KCNQ2/3钾通道电流特征及M_1受体的调节作用

目的观察KCNQ2/3通道电流特征及M1受体激活对该电流的调节作用。方法以CHO细胞作为表达体系,用脂质体共转染KCNQ2、KCNQ3钾离子通道及毒蕈碱型M1受体。全细胞膜片钳方法,观察KCNQ2/3电流特征,药理学阻断剂的作用及M1受体激活对电流的调节。结果KC-NQ2/3电流呈现慢激活、低阈值、非失活、电压依赖性的外向钾离子电流特点,其激活电压的阈值在-60 mV,半数激活电压值(-26.8±1.2)mV,其去活曲线可用双指数方程拟合,fτast约101 m s;sτlow约为309 m s。该电流对4-AP,Ba2+,TEA不敏感,L inop ird ine抑制KCNQ2/3电流IC50为(6.5±0.83)μmol.L-1。乙酰胆碱激活M1受体后会可逆性地抑制KCNQ2/3电流,其抑制的IC50为(0.7±0.05)μmol.L-1。结论KCNQ2/3通道作为神经细胞M通道的分子基础,其电流特征与M电流一致,L inop ird ine对其有较强阻断作用,神经递质乙酰胆碱通过激活M1受体明显抑制该通道电流。研究KCNQ2/3通道电流特征及受体调节规律,对于理解与中枢兴奋性有关的疾病如:惊厥、癫痫、阿尔采末病等发生机制有重要指导意义。     

钾通道调节剂的研究进展

综述各种钾通道蛋白的生物化学性质、功能及其作为药物靶点的新药开发，并分类介绍新近开发的钾通道开放剂和阻滞剂。钾通道的分子生物学研究日益广泛，钾通道开放剂与阻滞剂在治疗心肌、血管平滑肌、神经、免疫与分泌系统疾病中起着重要作用。     

神经系统疾病的新药靶点：KCNQ／M-钾通道

电压门控KCNQ（Kv7）K＋通道家族由5个成员组成，分别以同源或异源四聚体形式组装成慢激活和非失活型K＋通道。KCNQ／M-通道（主要是KCNQ2／3）在疼痛传递通路和中枢神经系统呈特异性分布。KCNQ／M-通道通过影响后超极化电位调节神经元的兴奋性，与神经病理痛、癫痫、学习与记忆，[第一段]     

KCNQ1通道的结构和功能

该文系统总结了近年来关于KCNQ1通道的研究进展。首先介绍了α、β亚基的结构以及二者之间的相互作用。其次详尽阐述了通道复合体在生物体内的生理功能,如:通道功能异常造成的多种遗传性心率失常疾病和在各种上皮细胞中的分泌功能。最后介绍了一些关于KCNQ1通道阻断剂和开放剂的研究。     

钾通道开放剂研究进展

有关钾通道的研究已有半个世纪的历史，以往对钾通道的认识多数局限在细胞膜的钾平衡电位上。另外，钾通道的多态性，特别是同一细胞上存在多种类型的钾通道，因而给本领域的研究带来了许多困难。近10年来，由于发现了一些动物毒素（如α－pamin，charybditoxin等）具有特异性的阻断钾通道作用，从而使研究者能揭示出不同类型钾通道的生理功能；加之实验技术及方法学的改进，尤其是全细胞电压钳及离子单通道记录技术的应用［’j，以及在过去5年里利用分子生物学技术，对有功能的细胞膜钾通道的克隆及基因表达，更加拓宽了人们对钾通道的了解…     

双孔钾通道TREK-1中枢神经系统药理学研究进展

双孔钾离子通道(two-pore domain potassium channels, K₂P)是20世纪90年代发现的一类钾离子通道亚家族,其中TREK-1是研究最为广泛的一种K₂P亚型。TREK-1广泛存在于机体内,特别是在中枢神经系统中高表达,它的主要作用是控制细胞兴奋性并维持膜电位低于去极化阈值,因此参与调节多种生理和病理过程。TREK-1也是多种疾病潜在的药物作用靶标,很多已上市药物可影响TREK-1的功能,但目前缺少特异性的TREK-1调节剂和以TREK-1为靶点的药物。本文在介绍TREK-1的结构、分布以及调控的基础上,重点介绍近年来TREK-1在神经保护、癫痫、抑郁以及麻醉等方面的药理学研究进展和相关的TREK-1调节剂的研究现状。     

钾通道药理的研究进展

钾通道药理的研究进展陈志刚王佩徐建华（浙江医科大学药学系药理学教研室，杭州３１００３１）中国图书分类号Ｒ９７１；Ｒ９７２．４；Ｒ９７４．３钾通道是一类分布很广的膜离子通道。８０年代以来，由于电压钳和单细胞通道记录等新技术的应用，对Ｋ＋通道的认识逐渐深...     

