受体及药物引起的M/KCNQ钾通道、钙激活氯通道及内向整流钾通道的调控作用及其生理学意义
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摘要
M型钾离子通道介导一种具有电压及时间依赖性、慢激活、慢去活、非失活的外向钾电流。由于最初发现其能被毒蕈碱M1受体激活后所强烈抑制,故因此得名。目前认为M通道的分子基础是由KCNQ家族成员(KCNQ1-5,又称KV7.1-7.5)的单或异二聚体所构成。M通道的激活阈值位于-60 mV左右,这一特征使得M通道能够在细胞静息膜电位水平附近开放。目前人们认为神经元中M电流可以发挥一种类似“细胞内在电压钳”的功能,即能对其静息膜电位、动作电位发放阈值等影响神经元兴奋性的因素进行调节,抑制神经元的过度兴奋。M通道在神经系统中有非常广泛的分布,其中在海马、皮层、外周感觉神经元以及一些交感神经元中均有明显表达。此外越来越多的实验证据表明,功能性的M通道在外周负责感觉传递的神经纤维中有表达,并且对神经纤维的兴奋性有重要调节作用。钙激活氯通道(CACC)是一类在包括神经元、平滑肌细胞及上皮细胞等诸多类型细胞有表达,并具有重要生理功能的离子通道。在神经系统中,嗅觉神经元、视锥与视杆细胞、味觉细胞及背根神经元中都存在功能性的CACC。CACC在神经细胞中的主要生理功能是促使神经元去极化,并对由其他离子通道引发的去极化起到加强作用,参与兴奋性的产生。直到最近才由三个实验室共同发现构成CACC的分子基础为跨膜蛋白16A (TMEM 16A),而这一发现使得人为通过基因手段调控CACC的功能与表达成为可能。
内向整流钾离子通道(Kir)包括至少7个亚家族(Kir1-7),这些通道在组织中分布广泛,能够保持细胞内外K+平衡、维持细胞膜电位,因此在动作电位复极化过程以及调节细胞兴奋性等方面发挥重要作用。近年来有文献报道,Kir2.0家族成员因其与细胞膜PIP2结合力强弱不同,因此在对一些通道调节剂的调节作用方面表现有所区别。
本研究论文以上述几种通道为出发点,利用电生理膜片钳、细胞内钙成像、小RNA干扰、原代神经元转染、双电极电压钳、激光扫描共聚焦显微镜以及动物行为学实验等技术手段,系统研究了以下几方面内容:(1)炎症介导因子缓激肽引发急性痛觉信号的离子机制;(2)炎症介导因子组胺引发KCNQ/M通道调节作用及其内在机制;(3)第一代抗组胺药物相比其他抗组胺药物在中毒剂量下更易引发严重神经系统不良反应的分子机制。论文具体内容如下:
第一部分缓激肽引发急性痛觉信号的离子机制
目的:研究缓激肽引发急性痛觉信号的离子机制,观察缓激肽对大鼠背根神经元中离子通道的调节作用。我们发现缓激肽能够抑制M型钾电流的同时激活一种内向离子电流。我们进行了一系列药理学实验和小RNA干扰实验来阐明缓激肽调节M电流的信号通路和这一内向电流的确切分子基础。通过电流钳实验我们又研究了这两种机制是否参与了缓激肽所引发的神经元兴奋性增高。最后通过动物行为学实验,我们在整体动物水平上研究了这两种机制在缓激肽引发动物急性疼痛行为中所发挥的作用。
方法:使用打孔全细胞膜片钳观察缓激肽对背根神经元离子电流的影响。使用细胞内钙成像测定细胞内钙离子浓度变化。利用激光扫描共聚焦显微镜动态观察PIP2特异性荧光探针YFP-tubby的变化过程。使用小RNA干扰技术结合神经元细胞核转染的方法探讨缓激肽引发内向电流的分子基础。利用电流钳观察缓激肽引发的神经元兴奋性,并最后通过整体动物行为学测试法,研究相应通道调节剂对缓激肽引发的急性疼痛的作用。
结果:(1)缓激肽抑制小背根神经元中的M电流:缓激肽能够剂量依赖性地抑制小背根神经元中的M电流,其抑制作用的EC50为60.0±16.3 nM。此外对背根神经元进行纯化培养后并不影响缓激肽的抑制作用。(2)缓激肽引发M电流抑制作用的信号途径:缓激肽能够在背根神经元引发PLCδ-PH-GFP出现荧光转位,而对PIP2特异性荧光探针YFP-tubby却无作用,说明缓激肽能够激活PLC,但细胞膜PIP2水平却没有明显变化。Ca2+成像实验显示缓激肽能够引发背根神经元细胞内Ca2+浓度明显增高。膜片钳实验进一步证实,缓激肽对M电流的抑制作用受到B2受体拮抗剂Hoe-140,IP3受体拮抗剂Xe-C,SERCA抑制剂thapsigargin及高浓度BAPTA电极内液的显著影响,但并不受PLA2阻断剂quinacrine和PKC抑制剂bisindolylmaleimide影响。(3)缓激肽激活TMEM16A依赖性的CACC:我们发现在缓激肽抑制M电流的同时会引发一种明显内向电流。这一内向电流对TRP通道阻断剂钌红及HCN特异性阻断剂ZD7288均不敏感。但当把电极内液换成低Cl-内液后,这一内向电流消失;而且Cl-通道阻断剂DIDS同样能够明显抑制这一电流的出现。提示这一内向电流是由Cl-通道开放而介导的。进一步的药理学实验证明,这种内向电流受Hoe-140,thapsigargin,Xe-C及高BAPTA电极内液的影响。提示这一内向电流为CACC所介导。此外利用siRNA技术使编码CACC的Tmem 16a基因沉默后,此内向电流显著降低,而缓激肽对M电流的抑制作用不受影响。(4) M通道的抑制与Ca2+激活Cl-通道的开放共同构成了缓激肽对感觉神经元的兴奋作用:在高Cl-内液环境下,缓激肽与M通道特异性抑制剂XE991均能明显增强背根神经元的兴奋性,而缓激肽的作用要强于XE991。此外二者均能使神经元细胞膜电位明显去极化。使用低Cl-内液去除CACC作用后,XE991同样能够引发神经元兴奋性,而此时若共同给予缓激肽+ XE991后,神经元兴奋性不再增加。在低Cl-内液环境下,若先给予缓激肽,再给予缓激肽+ XE991后,XE991能够在缓激肽引发兴奋性的基础上再增加兴奋性。(5) M通道增强剂能够拮抗缓激肽引发的动物疼痛表现:在动物行为学实验中,缓激肽能够引发受试动物出现明显疼痛反应。M通道增强剂retigabine和zinc pyrithione能够有效缓解缓激肽的致痛作用,然而Cl-阻断剂DIDS却对痛觉反应无明显作用。
结论:(1)缓激肽对小背根神经元中M电流具有剂量依赖性抑制作用。这种作用与其他非神经元作用无关。(2)缓激肽能够激活PLC,但并不能造成细胞膜PIP2整体水平出现明显下降;缓激肽还能引发细胞内Ca2+浓度升高。缓激肽抑制背根神经元中M电流的作用依赖于B2受体、PLC和其引发的细胞内Ca2+信号。(3)缓激肽在抑制M电流的同时引发的内向电流为Ca2+激活Cl-电流。这一电流由Tmem 16a基因所编码的Ca2+激活Cl-通道所介导。(4)缓激肽对M电流与Ca2+激活Cl-电流的作用共同参与了其引发的背根神经元兴奋性增高。(5) M通道的特异性增强剂能够明显缓解缓激肽引发受试动物出现的疼痛症状。说明M通道在缓激肽引发的急性疼痛中发挥重要作用。
第二部分细胞膜磷脂PIP2介导组胺对KCNQ/M通道电流调节机制的研究
目的:研究组胺对HEK293表达细胞与大鼠颈上交感神经元中KCNQ/M通道电流的调节作用,并对作用机制进行信号通路的研究,并且与已知的能调节KCNQ/M电流的M1受体与BK2受体激动剂oxo-M和缓激肽进行比较,从而对G蛋白偶联受体调节KCNQ/M电流的共同机制进行更深入的了解。
方法:利用膜片钳观察组胺对电流的调节作用,利用细胞内钙成像技术测定细胞内钙离子浓度变化,并使用一系列药理学方法配合激光扫描共聚焦显微镜作动态观察,详细研究组胺调节KCNQ/M电流的信号转导通路。
结果:(1)组胺通过激活组胺H1受体,抑制外源性表达于HEK293细胞的KCNQ2/Q3与大鼠颈上神经元的M通道电流:当HEK293细胞外源性表达KCNQ2/Q3通道与H1受体后,组胺(10μM)能够强烈抑制通道电流,抑制率达到77.9±3.5%,并且这种作用并不受H2受体拮抗剂西咪替丁影响(p > 0.05 vs. control),但却能被H1受体特异性抑制剂美吡拉敏所阻断(p < 0.01 vs. control)。组胺还能对大鼠颈上神经元的M通道电流有抑制作用,这种抑制作用同样能被美吡拉敏阻断(p < 0.01 vs. control),且这一作用具有剂量依赖性,通过对不同浓度组胺所引发的M电流抑制作用的量效曲线进行分析,组胺抑制作用的EC50 = 3.3±1.1μM。此外通过Western blot的方法,我们也发现在颈上神经元中的却存在H1受体。(2)组胺对KCNQ2/Q3电流的抑制作用是由PLC激活所介导:我们发现组胺对电流的抑制作用能被PLC阻断剂U-73122显著降低,只有7.1±0.6% (p < 0.01 vs. control);同样对于颈上神经元中的M电流来说,组胺的抑制作用也能被U-73122所取消(p < 0.01 vs. control)。(3)组胺对KCNQ/M电流的抑制作用是由于细胞膜磷脂PIP2水解所造成的:PLCδ1-PH-GFP是一种常用的标记PIP2的荧光探针。通过LSCM观察我们发现,组胺可以明显引起PLCδ1-PH-GFP从胞膜到胞浆的转位,胞膜荧光强度可以增加80.4±6.0%,且这一过程在组胺洗脱后可逆。组胺的这一作用能被H1受体特异性抑制剂美吡拉敏与PLC抑制剂U-73122所阻断(p < 0.01 vs. control)。如果使用膜片钳研究我们发现,当用PI4-Kinase抑制剂wortmannin灌流HEK293细胞以阻断PIP2再合成后,由组胺引起KCNQ2/Q3电流抑制后的恢复程度便由给予wortmannin前的88.4±6.2%显著降低到10.5±3.4% (p < 0.01 vs. control)。