电压门控钠通道NaV1.7与痛信号的产生

电压门控钠通道NaV1.7选择性高表达在伤害感受性脊髓背根神经节的感觉神经元上,在疼痛电信号的产生、传导和调控中具有重要的生理功能。伤害性感受器上的NaV1.7亦在慢性神经痛和炎症痛的病理生理过程中发挥关键作用。近年来的研究发现,人类遗传性疼痛症(如红斑性肢痛病)与NaV1.7钠离子通道基因SCN9A的某些功能增强型突变相关。最近Cox等首次报道了SCN9A突变将导致人先天痛觉完全丧失,而无痛症患者机体其它功能正常,提示NaV1.7将可能成为有效治疗疼痛而无副作用的一个新靶标。     

钠离子通道疾病及其抑制剂生物学功能研究进展

电压门控型钠离子通道(Voltage-gated sodium channel,VGSC)广泛分布于兴奋性细胞,是电信号扩大和传导的主要介质,在神经细胞以及心肌细胞兴奋传导等方面发挥重要作用。钠离子通道结构和功能的异常会改变细胞的兴奋性,从而导致多种疾病的发生,如神经性疼痛、癫痫,以及心律失常等。目前临床上多采用钠离子通道抑制剂治疗上述疾病。近些年,研究人员陆续从动物的毒液中分离纯化出具有调控钠离子通道功能的神经毒素。这些神经毒素多为化合物或小分子多肽。现已有医药研发公司将这些天然的神经毒素进行定向设计改造成钠离子通道靶向药物用于临床疾病的治疗。此外,来源于七鳃鳗Lampetra japonica口腔腺的富含半胱氨酸分泌蛋白(Cysteine-rich buccal gland protein,CRBGP)也首次被证明能够抑制海马神经元和背根神经元的钠离子电流。以下针对钠离子通道疾病及其抑制剂生物学功能的最新研究进展进行分析归纳。     

脊神经损伤后钠电流的变化及其离子通道机制

目的:检测脊神经切断大鼠背根节(DRG)神经元重复放电能力和钠电流的变化,并研究介导其电流变化的钠通道亚型的表达情况。方法:脊神经切断术后2～8d慢性痛大鼠模型背根节急性分离,对中等直径DRG神经元运用全细胞膜片钳技术记录神经元放电和钠电流的变化。对背根节神经元进行RT-PCR检测,分析其钠通道亚型的表达情况。结果:电流钳下,实验组DRG神经元在电流刺激下产生重复放电,而对照组神经元多诱发单个动作电位,电压钳记录发现实验组背根节神经元快钠电流和持续性钠电流幅值均明显大于对照组,PCR结果显示,Nav1.3、Nav1.7和Nav1.8通道亚型mRNA表达显著增高。结论:钠通道介导了脊神经受损模型的DRG神经元兴奋性增高,持续性钠电流可能通过调节阈下膜电位振荡的产生调节神经元兴奋性。     

脊神经损伤后钠电流的变化及其离子通道机制

目的：检测脊神经切断大鼠背根节（DRG）神经元重复放电能力和钠电流的变化,并研究介导其电流变化的钠通道亚型的表达情况。方法：脊神经切断术后2～8d慢性痛大鼠模型背根节急性分离,对中等直径DRG神经元运用全细胞膜片钳技术记录神经元放电和钠电流的变化。对背根节神经元进行RT-PCR检测,分析其钠通道亚型的表达情况。结果：电流钳下,实验组DRG神经元在电流刺激下产生重复放电,而对照组神经元多诱发单个动作电位,电压钳记录发现实验组背根节神经元快钠电流和持续性钠电流幅值均明显大于对照组,PCR结果显示,Nav1.3、Nav1.7和Nav1.8通道亚型mRNA表达显著增高。结论：钠通道介导了脊神经受损模型的DRG神经元兴奋性增高,持续性钠电流可能通过调节阈下膜电位振荡的产生调节神经元兴奋性。     

