demonstrated that GDF-15 decreased the excitability of small-diameter dorsal root ganglia(DRG)neurons.Furthermore,GDF-15 concentration-dependently suppressed tetrodotoxin-resistant sodium channel Nav1.8 currents,and shifted the steady-state inactivation curves of Nav1.8 in a hyperpolarizing direction.GDF-15 also reduced window currents and slowed down the recovery rate of Nav1.8 channels,suggesting that GDF-15 accelerated inactivation and slowed recovery of the channel.Immunohistochemistry results showed that activin receptor-like kinase-2(ALK2)was widely expressed in DRG medium-and small-diameter neurons,and some of them were Nav1.8-positive.Blockade of ALK2 prevented the GDF-15-induced inhibition of Nav1.8 currents and nociceptive behaviors.Inhibition of PKA and ERK,but not PKC,blocked the inhibitory effect of GDF-15 on Nav1.8 currents.These results suggest a functional link between GDF-15 and Nav1.8 in DRG neurons via ALK2 receptors and PKA associated with MEK/ERK,which mediate the peripheral analgesia of GDF-15.

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.

Na^+ Channels Activation Recovery from Use-dependent Block in Human Embryonic Kidney Cells and Effects of Gansong Volatile Oil on Na^+ Current Recovery

Objectives This experiment used whole-cell patch-clamp technique to investigate the course of recovery from use- dependent block of Na ＋ channels （Nav 1.5） in human embryonic kidney （HEK） cells, on which to verify the effects of volatile oil of Nardostachy chinesis Batal （Gansong）. Methods Two pulses generated by computer followed by a recovery pulse and a test pulse, the interval duration between the two pulses varied from 16 ms to 1 s, and holding potential is -80 mV to - 140 inV. The peak Na^＋ current for a given recovery time was normalized to the tully recovered peak current, and the normalized value was the plot as a function of the recovery time to study the effects of 3 ppm concentration Gansong volatile oil on recovery from use-dependent block of Navl. 5 in HEK. Results It showed that Gansong group, comparing with control group, delayed the time courses of recovery from use-dependent block [ （33.2± 5.77 ） ms for control group and （52.5± 6.08 ） ms for 3 ppm Gansong group, P 〈 0.05 ] In the presence of Gansong, inhibition of the Na^＋ current was enhanced by increasing frequency of depolarizing pulse from 56.5 ms to 16 ms. In the control group, the time course of recovery showed that recovery started at 19.5 ms and finished by 36.5 ms. In the presence of Gansong, the time course of recovery showed that recovery started at 36.5 ms and finished by 56.5 ms. Na^＋ currents recovered from the use-dependent block varying with holding potential （holding potential-dependent）. Conclusions The results suggested that Na ＋ currents recovered from the use-dependent block correlated with persistent time, holding potential. The Gansong volatile oil has inhibitive effect on the Na^＋ current recovery.

电针“内关”对缺血性心肌损伤大鼠钠离子通道相关蛋白的影响

目的：探讨电针＂内关＂对缺血性心肌的保护机制,阐明经穴效应循经特异性在心脏器官水平的钠离子通道的响应模式及特征。方法：将60只SPF级雄性大鼠随机分为空白组、模型组、非经非穴组、内关组、列缺组,每组12只。除空白组外,余组皮下注射异丙肾上腺素制备心肌缺血模型。内关组、列缺组、非经非穴组大鼠分别给予相应穴位的疏密波电针治疗,频率为2 Hz/20Hz,强度为2~3mA,每次电针20min,每天1次,连续7d。空白组与模型组仅在每天治疗时抓取固定。应用Western-blot法检测各组大鼠心肌电压-门控钠离子通道alpha（α）亚单位（Nav 1.5）、蛋白酪氨酸激酶（protein tyrosine kinase,PTKs）和蛋白酪氨酸磷酸酶（protein tyrosine phosphatases,PTPs）的蛋白表达水平。结果：模型组Nav 1.5和PTKs蛋白表达水平明显低于空白组（均P〈0.01）,内关组和列缺组较模型组表达水平增加（均P〈0.01）,内关组Nav 1.5和PTKs蛋白表达水平高于列缺组（均P〈0.01）;模型组和非经非穴组PTPs的蛋白表达均明显高于空白组（均P〈0.01）,内关组和列缺组PTPs的蛋白表达明显下调,均较模型组低（均P〈0.01）,内关组下调水平优于列缺组,两组比较差异具有统计学意义（P〈0.05）。结论：电针＂内关＂可能通过下调PTPs、上调Nav 1.5、PTKs的蛋白表达水平实现对钠离子通道电流以及钙超载的调节,进而改善心肌缺血状态,为经穴效应循经特异性理论提供实验基础。

Modulation of Na_v1.8 by Lysophosphatidic Acid in the Induction of Bone Cancer Pain

Given that lysophosphatidic acid（LPA） and the tetrodotoxin-resistant sodium channel Na_v1.8 are both involved in bone cancer pain, the present study was designed to investigate whether crosstalk between the LPA receptor LPA_1（also known as EDG2） and Na_v1.8 in the dorsal root ganglion（DRG） contributes to the induction of bone cancer pain. We showed that the EDG2 antagonist Ki16198 blocked the mechanical allodynia induced by intrathecal LPA in na？ve rats and attenuated mechanical allodynia in a rat model of bone cancer. EDG2 and Na_v1.8expression in L_（4-6）DRGs was upregulated following intrathecal or hindpaw injection of LPA. EDG2 and Na_v1.8expression in ipsilateral L_（4-6）DRGs increased with the development of bone cancer. Furthermore, we showed that EDG2 co-localized with Na_v1.8 and LPA remarkably enhanced Na_v1.8 currents in DRG neurons, and this was blocked by either a protein kinase C（PKC） inhibitor or a PKCe inhibitor. Overall, we demonstrated the modulation of Na_v1.8 by LPA in DRG neurons, and that this probably underlies the peripheral mechanism by which bone cancer pain is induced.

Design and synthesis of novel α-aminoamides derivatives as Nav1.7 inhibitors for antinociception

Three novel series of α-aminoamides derivatives were designed and synthesized based on ralfinamide,and their Nav1.7 inhibitory activities were evaluated using manual patch clamp electrophysiology. Active compounds inhibited Nav1.7 with half maximal inhibitory concentration(IC_(50)) values ranging from2.9 μmol/L to 21.4 μmol/L. Among them, the most potent compound 19h exhibited about 12-fold potency better than ralfinamide. The investigation of their structure-activity relationship gives a strategy to improve the Nav1.7 inhibition of ralfinamide analogues. Compound 19h was efficacious in antinociception in the mouse spared nerve injury(SNI) model of neuropathic pain without causing sedation in the open field test.