另一种PI4-Kinase抑制剂PAO同样使电流被组胺抑制后的恢复程度从给药前的95.4±4.6%显著降低到6.3±0.9% (p < 0.01 vs. control)。(4)组胺对HEK293细胞表达的KCNQ电流的抑制作用不依赖于细胞内钙信号:我们发现组胺能够引起明显的细胞内钙浓度升高,并且在无钙外液下依旧存在,但是这种变化可以被PLC抑制剂U-73122很大程度上取消。在全细胞膜片钳记录模式,当细胞内通过长时间由微电极向细胞内导入钙离子螯合内液以缓冲钙信号后,由组胺引发的KCNQ电流抑制作用不受任何影响(81.3±4.0%,p > 0.05 vs. control)。此外细胞内钙库耗竭剂thapsigargin也不能影响组胺的作用(88.6±3.9%,p > 0.05 vs. control)。(5)组胺不能引发颈上神经元中钙离子浓度升高:测定细胞内钙离子在给予组胺后的变化,并与M1受体与BK2受体激动剂oxo-M,缓激肽的作用进行比较。缓激肽能够引发很明显的细胞内钙离子浓度升高,但oxo-M与组胺却不能引发细胞内钙变化。缓激肽, oxo-M和组胺所引起的荧光强度的升高分别为0.04±0.008, 0.03±0.004和0.32±0.05 (p < 0.01)。(6)组胺对颈上神经元M电流的抑制不依赖于细胞内钙信号:当使用含有0.1 mM BAPTA (低BAPTA)的细胞内液对颈上神经元进行全细胞渗透后,组胺、oxo-M和缓激肽均能不同程度抑制M电流,其抑制程度分别为38.3±7.9%, 98.8±0.4%和85.3±4.4%。当使用含有20 mM BAPTA + 10 mM CaCl2 (高BAPTA)的细胞内液对颈上神经元进行全细胞渗透后,缓激肽的作用便会被显著降低到20.5±2.6% (p < 0.01 vs. control),而组胺、oxo-M的作用分别为32.1±3.1%和87.3±7.0%,与对照相比没有明显变化(p > 0.05 vs. control)。
结论:(1)组胺可以通过激活H1受体抑制外源性表达于HEK293细胞上的KCNQ2/Q3与大鼠颈上神经元中的M通道电流,且这种抑制作用具有剂量依赖性。(2)组胺对KCNQ/M电流的抑制作用与PLC的激活密切相关。(3)组胺能够引起细胞膜磷脂PIP2的水解,并且其对KCNQ电流的抑制作用很可能是由于细胞膜磷脂PIP2水解造成。(4)组胺虽能引起HEK293细胞中内钙浓度升高,但是其对KCNQ电流的抑制作用并不取决于钙信号。(5)组胺对颈上神经元中M电流的抑制也不依赖于细胞内钙信号,与已知的两种G蛋白偶联受体激动剂缓激肽与oxo-M的作用机制相比,更接近于oxo-M。因此H1受体在调解M电流的机制方面与已知的M1受体非常一致。这一发现充实并完善了G蛋白偶联受体调节KCNQ/M电流的信号途径,此外组胺对神经元M电流的抑制作用对研究其作为炎症介导因子与神经递质参与并引发神经元兴奋性的机制提供了新的方向。
第三部分抗组胺药物美吡拉敏直接抑制KCNQ/M电流并且引发大鼠颈上神经元去极化
目的:研究第一代H1受体抗组胺药美吡拉敏及苯海拉明对KCNQ/M通道电流的作用及其机制,并研究其对神经元兴奋性的影响,揭示第一代抗组胺药在服用过量下所引发的神经系统不良反应的分子机制。方法:利用电生理膜片钳技术,观察美吡拉敏及苯海拉明对KCNQ/M通道电流的作用,并对通道动力学特征变化进行分析;利用全细胞膜片钳细胞内渗透给药与outside-out膜片钳相结合的实验手段确定药物作用于通道的部位;利用电流钳模式记录药物对细胞膜电位以及细胞兴奋性的影响。
结果:(1)美吡拉敏抑制KCNQ2/Q3通道电流并影响其动力学特征:利用细胞转染的方法,将KCNQ2/Q3基因外源性导入HEK293细胞。第一代H1受体抗组胺药美吡拉敏(100μM)能够强烈抑制KCNQ2/Q3电流,其对KCNQ2/Q3尾电流的抑制率达到92.0±1.7%。这种抑制作用很快,并且能在很大程度上被洗脱。这种作用具有剂量依赖性,其IC50 = 12.5±1.8μM。而H2受体拮抗剂西咪替丁则对KCNQ2/Q3电流没有任何作用。(2)美吡拉敏对通道动力学特征的影响:美吡拉敏可以延长电流激活段时间参数而缩短电流去活段时间参数,并且将通道I-V曲线半数激活电压从-16.9±0.9 mV右移至-7.8±1.4 mV。(3)苯海拉明也具有相似的抑制作用,其对KCNQ2/Q3的抑制率达到66.9±5.9%,并且也可以将通道I-V曲线右移。(4)美吡拉敏对KCNQ通道家族(KCNQ1-5)的作用:除对KCNQ2/Q3电流的抑制作用外,美吡拉敏还能够分别抑制KCNQ1,KCNQ2,KCNQ3,KCNQ4,KCNQ3/5等KCNQ通道成员,并且这种抑制作用均具有剂量依赖性。(5)美吡拉敏从细胞膜外面直接抑制KCNQ2/Q3通道电流:首先我们利用全细胞膜片钳的方法向表达有KCNQ2/Q3的HEK293细胞内部渗透美吡拉敏,研究药物是否通过细胞内部的作用部位发挥对KCNQ通道的抑制作用。在对照情况下,如果利用正常细胞内液进行细胞渗透,经过12分钟后,KCNQ2/Q3电流大小会降低到原来的56.0±9.0%;而使用美吡拉敏进行渗透后,电流会降低到原来的54.8±11.6%,这与对照组相比没有显著差别(p > 0.05)。而与熟知的通过受体激活引发PIP2水解而抑制KCNQ/M通道的M1受体激动剂oxo-M比较后我们发现,oxo-M对电流的抑制作用会受到PLC抑制剂U-73122的影响,而美吡拉敏则不受U-73122的任何影响。通过膜外面向外(outside-out)膜片钳实验我们进一步发现,美吡拉敏可以非常迅速且明显地抑制outside-out膜片钳记录下HEK293细胞的KCNQ电流,抑制率为90.1±2.8%。(6)美吡拉敏抑制大鼠颈上神经元中的M电流:100μM美吡拉敏也同样能够抑制颈上神经元中的M电流,其抑制率达到76.7±2.6%,且这种作用也具有剂量依赖性,IC50 = 25.7±0.7μM。此外美吡拉敏也能够引发颈上神经元明显的去极化,可将细胞膜电位从–51.0±5.6 mV去极化至–38.9±7.3 mV。若使用M通道的特异性抑制剂linopirdine事先作用于细胞后,美吡拉敏便不能在linopirdine持续存在的情况下进一步引发去极化作用。(7)美吡拉敏对神经元兴奋性的影响:我们发现虽然M通道的特异性抑制剂linopirdine的却能够明显引发神经元兴奋性增高,但美吡拉敏却并不能增高兴奋性。
结论:(1)第一代H1受体抗组胺药美吡拉敏及苯海拉明能够明显抑制KCNQ/M电流,这种作用是由药物在细胞膜外侧直接阻断通道而引起。(2)美吡拉敏可以改变KCNQ通道激活与去活动力学特征,并引起通道I-V曲线右移。(3)美吡拉敏剂量依赖地抑制所有KCNQ家族成员(KCNQ1-5)。(4)美吡拉敏同样抑制大鼠颈上神经元M电流,并通过这种作用引发神经元明显去极化,但美吡拉敏却并不引起神经元兴奋性改变。第一代H1受体抗组胺药美吡拉敏和苯海拉明在大剂量情况下抑制KCNQ/M电流,并且引发神经元明显去极化的作用,很可能是导致其在中毒剂量引发病人抽搐、痉挛、癫痫等神经系统毒性作用的分子机制之一。
第四部分抗组胺药物选择性抑制Kir通道
目的:研究第一、二、三代H1受体抗组胺药对内向整流钾离子通道(Kir)的作用,试图揭示第一代H1受体抗组胺药在大剂量情况下所引起的神经系统毒性反应的分子机制。
方法:利用分子克隆、RNA体外转录以及细胞显微注射等技术,在非洲爪蟾卵母细胞中,外源性表达Kir2.1,2.3,3.4及Kir2.3突变体通道蛋白,再利用双电极电压钳记录卵母细胞全细胞电流。观察大剂量情况下各种H1受体抗组胺药对通道电流的作用。
结果:(1)抗组胺药对表达于卵母细胞Kir2.3通道电流的作用:在高钾外液孵育下的Kir2.3通道呈现明显的内向整流特性,其反转电位位于0 mV附近。外液给予第一代H1受体抗组胺药美吡拉敏(100μM)或苯海拉明(100μM)后,Kir2.3通道电流均受到明显抑制,其抑制率分别为25.0±2.9%和17.3±0.7%。而第二代及第三代H1受体抗组胺药以及H2受体阻断药阿司咪唑,地氯雷他定及西咪替丁对Kir2.3通道没有任何作用。通过对通道I-V曲线进行分析,美吡拉敏对Kir2.3的抑制作用无电压依赖性。美吡拉敏及苯海拉明能剂量依赖地抑制Kir2.3电流,其IC50分别为306.4μM和689.2μM。(2)抗组胺药对表达于卵母细胞Kir2.1通道电流的作用:第一代H1受体抗组胺药美吡拉敏(100μM)及苯海拉明(100μM)对Kir2.1电流没有明显影响。同样如果给予其他类型药物,包括阿司咪唑,地氯雷他定及西咪替丁,均对Kir2.1电流没有影响。(3)抗组胺药对Kir电流的选择性作用与通道―PIP2相互作用程度有关:如果利用分子生物学方法进行点突变,将Kir2.3位于213位的异亮氨酸置换为亮氨酸(即Kir2.3(I213L)),而增强通道与细胞膜磷脂PIP2的亲和力后,我们发现美吡拉敏对通道电流的抑制作用会被明显削弱。此外通过利用另一种与PIP2结合力较弱的Kir3.4进行实验后我们发现,美吡拉敏能明显抑制其电流,抑制率为30.3±4.6%。因此美吡拉敏对Kir电流抑制率大小顺序为Kir3.4 > Kir2.3 > Kir2.3 (I213L) > Kir2.1,而这与以往关于关于Kir通道与PIP2亲和力所报道的顺序一致。(4)第一代H1受体抗组胺药通过抑制Kir2.3电流引发卵母细胞膜电位去极化:在表达了Kir2.3通道后,卵母细胞膜电位为-95.2±2.3 mV。外液给予美吡拉敏后,可以引起细胞膜电位逐渐去极化,达稳态后,膜电位去极化至-57.1±6.1 mV。苯海拉明也具有相似效应,可将卵母细胞膜电位去极化至-70.0±3.4 mV。