电压门控钠离子通道β亚基的研究

电压门控钠离子通道对Na+的选择性通透是神经元等兴奋性细胞产生动作电位的基础。该通道为跨膜蛋白,主要是由形成孔道的α亚基和一个或几个辅助性的β亚基组成,近年来发现,β亚基对α亚基的调节主要是在调节钠通道的膜上表达和亚细胞定位方面。由于β亚基的突变不仅能够引起动作电位的传导异常,导致神经元功能障碍,引发多种心脏系统疾病,包括恶性心律失常、Brugada综合征、QT间期延长综合征及其他传导性疾病,还能引起亨廷顿病(Huntigton’s diaease,HD)等神经系统疾病。本文就近几年钠离子通道β亚基生理功能的研究及其突变体与疾病的关系等方面作一阐述。     

昆虫钠离子通道的研究进展

昆虫只有一个或两个电压门控钠离子通道α亚基基因,但两种转录后修饰(选择性剪切和RNA编辑)实现了昆虫钠离子通道的功能多样性。昆虫β辅助亚基TipE和TEH1-4在钠离子通道表达和调控中也起着重要作用。电压门控钠离子通道在动作电位的产生和传递中至关重要,是多种天然和人工合成神经毒素及杀虫剂的作用靶标,包括广泛使用的拟除虫菊酯类、茚虫威和氰氟虫腙等杀虫剂。其中,拟除虫菊酯类杀虫剂通过调控昆虫钠离子通道的失活和去激活,延长跨膜钠离子流的时间,引起神经兴奋性传导障碍;茚虫威和氰氟虫腙阻断昆虫中枢和外周神经系统神经元的动作电位传导,这些神经毒剂都能干扰昆虫钠离子通道的正常功能。昆虫钠离子通道一般存在两个拟除虫菊酯类杀虫剂结合位点,但不同物种钠离子通道与拟除虫菊酯的结合位点存在一定差异。据此,本文就昆虫钠离子通道及其与杀虫剂的相互作用加以综述,有望推动昆虫神经受体研究,且对鉴定昆虫抗药性相关突变位点和研发高效的杀虫剂均具有重要参考价值。     

电压门控钠离子通道疾病的研究进展

细胞膜上的电压门控钠离子通道（Voltage-gated Sodium Channels,VGSCs）是细胞形成动作电位过程中重要的组成构件,由一个大的α亚基和一个或多个不同的β亚基组成,中央是具高度选择性只允许钠离子通过的亲水通道。电压门控钠离子通道在调节细胞膜电位、维持细胞离子稳态、细胞增殖和凋亡等生理过程中发挥着重要作用,因而钠离子通道自身的异变或是相关基因的变异都可能引起一系列身体病变。本文主要介绍了电压门控钠离子通道的结构与功能,阐述了其与癌细胞侵袭转移和神经病理性疼痛的关系,并介绍了几种典型的由钠离子通道基因变异引起的疾病。随着对电压门控钠离子通道及其异常分子机制研究的不断深入,新成果将为生理学、药理学和病理学等领域的研究提供理论基础和新的研究思路,为离子通道疾病的临床预防、诊断与治疗找到新途径。     

痛觉相关钠离子通道研究进展

疼痛是一种与组织损伤或潜在的损伤相关的不愉快的主观感觉和情感体验,是机体受到伤害性刺激后产生的一种防御反应。伤害性感觉神经元细胞膜上的电压门控钠离子通道是细胞表面一类跨膜糖蛋白,负责可兴奋细胞动作电位的产生和传导,并且在炎性痛、神经病理性疼痛和功能性痛的产生、传导以及维持上起到了重要的作用,成为近年来疼痛病理生理机制研究和疼痛治疗的分子靶标。本文将就痛觉相关钠离子通道的类型,结构,及其表达和功能的改变与疼痛的关系进行综述。     