靶向电压门控KCNQ/M钾通道的调节剂对神经精神疾病的潜在治疗作用

1980年David Brown博士首次发现了M型乙酰胆碱受体激动剂可以抑制一种钾通道电流,并将其命名为M型钾电流（IM）十八年后科学家们克隆了导致新生儿癫痫的KCNQ2和KCNQ3突变基因,并且证明KCNQ2和KCNQ3亚基异源组装介导M型钾通道电流。电压门控KCNQ2/3钾通道（亦称M通道）功能降低可导致神经元的过度兴奋,诱发癫痫及慢性疼痛。因此,研发激活KCNQ2/3通道功能的开放剂可用于治疗癫痫和疼痛等神经疾病。著名的KCNQ通道开放剂瑞替加滨retigabine（商品名Potiga）于2011年被美国食品与药品监督管理局批准治疗癫痫。我们最近参与研发了一类新型的KCNQ2/3特异性开放剂—吡唑并[1,5-a]嘧啶酮类化合物。在动物模型上,该开放剂具有抗癫痫和镇痛的效果。最近的研究工作提示,KCNQ通道开放剂还对抑郁症、焦虑症和精神分裂症等动物模型有效,而KCNQ通道抑制剂可以提高动物的学习与记忆能力。目前的研究进展提示,电压门控KCNQ/M通道是治疗神经精神类疾病的靶点,靶向该通道的调节剂对多种神经精神疾病具有潜在的治疗价值。     

钾通道在维拉帕米抑制上皮钠通道中的作用

目的探讨钾通道在维拉帕米引起的肺水肿中的作用。方法应用尤斯灌流室技术检测钾通道阻滞剂对维拉帕米抑制上皮钠通道的作用。Clofilium,clotrimazole以及glibenclamide(分别为KvLQT1,KCa和KATP通道的阻滞剂)加入H441单层细胞基底侧后,记录短路电流。同时,在穿孔细胞层中观察了维拉帕米对Na+/K+泵的作用。结果在穿孔H441细胞层中观察到维拉帕米对上皮钠通道和Na+/K+泵均有抑制作用。Clofilium,clotri mazole以及glibenclamide分别抑制短路电流至54.4±4.6%,32.1±5.2%,和20.5±1.1%(n=5)。用上述三种钾通道阻滞剂预处理后,维拉帕米对短路电流的作用由对照组的45.9±4.8%分别降低至23.7±4.3%,34.2±2.6%和36.0±2.8%(n=4)。Clofilium显示了对短路电流的最大抑制,与其它两种抑制剂相比具有统计学差异。应用clofilium后加入维拉帕米与对照组相比,其抑制率具有明显统计学差异(P<0.05)。结论这些结果表明KCa,KATP,尤其是KvLQT1通道在肺上皮细胞维拉帕米引起的非心源性肺水肿的Na+和Cl-转运的病理生理过程中发挥重要作用。     

钾通道开放剂药理作用研究进展

目的:综述钾通道开放剂药理作用的研究进展.方法:依据文献,介绍钾通道开放剂的药理作用.结果:钾通道开放剂药理作用广泛,但选择性较差.结论:钾通道开放剂具有良好的临床应用前景,应进一步开发选择性较高的钾通道开放剂.     

The acrylamide (S)-1 differentially affects Kv7 (KCNQ) potassium channels

The family of Kv7 (KCNQ) potassium channels consists of five members. Kv7.2 and 3 are the primary molecular correlates of the M-current, but also Kv7.4 and Kv7.5 display M-current characteristics. M-channel modulators include blockers (e.g., linopirdine) for cognition enhancement and openers (e.g., retigabine) for treatment of epilepsy and neuropathic pain. We investigated the effect of a Bristol-Myers Squibb compound (S)-N-[1-(3-morpholin-4-yl-phenyl)-ethyl]-3-phenyl-acrylamide [(S)-1] on cloned human Kv7.1-5 potassium channels expressed in Xenopus laevis oocytes. Using two-electrode voltage-clamp recordings we found that (S)-1 blocks Kv7.1 and Kv7.1/KCNE1 currents. In contrast, (S)-1 produced a hyperpolarizing shift of the activation curve for Kv7.2, Kv7.2/Kv7.3, Kv7.4 and Kv7.5. Further, the compound enhanced the maximal current amplitude at all potentials for Kv7.4 and Kv7.5 whereas the combined activation/block of Kv7.2 and Kv7.2/3 was strongly voltage-dependent. The tryptophan residue 242 in S5, known to be crucial for the effect of retigabine, was also shown to be critical for the enhancing effect of (S)-1 and BMS204352. Furthermore, no additive effect on Kv7.4 current amplitude was observed when both retigabine and (S)-1 or BMS204352 were applied simultaneously. In conclusion, (S)-1 differentially affects the Kv7 channel subtypes and is dependent on a single tryptophan for the current enhancing effect in Kv7.4.     