结论:(1)第一代H1受体抗组胺药美吡拉敏和苯海拉明能够明显抑制Kir2.3通道电流,且作用具有剂量依赖性,而第二、三代H1受体抗组胺药以及H2受体拮抗剂阿司咪唑,地氯雷他定及西咪替丁对Kir2.3没有任何作用。(2) Kir2.1通道对任何抗组胺药均不敏感。(3)美吡拉敏对Kir2.1与Kir2.3通道之间作用的明显差别很可能源于通道与PIP2的亲和力大小。美吡拉敏也同样能抑制与PIP2亲和力较弱的Kir3.4通道电流。(4)通过抑制表达于爪蟾卵母细胞的Kir2.3通道电流,美吡拉敏可以明显引发卵母细胞去极化。因此第一代H1受体抗组胺药对Kir2.3通道的抑制作用很可能也是其服用过量或中毒情况下引发神经系统毒性作用的分子机制之一。
总结
1缓激肽能够剂量依赖地抑制存在于小背根神经元中的M电流,缓激肽通过作用于其B2受体,能够激活PLC,导致由IP3介导的内Ca2+释放。由缓激肽所引发的细胞内Ca2+信号能够同时引发M电流的抑制与Ca2+激活Cl-通道的开放。这两种通道的共同作用足以引发背根神经元的去极化并导致其兴奋性的增高。在动物行为学实验中,M通道的特异性增强剂能够显著缓解由缓激肽所引发的疼痛效应。上述实验结果揭示了缓激肽引发炎症性疼痛的详细分子机制,并为炎症性疼痛的治疗提供了崭新方向。
2组胺能够抑制HEK293细胞与大鼠颈上神经元中的KCNQ/M通道电流,这一作用是通过H1受体―PLC信号通路引发的PIP2水解所造成的,而与细胞内钙信号无关。与作用已知的两种G蛋白偶联受体激动剂缓激肽与oxo-M相比,组胺对M通道抑制作用的机制更接近于oxo-M。因此H1受体在调解M电流的机制方面与M1受体非常一致。
3第一代抗组胺药物美吡拉敏与苯海拉明能够剂量依赖性的抑制外源性表达于HEK293细胞中的KCNQ2/Q3通道电流。美吡拉敏影响通道的激活、失活动力学,并使其I-V曲线显著右移。美吡拉敏除对KCNQ2/Q3电流有抑制作用外,还能剂量依赖地抑制所有KCNQ通道家族成员(包括KCNQ1-4和KCNQ3/Q5)。美吡拉敏还能抑制大鼠颈上神经元中的M电流,并由此引发神经元显著去极化。美吡拉敏和苯海拉明对KCNQ/M通道抑制作用的IC50均位于临床上所观察到的中毒血药浓度范围之间。因此第一代抗组胺药物对M通道的抑制及由此引发的神经元去极化效应很可能导致其中毒剂量下病人所出现的神经系统毒性症状。
4第一代抗组胺药物美吡拉敏与苯海拉明能够剂量依赖地抑制外源性表达于卵母细胞中的Kir2.3通道电流,并能由此产生去极化效应;而第二与第三代抗组胺药物阿斯咪唑及地氯雷他定则无作用。上述所有药物则对Kir2.1通道电流无作用。美吡拉敏同样能够抑制Kir3.4通道电流。美吡拉敏对Kir通道的选择性抑制作用很可能与通道和细胞膜磷脂PIP2的亲和力存在一定关系。
M type potassium channel conducts a voltage and time dependent, slowly activating, slowly deactivating and non-inactivating outward current. It is such named since it can be robustly inhibited by muscarinic M1 receptor activation. At present, it is generally accepted that the molecular basis of M channel is constituted by homo- and hetero-multimers of KCNQ (KCNQ1-5) subunits. M channel opens at a threshold below -60 mV and this feature allows for a fraction of these channels to be open at or near the resting membrane potential of a neuron. It is suggested that M current in neurons can serve as an intrinsic“voltage-clamp”mechanism controlling the resting membrane potential and the threshold for action potential firing thus repressing the over excitability of neurons. M channel is extensively distributed in nervous system, including hippocampus, cortical, sensory and some sympathetic neurons. Moreover, growing evidence suggests that functional M channels are expressed in the peripheral sensory fibers and their activity strongly contributes to the fiber excitability in vivo.
Ca2+ activated Cl- channels (CACC) are a prominent group of ion channels robustly expressed in many mammalian cell types including neurons, smooth muscles and epithelia. In the nervous system, functional CaCCs are best characterized in neurons mediating different sensory inputs, such as olfactory neurons, photosensitive rods and cons, taste calls and DRG neurons. CaCCs play a role in depolarization or amplification of a depolarizing input produced by other channels in the nervous system. The molecular identity of CaCC remained controversial until very recently when three groups independently identified a member of the transmembrane protein 16A (TMEM16A) protein family as a CaCC subunit. This important finding makes it possible to alter CaCCs in native cells via genetic manipulations
Inwardly rectifying potassium (Kir) channel family contains at least 7 subfamilies (Kir1-7). These channels are extensively distributed in various tissues and serve a role in maintaining K+ equilibrium and keeping membrane potential. These channels play an important role in the repolarization phase of action potential and regulation of cell excitability. Furthermore, recent studies indicate PIP2 may play an important role in modulation of Kir2.0 channel function by several modulators.
Based upon the above mentioned channels, in my thesis, we use techniques such as patch clamp, calcium imaging, siRNA, native neuron transfection, TEVC, LSCM and animal behavioral assay, etc. to systematically study the following subjects: (1) Ionic mechanisms for the acute nociceptive signals induced by inflammatory mediator bradykinin (BK); (2) Mechanism underlying inflammatory mediator histamine regulation of KCNQ/M channels; (3) Mechanism underlying the first-generation antihistamine induced severe neuronal adverse effects.