白介素1β对大鼠皮层神经元钠电流的急性作用

白介素1β(Interleukin-1β,IL-1β)是重要的促炎细胞因子,在多种中枢神经系统的损伤和疾病过程中发挥关键作用。电压门控的钠通道是神经元中最重要的离子通道之一,是产生再生性动作电位的基础,决定了神经元的兴奋性等电学性质,也与多种中枢疾病过程相关。然而,现在还没有直接关于IL-1β与中枢钠通道的相互关系的研究。在该研究中,使用全细胞膜片钳记录测定了IL-1β对培养的皮层神经元钠电流的急性作用,并分析了由此对动作电位的影响。结果显示,IL-1β对钠电流幅度只有较小的抑制,而显著降低钠通道的半激活电压,不改变激活的斜率因子和失活性质,这个作用引起动作电位阈值显著降低。这些结果提示在损伤和疾病过程中,快速释放的IL-1β可能会增加神经元兴奋性,从而恶化神经损伤过程。     

电压门控钠离子通道与疼痛症

疼痛是一种常见的疾病和临床症状,有时会严重影响患者的生活质量,因此,疼痛的研究、治疗具有重要的实际意义。电压门控钠离子通道在神经元动作电位的起始和传导中起着关键作用,尤其是亚型Nav1.3、Nav1.7、Nav1.8和Nav1.9,它们广泛存在于背根神经节中,参与了疼痛的形成。其中,Nav1.7的基因突变会导致多种遗传性疾病。因此,这些亚型是潜在的、理想的疼痛治疗靶点。主要对电压门控钠离子通道与疼痛有关的最新研究进展进行了综述。     

Mapping protein interactions of sodium channel NaV1.7 using epitope‐tagged gene‐targeted mice

The voltage‐gated sodium channel Na_V1.7 plays a critical role in pain pathways. We generated an epitope‐tagged Na_V1.7 mouse that showed normal pain behaviours to identify channel‐interacting proteins. Analysis of Na_V1.7 complexes affinity‐purified under native conditions by mass spectrometry revealed 267 proteins associated with Nav1.7 in vivo. The sodium channel β3 (Scn3b), rather than the β1 subunit, complexes with Nav1.7, and we demonstrate an interaction between collapsing‐response mediator protein (Crmp2) and Nav1.7, through which the analgesic drug lacosamide regulates Nav1.7 current density. Novel Na_V1.7 protein interactors including membrane‐trafficking protein synaptotagmin‐2 (Syt2), L‐type amino acid transporter 1 (Lat1) and transmembrane P24‐trafficking protein 10 (Tmed10) together with Scn3b and Crmp2 were validated by co‐immunoprecipitation (Co‐IP) from sensory neuron extract. Nav1.7, known to regulate opioid receptor efficacy, interacts with the G protein‐regulated inducer of neurite outgrowth (Gprin1), an opioid receptor‐binding protein, demonstrating a physical and functional link between Nav1.7 and opioid signalling. Further information on physiological interactions provided with this normal epitope‐tagged mouse should provide useful insights into the many functions now associated with the Na_V1.7 channel.     

Voltage-gated sodium channels and pain pathways

Acute, inflammatory, and neuropathic pain can all be attenuated or abolished by local treatment with sodium channel blockers such as lidocaine. The peripheral input that drives pain perception thus depends on the presence of functional voltage-gated sodium channels. Remarkably, two voltage-gated sodium channel genes (Nav1.8 and Nav1.9) are expressed selectively in damage-sensing peripheral neurons, while a third channel (Nav1.7) is found predominantly in sensory and sympathetic neurons. An embryonic channel (Nav1.3) is also upregulated in damaged peripheral nerves and associated with increased electrical excitability in neuropathic pain states. A combination of antisense and knock-out studies support a specialized role for these sodium channels in pain pathways, and pharmacological studies with conotoxins suggest that isotype-specific antagonists should be feasible. Taken together, these data suggest that isotype-specific sodium channel blockers could be useful analgesics.     