KCNQ potassium channels in sensory system and neural circuits

M channels, an important regulator of neural excitability, are composed of four subunits of the Kv7 (KCNQ) K⁺ channel family. M channels were named as such because their activity was suppressed by stimulation of muscarinic acetylcholine receptors. These channels are of particular interest because they are activated at the subthreshold membrane potentials. Furthermore, neural KCNQ channels are drug targets for the treatments of epilepsy and a variety of neurological disorders, including chronic and neuropathic pain, deafness, and mental illness. This review will update readers on the roles of KCNQ channels in the sensory system and neural circuits as well as discuss their respective mechanisms and the implications for physiology and medicine. We will also consider future perspectives and the development of additional pharmacological models, such as seizure, stroke, pain and mental illness, which work in combination with drug-design targeting of KCNQ channels. These models will hopefully deepen our understanding of KCNQ channels and provide general therapeutic prospects of related channelopathies.     

KCNQ potassium channels: drug targets for the treatment of epilepsy and pain

Epilepsy and neuropathic pain are disorders characterised by excessive neuronal activity. These disorders are currently managed by drugs that are capable of dampening neuronal excitability, including voltage-gated sodium channel blockers, voltage-operated calcium channel modulators and modulators of inhibitory GABAergic neurotransmission. However, these drugs are rarely 100% efficacious and their use is often associated with limiting side effects. Thus, there is a clear medical need for novel agents to treat these diseases. One potential mechanism that has not yet been exploited is potassium (K⁺) channel opening. A significant (and growing) body of genetic, molecular, physiological and pharmacological evidence now exists to indicate that KCNQ-based currents represent particularly interesting targets for the treatment of diseases such as epilepsy and neuropathic pain. Evidence supporting these K⁺ channels as novel drug targets will be reviewed in the following article. Worldwide patent activity relating to KCNQ channels and KCNQ-modulating drugs and their uses will also be summarised.     

细胞外pH值对KCNQ2/3钾离子通道的调节

目的　研究细胞外pH值改变对KCNQ2 /3钾离子通道的调节。方法　用体外转录法将KCNQ2 /3钾离子通道的mRNA,微注射于爪蟾卵母细胞中,表达KCNQ2 /3电流。用双电极电压钳方法 (TEVC)观察细胞外pH值对KCNQ2 /3通道的调节。结果　细胞外pH减小,抑制KCNQ2 /3电流,影响激活电压,而且这种调节有一定的电压和浓度依赖性。在阈电压附近 -60mV、pH 6 8时对KCNQ2 /3通道的影响最明显。pH值改变还会调节通道开放时程,影响通道激活时间常数。结论　在维持神经细胞静息电位、调节神经兴奋性方面起重要作用的KCNQ2 /3通道,受细胞外pH值调节,这种调节作用可能在神经细胞生理性兴奋和病理性变化中如:惊厥、癫痫、休克有重要意义。     

药物对hERG钾通道作用机制研究进展

人ether-a-go-go-related gene(hERG)钾通道表达了延迟整流钾电流的快激活成分,对动作电位的复极至关重要。hERG钾电流不仅是抗心律失常作用的主要靶点,也是诸多药物增加尖端扭转型室速和心源性猝死风险的关键位点,而该电流的降低和(或)升高与基因突变或药物阻滞作用密切相关。随着对药物与hERG钾通道相互作用机制研究的深入,药物与通道孔道区蛋白结合位点的作用及其对通道转运的影响逐步被揭示,但这些药物对hERG作用的临床应用仍有待评价。     

The new KCNQ2 activator 4-Chlor-N-(6-chlor-pyridin-3-yl)-benzamid displays anticonvulsant potential

Background and Purpose

KCNQ2-5 channels are voltage-gated potassium channels that regulate neuronal excitability and represent suitable targets for the treatment of hyperexcitability disorders. The effect of Chlor-N-(6-chlor-pyridin-3-yl)-benzamid was tested on KCNQ subtypes for its ability to alter neuronal excitability and for its anticonvulsant potential.