Part 1 Ionic mechanisms for the acute nociceptive signals induced by bradykinin
Objective: We aim to investigate the mechanisms of acute nociceptive signaling induced by bradykinin. We characterized major membrane currents directly affected by BK in nociceptive sensory neurons, investigated their molecular identities, signaling cascades coupling their regulation to BK receptors and the contribution of these currents to the excitability of nociceptive neurons and to the BK-induced nocifensive behaviour in rats.
Methods: Perforated patch clamp (voltage clamp) was used to monitor the effects of BK on ion channels in DRG neurons. Current clamp was used to record the neuron excitability. Calcium photometry was used to measure the changes of intracellular Ca2+ concentration. Membrane PIP2 hydrolysis was monitored by confocal microscope. Whole animal behavioral study was carried out to monitor the nocifensive behavior of rats upon BK application.
Results: (1) BK inhibits M current in small DRG neurons: BK caused a prominent concentration-dependent inhibition of M current in small DRG neurons, with an EC50 of 60.0±16.3 nM. The inhibitory effect was independent of non-neuronal cells since purified neuron culture did not affect BK’s response. (2) Signaling pathway of BK-induced M current inhibition: BK produced significant phospholipase C (PLC) activation since PLCδ-PH-GFP, a fluorescent probe that binds PIP2 and IP3, translocated from the membrane to the cytosol after BK application. However, membrane PIP2 concentration did not drop significantly since a more specific PIP2 probe YFP-tubby failed to translocate to the cytosol following BK application. Ca2+ imaging experiments showed that BK induced significant intracellular Ca2+ rises in DRG neurons. Patch clamp studies further revealed that the inhibitory effect of BK was significantly attenuated by B2R antagonist Hoe-140, PLC inhibitor edelfosine, IP3 receptor antagonist Xe-C, SERCA inhibitor thapsigargin and high BAPTA internal solution, but was not affected by PKC inhibitor bisindolylmaleimide and PLA2 inhibitor quinacrine. (3) BK activates TMEM16A-dependent Cl- channels: BK can induce a prominent inward current accompanied by M current inhibition. This inward current was not due to TRPV1 or HCN channel opening since ruthenium red and ZD7288 did not affect it. However, this inward current was abolished by Cl- channel blocker DIDS and low Cl- internal solution. Further experiments showed that the inward current was also abolished by B2R antagonist Hoe-140, PLC inhibitor edelfosine, IP3 receptor antagonist Xe-C, SERCA inhibitor thapsigargin and high BAPTA internal solution. The above results suggest that the inward current was conducted by Ca2+ activated Cl- channels (CACC). The gene encoding essential CaCC subunit has recently been identified as Tmem 16a. Experiments using siRNA against Tmem 16a significantly attenuated BK induced inward current, revealing that Tmem 16a is a molecular correlate of the BK-induced Cl- current in DRG neurons. (4) M-current inhibition and CaCC activation contribute to and can account for the excitatory effect of BK: BK and XE991 could both significantly increase DRG neuron excitability and cause depolarization. In low Cl- intracellular solution which abolished BK-induced inward current, co-application of BK + XE991 failed to produce further excitation upon XE991 application. However, XE991 could further excite the neuron in the presence of BK. (5) M channel openers can avert nocifensive behaviour induced by BK: Intraplantar injection of BK (10 nM/site) into the rat hind paw produced strong nocifensive behaviour, which was significantly alleviated by specific M channel openers retigabine or zinc pyrithione. However, the Cl- channel blocker DIDS had no clear effect on the BK-induced nocifensive behaviour and co-application of retigabine and DIDS had only a marginal additivity.