Characterization of a new class of potent inhibitors of the voltage-gated sodium channel Nav1.7

Voltage-gated sodium channels (Nav1) transmit pain signals from peripheral nociceptive neurons, and blockers of these channels have been shown to ameliorate a number of pain conditions. Because these drugs can have adverse effects that limit their efficacy, more potent and selective Nav1 inhibitors are being pursued. Recent human genetic data have provided strong evidence for the involvement of the peripheral nerve sodium channel subtype, Nav1.7, in the signaling of nociceptive information, highlighting the importance of identifying selective Nav1.7 blockers for the treatment of chronic pain. Using a high-throughput functional assay, novel Nav1.7 blockers, namely, the 1-benzazepin-2-one series, have recently been identified. Further characterization of these agents indicates that, in addition to high-affinity inhibition of Nav1.7 channels, selectivity against the Nav1.5 and Nav1.8 subtypes can also be achieved within this structural class. The most potent, nonselective member of this class of Nav1.7 blockers has been radiolabeled with tritium. [3H]BNZA binds with high affinity to rat brain synaptosomal membranes (Kd = 1.5 nM) and to membranes prepared from HEK293 cells stably transfected with hNav1.5 (Kd = 0.97 nM). In addition, and for the first time, high-affinity binding of a radioligand to hNav1.7 channels (Kd = 1.6 nM) was achieved with [3H]BNZA, providing an additional means for identifying selective Nav1.7 channel inhibitors. Taken together, these data suggest that members of the novel 1-benzazepin-2-one structural class of Nav1 blockers can display selectivity toward the peripheral nerve Nav1.7 channel subtype, and with appropriate pharmacokinetic and drug metabolism properties, these compounds could be developed as analgesic agents.     

The role of sodium channels in neuropathic pain

Our knowledge of the ion channels, receptors and signalling mechanisms involved in pain pathophysiology, and which specific channels play a role in subtypes of pain such as neuropathic and inflammatory pain, has expanded considerably in recent years. It is now clear that in the neuropathic state the expression of certain channels is modified, and that these changes underlie the plasticity of responses that occur to generate inappropriate pain signals from normally trivial inputs. Pain is modulated by a subset of the voltage-gated sodium channels, including Nav1.3, Nav1.7, Nav1.8 and Nav1.9. These isoforms display unique expression patterns within specific tissues, and are either up- or down-regulated upon injury to the nervous system. Here we describe our current understanding of the roles of sodium channels in pain and nociceptive information processing, with a particular emphasis on neuropathic pain and drugs useful for the treatment of neuropathic pain that act through mechanisms involving block of sodium channels. One of the future challenges in the development of novel sodium channel blockers is to design and synthesise isoform-selective channel inhibitors. This should provide substantial benefits over existing pain treatments.     

Matrix Metalloproteinase (MMP) Proteolysis of the Extracellular Loop of Voltage-gated Sodium Channels and Potential Alterations in Pain Signaling

Congenital insensitivity to pain (CIP) or congenital analgesia is a rare monogenic hereditary condition. This disorder is characterized by the inability to perceive any form of pain. Nonsense mutations in Nav.1.7, the main pain signaling voltage-gated sodium channel, lead to its truncations and, consequently, to the inactivation of the channel functionality. However, a non-truncating homozygously inherited missense mutation in a Bedouin family with CIP (Nav1.7-R907Q) has also been reported. Based on our currently acquired in-depth knowledge of matrix metalloproteinase (MMP) cleavage preferences, we developed the specialized software that predicts the presence of the MMP cleavage sites in the peptide sequences. According to our in silico predictions, the peptide sequence of the exposed extracellular unstructured region linking the S5–S6 transmembrane segments in the DII domain of the human Nav1.7 sodium channel is highly sensitive to MMP-9 proteolysis. Intriguingly, the CIP R907Q mutation overlaps with the predicted MMP-9 cleavage site sequence. Using MMP-9 proteolysis of the wild-type, CIP, and control peptides followed by mass spectrometry of the digests, we demonstrated that the mutant sequence is severalfold more sensitive to MMP-9 proteolysis relative to the wild type. Because of the substantial level of sequence homology among sodium channels, our data also implicate MMP proteolysis in regulating the cell surface levels of the Nav1.7, Nav1.6, and Nav1.8 channels, but not Nav1.9. It is likely that the aberrantly accelerated MMP-9 proteolysis during neurogenesis is a biochemical rational for the functional inactivation in Nav1.7 and that the enhanced cleavage of the Nav1.7-R907Q mutant is a cause of CIP in the Bedouin family.     