Experimental Approach

The effect of 4-Chlor-N-(6-chlor-pyridin-3-yl)-benzamid was evaluated using whole-cell voltage-clamp recordings from CHO cells and Xenopus laevis oocytes expressing different types of KCNQ channels. Epileptiform afterdischarges were recorded in fully amygdala-kindled rats in vivo. Neuronal excitability was assessed using field potential and whole cell recording in rat hippocampus in vitro.

Key Results

4-Chlor-N-(6-chlor-pyridin-3-yl)-benzamid caused a hyperpolarizing shift of the activation curve and a pronounced slowing of deactivation in KCNQ2-mediated currents, whereas KCNQ3/5 heteromers remained unaffected. The effect was also apparent in the Retigabine-insensitive mutant KCNQ2-W236L. In fully amygdala-kindled rats, it elevated the threshold for induction of afterdischarges and reduced seizure severity and duration. In hippocampal CA1 cells, 4-Chlor-N-(6-chlor-pyridin-3-yl)-benzamid strongly damped neuronal excitability caused by a membrane hyperpolarization and a decrease in membrane resistance and induced an increase of the somatic resonance frequency on the single cell level, whereas synaptic transmission was unaffected. On the network level, 4-Chlor-N-(6-chlor-pyridin-3-yl)-benzamid caused a significant reduction of γ and θ oscillation peak power, with no significant change in oscillation frequency.

Conclusion and Implications

Our data indicate that 4-Chlor-N-(6-chlor-pyridin-3-yl)-benzamid is a potent KCNQ activator with a selectivity for KCNQ2 containing channels. It strongly reduces neuronal excitability and displays anticonvulsant activity in vivo.     

Discovery of a retigabine derivative that inhibits KCNQ2 potassium channels

Aim:

Retigabine, an activator of KCNQ2-5 channels, is currently used to treat partial-onset seizures. The aim of this study was to explore the possibility that structure modification of retigabine could lead to novel inhibitors of KCNQ2 channels, which were valuable tools for KCNQ channel studies.

Methods:

A series of retigabine derivatives was designed and synthesized. KCNQ2 channels were expressed in CHO cells. KCNQ2 currents were recorded using whole-cell voltage clamp technique. Test compound in extracellular solution was delivered to the recorded cell using an ALA 8 Channel Solution Exchange System.

Results:

A total of 23 retigabine derivatives (HN31-HN410) were synthesized and tested electrophysiologically. Among the compounds, HN38 was the most potent inhibitor of KCNQ2 channels (its IC₅₀ value=0.10±0.05 μmol/L), and was 7-fold more potent than the classical KCNQ inhibitor XE991. Further analysis revealed that HN38 (3 μmol/L) had no detectable effect on channel activation, but accelerated deactivation at hyperpolarizing voltages. In contrast, XE991 (3 μmol/L) did not affect the kinetics of channel activation and deactivation.

Conclusion:

The retigabine derivative HN38 is a potent KCNQ2 inhibitor, which differs from XE991 in its influence on the channel kinetics. Our study provides a new strategy for the design and development of potent KCNQ2 channel inhibitors.     

The therapeutic potential of neuronal KV7 (KCNQ) channel modulators: an update

Background: Neuronal KCNQ channels (K_V7.2-5) represent attractive targets for the development of therapeutics for chronic and neuropathic pain, migraine, epilepsy and other neuronal hyperexcitability disorders, although there has been only modest progress in translating this potential into useful therapeutics. Objective: Compelling evidence of the importance of K_V7 channels as neuronal regulatory elements, readily amenable to pharmacological modulation, has sustained widespread interest in these channels as drug targets. This review will update readers on key aspects of the characterization of these important ion channel targets, and will discuss possible current barriers to their exploitation for CNS therapeutics. Methods: This article is based on a review of recent literature, with a focus on data pertaining to the roles of these channels in neurophysiology. In addition, I review some of the regulatory elements that influence the channels and how these may relate to channel pharmacology, and present a review of recent advances in neuronal K_V7 channel pharmacology. Conclusions: These channels continue to be valid and approachable targets for CNS therapeutics. However, we may need to understand more about the roles of neuronal K_V7 channels during the development of disease states, as well as to pay more attention to a detailed analysis of the molecular pharmacology of the different channel subfamily members and the modes of interaction of individual modulators, in order to successfully target these channels for therapeutic development.