Conclusions: (1) BK concentration-dependently inhibits M current in small DRG neurons. The action of BK on M current is not mediated by non-neuronal cells but due to a primary effect on DRG neurons. (2) BK activates PLC but does not produce a global depletion of the membrane PIP2. BK can also induce intracellular Ca2+ mobilization. Pharmacological studies reveal that the inhibitory effect of BK on M current is due to B2R-PLC and IP3-mediated intracellular Ca2+ rise. (3) A series of pharmacological experiments show that the inward current induced by BK is conducted by CACC. Furthermore, siRNA experiments reveal that the recently discovered Tmem 16a underlies molecular correlate of CACC in DRG neurons. (4) Current clamp studies show that BK causes hyperexcitability in DRG neurons. By comparing the effects of BK and Xe991 in high and low Cl- containing internal solution, we show that M current and CACC are both involved in BK’s excitatory effect. (5) BK induced nocifensive behavior in rat is significantly attenuated by M current enhancers, suggesting inhibition of M channels in the peripheral sensory terminals can indeed produce firing of action potentials in vivo.
Part 2 Phosphatidylinositol 4, 5-bisphosphate hydrolysis mediates histamine-induced KCNQ/M current inhibition
Objective: We aim to study the effects of histamine on KCNQ/M channel current in HEK293 cells and rat SCG neurons. We further explore the signaling pathway and compare the effects of histamine on SCG neurons with those of the well characterized M1 and B2 receptor agonists, oxo-M and BK. The final goal will be to understand the common mechanisms underlying GPCR modulation of KCNQ/M channels in more detail.
Methods: Patch clamp technique was used to study the effect of histamine on channel currents and calcium photometry was used to measure the changes of intracellular Ca2+ concentration. Pharmacology methods, in combination with LSCM, were used to study the signaling pathway underlying histamine modulation of KCNQ/M channel in detail.
Results: (1) Histamine suppresses KCNQ/M current in HEK293 cells and rat SCG neurons via activation of H1 receptor: Histamine (10μM) strongly inhibited KCNQ2/Q3 channel currents heterologusly expressed in HEK293 cells and the inhibition was 77.9±3.5%. The inhibitory effect was not affected by H2 receptor antagonist cimetidine (p > 0.05 vs. control) but was attenuated by H1 receptor antagonist mepyramine (p < 0.01 vs. control). Histamine could also inhibit M channel current in rat SCG neurons in a concentration dependent manner and this effect was also attenuated by mepyramine (p < 0.01 vs. control). The EC50 deduced from the concentration response relationship was 3.3±1.1μM. Moreover, the existence of H1 receptor in SCG neurons was confirmed by Western blot. (2) Histamine suppresses KCNQ/M current via activation of PLC: The inhibitory effect of histamine was significantly reduced to 7.1±0.6% (p < 0.01 vs. control) by a PLC antagonist U-73122. Meanwhile, the inhibitory effect of histamine on M current in SCG neurons was also significantly abolished by U-73122 (p < 0.01 vs. control). (3) Histamine-induced inhibition of KCNQ2/Q3 currents is the result of membrane PIP2 hydrolysis: Histamine produced an obvious translocation of the PLCδ1-PH-GFP probe from the membrane to the cytosol and the effect was reversible upon histamine washout. The rise of fluorescence signals in the cytosol evoked by application of histamine was 80.4±6.0%. This effect was blocked by mepyramine and U-73122 (p < 0.01 vs. control). Patch clamp studies showed that the recovery of the current inhibited by histamine was significantly reduced to 10.5±3.4% (p < 0.01 vs. control) when treated with PI4-Kinase inhibitor wortmannin, compared with 88.4±6.2% in control conditions. In addition, PAO, another PI4-Kinase inhibitor, also significantly reduced the recovery to 6.3±0.9% (p < 0.01 vs. control) compared with 95.4±4.6% in control conditions. (4) Histamine inhibits KCNQ2/Q3 currents in HEK293 cells deprived of [Ca2+]i rises: Histamine (10μM) could induce an obvious [Ca2+]i rise and this effect still existed in Ca2+-free extracellular solution but largely abolished by U-73122. In whole-cell patch clamp configuration, the histamine induced KCNQ current inhibition in HEK293 cells was not altered (81.3±4.0%, p > 0.05 vs. control) when cells were dialyzed with high BAPTA intracellular solution. Besides, thapsigargin, a sarcoplasmic-endoplasmic reticulum Ca2+ pump inhibitor, which can empty the Ca2+ stores, did not affect the effect of histamine, either (88.6±3.9%, p > 0.05 vs. control). (5) H1 receptor stimulation does not evoke [Ca2+]i signal in SCG neurons: Calcium photometry was used to monitor the intracellular Ca2+ changes upon histamine application and the effect of histamine was compared with M1 and BK2 receptor agonist oxo-M and BK. BK could evoke an obvious [Ca2+]i rise, while oxo-M and histamine could not. The rises in the F340/380 ratio produced by histamine, oxo-M and BK were 0.04±0.008, 0.03±0.004 and 0.32±0.05. (6) Histamine does not require [Ca2+]i signal for M current inhibition in SCG neurons: Application of histamine, oxo-M and BK suppressed M currents by 38.3±7.9%, 98.8±0.4% and 85.3±4.4%, respectively, when neurons were dialyzed with low BAPTA internal solution (containing 0.1 mM BAPTA). When neurons were dialyzed with high BAPTA internal solutions, the suppression of M currents by histamine and oxo-M was 32.1±3.1% and 87.3±7.0%, respectively, which was not significantly altered (p > 0.05 vs. control). In contrast, the inhibitory effect of BK was significantly reduced to 20.5±2.6% (p < 0.01 vs. control).
Conclusions: (1) Histamine inhibits KCNQ2/Q3 and M channel currents in heterologusly expressed HEK293 cells and rat SCG neurons by H1 receptor activation and this inhibitory effect shows dose dependence. (2) The inhibitory effect of histamine is closely related to PLC activation. (3) Histamine can induce membrane PIP2 hydrolysis and its inhibitory effect on KCNQ currents may likely be due to the hydrolysis of membrane PIP2. (4) The inhibitory effect of histamine on KCNQ currents in HEK293 cells does not rely on intracellular Ca2+ signals despite the fact that it can mobilize intracellular Ca2+ in these cells. (5) The inhibitory effect of histamine on M currents in SCG neurons is also independent of intracellular Ca2+ signals. This effect is similar with that of the oxo-M. Thus activation of H1 receptor inhibits M currents in rat SCG neurons through a similar mechanism with that of M1 receptor. This finding further enriches our understanding of signaling pathway underlying GPCR modulation of KCNQ/M channel. Moreover, the inhibitory effect of histamine on M current may provide new insights into the understanding of the excitatory effects of histamine on neurons.