Temperature dependence of erythromelalgia mutation L858F in sodium channel Nav1.7

Background

The disabling chronic pain syndrome erythromelalgia (also termed erythermalgia) is characterized by attacks of burning pain in the extremities induced by warmth. Pharmacological treatment is often ineffective, but the pain can be alleviated by cooling of the limbs. Inherited erythromelalgia has recently been linked to mutations in the gene SCN9A, which encodes the voltage-gated sodium channel Nav1.7. Nav1.7 is preferentially expressed in most nociceptive DRG neurons and in sympathetic ganglion neurons. It has recently been shown that several disease-causing erythromelalgia mutations alter channel-gating behavior in a manner that increases DRG neuron excitability.

Results

Here we tested the effects of temperature on gating properties of wild type Nav1.7 and mutant L858F channels. Whole-cell voltage-clamp measurements on wild type or L858F channels expressed in HEK293 cells revealed that cooling decreases current density, slows deactivation and increases ramp currents for both mutant and wild type channels. However, cooling differentially shifts the midpoint of steady-state activation in a depolarizing direction for L858F but not for wild type channels.

Conclusion

The cooling-dependent shift of the activation midpoint of L858F to more positive potentials brings the threshold of activation of the mutant channels closer to that of wild type Nav1.7 at lower temperatures, and is likely to contribute to the alleviation of painful symptoms upon cooling in affected limbs in patients with this erythromelalgia mutation.

     

Inherited Pain: SODIUM CHANNEL NAV1.7 A1632T MUTATION CAUSES ERYTHROMELALGIA DUE TO A SHIFT OF FAST INACTIVATION*

Inherited erythromelalgia (IEM) causes debilitating episodic neuropathic pain characterized by burning in the extremities. Inherited “paroxysmal extreme pain disorder” (PEPD) differs in its clinical picture and affects proximal body areas like the rectal, ocular, or jaw regions. Both pain syndromes have been linked to mutations in the voltage-gated sodium channel Nav1.7. Electrophysiological characterization shows that IEM-causing mutations generally enhance activation, whereas mutations leading to PEPD alter fast inactivation. Previously, an A1632E mutation of a patient with overlapping symptoms of IEM and PEPD was reported (Estacion, M., Dib-Hajj, S. D., Benke, P. J., Te Morsche, R. H., Eastman, E. M., Macala, L. J., Drenth, J. P., and Waxman, S. G. (2008) NaV1.7 Gain-of-function mutations as a continuum. A1632E displays physiological changes associated with erythromelalgia and paroxysmal extreme pain disorder mutations and produces symptoms of both disorders. J. Neurosci. 28, 11079–11088), displaying a shift of both activation and fast inactivation. Here, we characterize a new mutation of Nav1.7, A1632T, found in a patient suffering from IEM. Although transfection of A1632T in sensory neurons resulted in hyperexcitability and spontaneous firing of dorsal root ganglia (DRG) neurons, whole-cell patch clamp of transfected HEK cells revealed that Nav1.7 activation was unaltered by the A1632T mutation but that steady-state fast inactivation was shifted to more depolarized potentials. This is a characteristic normally attributed to PEPD-causing mutations. In contrast to the IEM/PEPD crossover mutation A1632E, A1632T failed to slow current decay (i.e. open-state inactivation) and did not increase resurgent currents, which have been suggested to contribute to high-frequency firing in physiological and pathological conditions. Reduced fast inactivation without increased resurgent currents induces symptoms of IEM, not PEPD, in the new Nav1.7 mutation, A1632T. Therefore, persistent and resurgent currents are likely to determine whether a mutation in Nav1.7 leads to IEM or PEPD.     

Erythromelalgia mutation L823R shifts activation and inactivation of threshold sodium channel Nav1.7 to hyperpolarized potentials

Erythromelalgia (also termed erythermalgia) is a neuropathic pain syndrome, characterized by severe burning pain combined with redness in the extremities, triggered by mild warmth. The inherited form of erythromelalgia (IEM) has recently been linked to mutations in voltage-gated sodium channel Nav1.7, which is expressed in peripheral nociceptors. Here, we used whole-cell voltage-clamp recordings in HEK293 cells to characterize the IEM mutation L823R, which introduces an additional positive charge into the S4 voltage sensor of domain II. The L823R mutation produces a ∼15 mV hyperpolarizing shift in the midpoint of activation and also affects the activation slope factor. Closing of the channel from the open state (deactivation) is slowed, increasing the likelihood of the channel remaining in the open state. The L823R mutation induces a ∼10 mV hyperpolarizing shift in fast-inactivation. L823R is the only naturally-occurring IEM mutation studied thus far to shift fast-inactivation to more negative potentials. We conclude that introduction of an additional charge into the S4 segment of domain II of Nav1.7 leads to a pronounced hyperpolarizing shift of activation, a change that is expected to increase nociceptor excitability despite the hyperpolarizing shift in fast-inactivation, which is unique among the IEM mutations.     

Size matters: Erythromelalgia mutation S241T in Nav1.7 alters channel gating

The Nav1.7 sodium channel is preferentially expressed in most nociceptive dorsal root ganglion neurons and in sympathetic neurons. Inherited erythromelalgia (IEM, also known as erythermalgia), an autosomal dominant neuropathy characterized by burning pain in the extremities in response to mild warmth, has been linked to mutations in Nav1.7. Recently, a substitution of Ser-241 by threonine (S241T) in the domain I S4-S5 linker of Nav1.7 was identified in a family with IEM. To investigate the possible causative role of this mutation in the pathophysiology of IEM, we used whole-cell voltage-clamp analysis to study the effects of S241T on Nav1.7 gating in HEK293 cells. We found a hyperpolarizing shift of activation midpoint by 8.4 mV, an accelerated time to peak, slowing of deactivation, and an increase in the current in response to small, slow depolarizations. Additionally, S241T produced an enhancement of slow inactivation, shifting the midpoint by -12.3 mV. Because serine and threonine have similar biochemical properties, the S241T substitution suggested that the size of the side chain at this position affected channel gating. To test this hypothesis, we investigated the effect of S241A and S241L substitutions on the gating properties of Nav1.7. Although S241A did not alter the properties of the channel, S241L mimicked the effects of S241T. We conclude that the linker between S4 and S5 in domain I of Nav1.7 modulates gating of this channel, and that a larger side chain at position 241 interferes with its gating mechanisms.     

Chemical shift perturbation mapping of the Ubc9-CRMP2 interface identifies a pocket in CRMP2 amenable for allosteric modulation of Nav1.7 channels

Drug discovery campaigns directly targeting the voltage-gated sodium channel NaV1.7, a highly prized target in chronic pain, have not yet been clinically successful. In a differentiated approach, we demonstrated allosteric control of trafficking and activity of NaV1.7 by prevention of SUMOylation of collapsin response mediator protein 2 (CRMP2). Spinal administration of a SUMOylation incompetent CRMP2 (CRMP2 K374A) significantly attenuated pain behavior in the spared nerve injury (SNI) model of neuropathic pain, underscoring the importance of SUMOylation of CRMP2 as a pathologic event in chronic pain. Using a rational design strategy, we identified a heptamer peptide harboring CRMP2’s SUMO motif that disrupted the CRMP2-Ubc9 interaction, inhibited CRMP2 SUMOylation, inhibited NaV1.7 membrane trafficking, and specifically inhibited NaV1.7 sodium influx in sensory neurons. Importantly, this peptide reversed nerve injury-induced thermal and mechanical hypersensitivity in the SNI model, supporting the practicality of discovering pain drugs by indirectly targeting NaV1.7 via prevention of CRMP2 SUMOylation. Here, our goal was to map the unique interface between CRMP2 and Ubc9, the E2 SUMO conjugating enzyme. Using computational and biophysical approaches, we demonstrate the enzyme/substrate nature of Ubc9/CRMP2 binding and identify hot spots on CRMP2 that may form the basis of future drug discovery campaigns disrupting the CRMP2-Ubc9 interaction to recapitulate allosteric regulation of NaV1.7 for pain relief.