Part 3 Antihistamine mepyramine directly inhibits KCNQ/M channel and depolarizes rat superior cervical ganglion neurons Objective: We aim to study the effects of the first-generation
antihistamine mepyramine and diphenhydramine on KCNQ/M channel currents and the underlying mechanisms. We also aim to study their effects on neuron excitability. We try to unravel the molecular mechanism underlying the neuronal toxicity induced by overdose of first-generation antihistamines.
Methods: Patch clamp technique was used to study the effect of mepyramine and diphenhydramine on KCNQ/M channel currents and the changes in channel kinetics were analyzed. Whole-cell patch clamp for intracellular drug application and outside-out patch were used to determine the action side of the drug. Current clamp recording mode was used to monitor the effects of drugs on membrane potentials and neuron excitability.
Results: (1) Mepyramine inhibits KCNQ2/Q3 channel currents and affects channel gating properties: KCNQ2/Q3 channels were expressed heterologusly in HEK293 cells by means of transfection. The first-generation antihistamine mepyramine could strongly inhibit KCNQ2/Q3 currents and the inhibition rate reached 92.0±1.7%. The inhibition developed rapidly and could be largely washed out. This effect showed concentration dependency and IC50 was 12.5±1.8μM. However, the H2 receptor antagonist cimetidine did not affect KCNQ2/Q3 currents. (2) The effect of mepyramine on channel kinetics: Mepyramine could prolong activation and shorten deactivation kinetics of KCNQ2/Q3 channels and right shift the half-maximal activation (V50) from -16.9±0.9 mV to -7.8±1.4 mV. (3) Diphenhydramine shows similar inhibitory effect. The inhibition produced by 100μM diphenhydramine was 66.9±5.9% and the I-V curve was shifted to the right. (4) Effect of mepyramine on KCNQ families (KCNQ1-5): Mepyramine could inhibit homomeric KCNQ1, KCNQ2, KCNQ3, KCNQ4 and heteromeric KCNQ3/5 channel currents and the inhibitory effects showed concentration dependence. (5) Mepyramine directly inhibits KCNQ2/Q3 channel current from outside of the cell membrane: We included mepyramine in the recording pipette for whole-cell recording to see if direct introduction of mepyramine into the cell would result in KCNQ2/Q3 current inhibition in HEK293 cells. In control and mepyramine treated groups, KCNQ2/Q3 current was decreased to 56.0±9.0% and 54.8±11.6% of its initial level after 12 min dialysis. There was no significant difference between these two groups (p > 0.05). After rundown of KCNQ2/Q3 currents, the inhibitory effect of oxo-M, an M1 receptor agonist that hydrolyzes PIP2 on KCNQ2/Q3 current, was significantly reduced, while effect of mepyramine was not. Outside-out patch clamp studies revealed that mepyramine could quickly and significantly inhibit KCNQ currents (90.1±2.8%) in the membrane excised from HEK293 cells. (6) Mepyramine inhibits M current in rat SCG neurons: Meyramine also inhibited M current in SCG neurons, the inhibition was 76.7±2.6% and showed concentration dependence (IC50 = 25.7±0.7μM). In addition, mepyramine also significantly depolarized the SCG neurons from–51.0±5.6 mV to–38.9±7.3 mV. Mepyramine could not induce depolarization in the continued presence of a specific M channel blocker, linopirdine. (7) Effect of mepyramine on evoked neuronal action potentials: We found that linopirdine could induce remarkable hyperexcitability in SCG neurons. On the contrary, mepyramine did not show any effect on the excitability of neurons.
Conclusions: (1) The first-generation antihistamine mepyramine and diphenhydramine can significantly inhibit KCNQ/M currents and this effect is due to direct drug inhibition from the extracellular side. (2) Mepyramine alters KCNQ channel activation and deactivation kinetics and shifts the I-V curve to the right. (3) Mepyramine concentration-dependently inhibits all members of KCNQ family (KCNQ1-5). (4) Mepyramine also inhibits M current in rat SCG neurons and induces depolarization in neurons. However, mepyramine does not evoke hyperexcitability in neurons. These actions are likely to be involved in the adverse neuroexcitatory effects including seizers, convulsion and epilepsy, observed in patients intoxicated by overdose of first-generation antihistamines.
Part 4 Selective inhibition of Kir currents by antihistamines
Objective: We aim to study the effects of the first, second and the third-generation antihistamines on inwardly rectifying potassium (Kir) channels and try to reveal the molecular mechanisms underlying neurotoxic effects by overdose of H1 receptor antagonists.
Methods: DNA preparation, RNA in vitro transcription and microinjection methods were used to express Kir2.1, 2.3 and 3.4 channels in Xenopus oocyte. Two electrode voltage clamp (TEVC) was used to record the whole cell currents in oocyte and to observe the effects of each type of antihistamines on channel currents.
Results: (1) Effect of antihistamines on Kir2.3 currents expressed in Xenopus oocyte: Kir2.3 channel showed a substantial inwardly rectifying property in a high extra-cellular K+ bath solution. The reverse potential was near 0 mV. Extra-cellular perfusion with 100μM mepyramine or diphenhydramine, two histamine H1 receptor antagonist of the first-generation antihistamine, both reduced the current amplitude by 25.0±2.9% and 17.3±0.7%, respectively. However, the second and third-generation H1 receptor antihistamines astemizole, desloratadine and H2 receptor antagonist cimetidine had no effect on Kir2.3 channel current. When deduced from the I-V curve, mepyramine-induced inhibition of Kir2.3 currents was not voltage-dependent. The Kir2.3 current inhibitions induced by mepyramine and diphenhydramine showed concentration dependence and IC50 was 306.4μM and 689.2μM, respectively. (2) Effect of antihistamines on Kir2.1 currents expressed in Xenopus oocyte: Mepyramine and diphenhydramine, at concentrations of 100μM, had little or no inhibitory effect on Kir2.1 channel currents. Similar results were also obtained from other drugs. Astemizole, desloratadine and cimetidine had no effect on Kir2.1 currents. (3) Selective inhibition of antihistamines on Kir currents depends on characteristic channel-PIP2 interaction: We found that the effect of mepyramine could be significantly abolished by a point mutation of Kir2.3, Kir2.3 (I213L), which could confer a stronger channel-PIP2 interaction to Kir2.3 channel. In addition, mepyramine could also inhibit Kir3.4, another member of the Kir subfamily, which has also been shown to be a Kir channel interacting weakly with PIP2 as Kir2.3. Our results indicated the rank order for current inhibition among Kir channels as follows: Kir3.4 > Kir2.3 > Kir2.3 (I213L) > Kir2.1. This result coincided with our previous results showing the rank order of the strength of channel-PIP2 interactions among Kir channels. (4) Inhibition of Kir2.3 current by the first-generation antihistamines leads to the depolarization of resting membrane potential of oocytes: After expressing Kir2.3 channels, the oocytes displayed resting membrane potential (Vm) values of -95.2±2.3 mV. Application of mepyramine (300μM) gradually and significantly depolarized Vm to -57.1±6.1 mV. Similar result was also obtained for 300μM diphenhydramine, which reduced the resting Vm value to -70.0±3.4mV.
Conclusions: (1) The first-generation antihistamines mepyramine and diphenhydramine significantly and concentration dependently inhibit Kir2.3 channel current. However, the second and third-generation H1 receptor antihistamine and H2 receptor antagonist astemizole, desloratadine and cimetidine have no effect on Kir2.3 currnet. (2) Kir2.1 channel is insensitive to any antihistamines. (3) The obvious discrepancy of the effect of mepyramine between Kir2.1 and Kir2.3 is due to channel-PIP2 interactions. Mepyramine also inhibits Kir3.4 channel, which shows weak channel-PIP2 interaction. (4) Mepyramine induces a significant depolarization in oocytes by inhibiting Kir2.3 channel. Thus, Kir2.3 channel inhibition may likely be involved in the molecular mechanisms underlying overdose first-generation antihistamine induced neurotoxic effects.
SUMMARY
1 BK inhibits M-type K+ channel and activates Ca2+-activated Cl- channel in small DRG neurons. BK, acting through its B2 receptors, PLC and releasing of Ca2+ ions from intracellular stores, robustly inhibits M-type K+ channels and activates TMEM 16A-dependent Ca2+-activated Cl- channels. Summation of these two effects adequately accounts for the depolarization and increase in action potential firing induced by BK in DRG neurons. Preclusion of BK-induced inhibition of M channels with specific openers strongly attenuates the nociceptive effect of BK in vivo. The above results reveal the detailed molecular mechanism underlying BK-induced inflammatory pain and provide new directions for the treatment of inflammatory pain.
2 Histamine, via activating H1 receptor, PLC and hydrolyzing PIP2, inhibits KCNQ/M channel in HEK293 cells and rat SCG neurons. This process does not involve Ca2+ signal. By comparing the effects of histamine on SCG neurons with those of the well-characterized M1 and B2 receptor agonist, oxo-M, and BK, we suggest that histamine inhibits M current in a similar mechanism with oxo-M.
3 The first-generation antihistamine mepyramne and diphenhydramine concentration dependently inhibit KCNQ2/Q3 channel exogenously expressed in HEK293 cells. Meyramine alters channel activation and deactivation kinetics and shift the I-V curve to the right. In addition, mepyramine concentration dependently inhibits all KCNQ channels, including KCNQ1-4 and KCNQ3/Q5. Moreover, mepyrmaine, by inhibiting M channel current in rat SCG neurons, induces significant membrane potential depolarization in neurons. The IC50 for the inhibitory effect of mepyramine lies in the range of plasma drug concentration of intoxicated patients. Thus, KCNQ/M channel inhibition and the subsequent marked depolarization of neuron membrane potential may participate in adverse effects observed in patients intoxicated by overdose with first-generation antihistamines.
4 The first-generation antihistamine mepyramne and diphenhydramine dose dependently inhibit Kir2.3 channel current expressed in Xenopus oocytes and induce membrane depolarization, while the second and third-generation antihistamine astemizole and desloratadine do not. Kir2.1 current is insensitive to any of these drugs. Mepyrmaine also inhibits Kir3.4 current. We speculate that the specific Kir channel inhibition produced by the first-generation antihistamine is likely to be determined by the strength of channel-PIP2 interactions.
内向整流钾离子通道(Kir)包括至少7个亚家族(Kir1-7),这些通道在组织中分布广泛,能够保持细胞内外K+平衡、维持细胞膜电位,因此在动作电位复极化过程以及调节细胞兴奋性等方面发挥重要作用。近年来有文献报道,Kir2.0家族成员因其与细胞膜PIP2结合力强弱不同,因此在对一些通道调节剂的调节作用方面表现有所区别。
本研究论文以上述几种通道为出发点,利用电生理膜片钳、细胞内钙成像、小RNA干扰、原代神经元转染、双电极电压钳、激光扫描共聚焦显微镜以及动物行为学实验等技术手段,系统研究了以下几方面内容:(1)炎症介导因子缓激肽引发急性痛觉信号的离子机制;(2)炎症介导因子组胺引发KCNQ/M通道调节作用及其内在机制;(3)第一代抗组胺药物相比其他抗组胺药物在中毒剂量下更易引发严重神经系统不良反应的分子机制。论文具体内容如下:
第一部分缓激肽引发急性痛觉信号的离子机制
目的:研究缓激肽引发急性痛觉信号的离子机制,观察缓激肽对大鼠背根神经元中离子通道的调节作用。我们发现缓激肽能够抑制M型钾电流的同时激活一种内向离子电流。我们进行了一系列药理学实验和小RNA干扰实验来阐明缓激肽调节M电流的信号通路和这一内向电流的确切分子基础。通过电流钳实验我们又研究了这两种机制是否参与了缓激肽所引发的神经元兴奋性增高。最后通过动物行为学实验,我们在整体动物水平上研究了这两种机制在缓激肽引发动物急性疼痛行为中所发挥的作用。
方法:使用打孔全细胞膜片钳观察缓激肽对背根神经元离子电流的影响。使用细胞内钙成像测定细胞内钙离子浓度变化。利用激光扫描共聚焦显微镜动态观察PIP2特异性荧光探针YFP-tubby的变化过程。使用小RNA干扰技术结合神经元细胞核转染的方法探讨缓激肽引发内向电流的分子基础。利用电流钳观察缓激肽引发的神经元兴奋性,并最后通过整体动物行为学测试法,研究相应通道调节剂对缓激肽引发的急性疼痛的作用。
结果:(1)缓激肽抑制小背根神经元中的M电流:缓激肽能够剂量依赖性地抑制小背根神经元中的M电流,其抑制作用的EC50为60.0±16.3 nM。此外对背根神经元进行纯化培养后并不影响缓激肽的抑制作用。(2)缓激肽引发M电流抑制作用的信号途径:缓激肽能够在背根神经元引发PLCδ-PH-GFP出现荧光转位,而对PIP2特异性荧光探针YFP-tubby却无作用,说明缓激肽能够激活PLC,但细胞膜PIP2水平却没有明显变化。Ca2+成像实验显示缓激肽能够引发背根神经元细胞内Ca2+浓度明显增高。膜片钳实验进一步证实,缓激肽对M电流的抑制作用受到B2受体拮抗剂Hoe-140,IP3受体拮抗剂Xe-C,SERCA抑制剂thapsigargin及高浓度BAPTA电极内液的显著影响,但并不受PLA2阻断剂quinacrine和PKC抑制剂bisindolylmaleimide影响。(3)缓激肽激活TMEM16A依赖性的CACC:我们发现在缓激肽抑制M电流的同时会引发一种明显内向电流。这一内向电流对TRP通道阻断剂钌红及HCN特异性阻断剂ZD7288均不敏感。但当把电极内液换成低Cl-内液后,这一内向电流消失;而且Cl-通道阻断剂DIDS同样能够明显抑制这一电流的出现。提示这一内向电流是由Cl-通道开放而介导的。进一步的药理学实验证明,这种内向电流受Hoe-140,thapsigargin,Xe-C及高BAPTA电极内液的影响。提示这一内向电流为CACC所介导。此外利用siRNA技术使编码CACC的Tmem 16a基因沉默后,此内向电流显著降低,而缓激肽对M电流的抑制作用不受影响。(4) M通道的抑制与Ca2+激活Cl-通道的开放共同构成了缓激肽对感觉神经元的兴奋作用:在高Cl-内液环境下,缓激肽与M通道特异性抑制剂XE991均能明显增强背根神经元的兴奋性,而缓激肽的作用要强于XE991。此外二者均能使神经元细胞膜电位明显去极化。使用低Cl-内液去除CACC作用后,XE991同样能够引发神经元兴奋性,而此时若共同给予缓激肽+ XE991后,神经元兴奋性不再增加。在低Cl-内液环境下,若先给予缓激肽,再给予缓激肽+ XE991后,XE991能够在缓激肽引发兴奋性的基础上再增加兴奋性。(5) M通道增强剂能够拮抗缓激肽引发的动物疼痛表现:在动物行为学实验中,缓激肽能够引发受试动物出现明显疼痛反应。M通道增强剂retigabine和zinc pyrithione能够有效缓解缓激肽的致痛作用,然而Cl-阻断剂DIDS却对痛觉反应无明显作用。
结论:(1)缓激肽对小背根神经元中M电流具有剂量依赖性抑制作用。这种作用与其他非神经元作用无关。(2)缓激肽能够激活PLC,但并不能造成细胞膜PIP2整体水平出现明显下降;缓激肽还能引发细胞内Ca2+浓度升高。缓激肽抑制背根神经元中M电流的作用依赖于B2受体、PLC和其引发的细胞内Ca2+信号。(3)缓激肽在抑制M电流的同时引发的内向电流为Ca2+激活Cl-电流。这一电流由Tmem 16a基因所编码的Ca2+激活Cl-通道所介导。(4)缓激肽对M电流与Ca2+激活Cl-电流的作用共同参与了其引发的背根神经元兴奋性增高。(5) M通道的特异性增强剂能够明显缓解缓激肽引发受试动物出现的疼痛症状。说明M通道在缓激肽引发的急性疼痛中发挥重要作用。
第二部分细胞膜磷脂PIP2介导组胺对KCNQ/M通道电流调节机制的研究
目的:研究组胺对HEK293表达细胞与大鼠颈上交感神经元中KCNQ/M通道电流的调节作用,并对作用机制进行信号通路的研究,并且与已知的能调节KCNQ/M电流的M1受体与BK2受体激动剂oxo-M和缓激肽进行比较,从而对G蛋白偶联受体调节KCNQ/M电流的共同机制进行更深入的了解。
方法:利用膜片钳观察组胺对电流的调节作用,利用细胞内钙成像技术测定细胞内钙离子浓度变化,并使用一系列药理学方法配合激光扫描共聚焦显微镜作动态观察,详细研究组胺调节KCNQ/M电流的信号转导通路。
结果:(1)组胺通过激活组胺H1受体,抑制外源性表达于HEK293细胞的KCNQ2/Q3与大鼠颈上神经元的M通道电流:当HEK293细胞外源性表达KCNQ2/Q3通道与H1受体后,组胺(10μM)能够强烈抑制通道电流,抑制率达到77.9±3.5%,并且这种作用并不受H2受体拮抗剂西咪替丁影响(p > 0.05 vs. control),但却能被H1受体特异性抑制剂美吡拉敏所阻断(p < 0.01 vs. control)。组胺还能对大鼠颈上神经元的M通道电流有抑制作用,这种抑制作用同样能被美吡拉敏阻断(p < 0.01 vs. control),且这一作用具有剂量依赖性,通过对不同浓度组胺所引发的M电流抑制作用的量效曲线进行分析,组胺抑制作用的EC50 = 3.3±1.1μM。此外通过Western blot的方法,我们也发现在颈上神经元中的却存在H1受体。(2)组胺对KCNQ2/Q3电流的抑制作用是由PLC激活所介导:我们发现组胺对电流的抑制作用能被PLC阻断剂U-73122显著降低,只有7.1±0.6% (p < 0.01 vs. control);同样对于颈上神经元中的M电流来说,组胺的抑制作用也能被U-73122所取消(p < 0.01 vs. control)。(3)组胺对KCNQ/M电流的抑制作用是由于细胞膜磷脂PIP2水解所造成的:PLCδ1-PH-GFP是一种常用的标记PIP2的荧光探针。通过LSCM观察我们发现,组胺可以明显引起PLCδ1-PH-GFP从胞膜到胞浆的转位,胞膜荧光强度可以增加80.4±6.0%,且这一过程在组胺洗脱后可逆。组胺的这一作用能被H1受体特异性抑制剂美吡拉敏与PLC抑制剂U-73122所阻断(p < 0.01 vs. control)。如果使用膜片钳研究我们发现,当用PI4-Kinase抑制剂wortmannin灌流HEK293细胞以阻断PIP2再合成后,由组胺引起KCNQ2/Q3电流抑制后的恢复程度便由给予wortmannin前的88.4±6.2%显著降低到10.5±3.4% (p < 0.01 vs. control)。另一种PI4-Kinase抑制剂PAO同样使电流被组胺抑制后的恢复程度从给药前的95.4±4.6%显著降低到6.3±0.9% (p < 0.01 vs. control)。(4)组胺对HEK293细胞表达的KCNQ电流的抑制作用不依赖于细胞内钙信号:我们发现组胺能够引起明显的细胞内钙浓度升高,并且在无钙外液下依旧存在,但是这种变化可以被PLC抑制剂U-73122很大程度上取消。在全细胞膜片钳记录模式,当细胞内通过长时间由微电极向细胞内导入钙离子螯合内液以缓冲钙信号后,由组胺引发的KCNQ电流抑制作用不受任何影响(81.3±4.0%,p > 0.05 vs. control)。此外细胞内钙库耗竭剂thapsigargin也不能影响组胺的作用(88.6±3.9%,p > 0.05 vs. control)。(5)组胺不能引发颈上神经元中钙离子浓度升高:测定细胞内钙离子在给予组胺后的变化,并与M1受体与BK2受体激动剂oxo-M,缓激肽的作用进行比较。缓激肽能够引发很明显的细胞内钙离子浓度升高,但oxo-M与组胺却不能引发细胞内钙变化。缓激肽, oxo-M和组胺所引起的荧光强度的升高分别为0.04±0.008, 0.03±0.004和0.32±0.05 (p < 0.01)。(6)组胺对颈上神经元M电流的抑制不依赖于细胞内钙信号:当使用含有0.1 mM BAPTA (低BAPTA)的细胞内液对颈上神经元进行全细胞渗透后,组胺、oxo-M和缓激肽均能不同程度抑制M电流,其抑制程度分别为38.3±7.9%, 98.8±0.4%和85.3±4.4%。当使用含有20 mM BAPTA + 10 mM CaCl2 (高BAPTA)的细胞内液对颈上神经元进行全细胞渗透后,缓激肽的作用便会被显著降低到20.5±2.6% (p < 0.01 vs. control),而组胺、oxo-M的作用分别为32.1±3.1%和87.3±7.0%,与对照相比没有明显变化(p > 0.05 vs. control)。
结论:(1)组胺可以通过激活H1受体抑制外源性表达于HEK293细胞上的KCNQ2/Q3与大鼠颈上神经元中的M通道电流,且这种抑制作用具有剂量依赖性。(2)组胺对KCNQ/M电流的抑制作用与PLC的激活密切相关。(3)组胺能够引起细胞膜磷脂PIP2的水解,并且其对KCNQ电流的抑制作用很可能是由于细胞膜磷脂PIP2水解造成。(4)组胺虽能引起HEK293细胞中内钙浓度升高,但是其对KCNQ电流的抑制作用并不取决于钙信号。(5)组胺对颈上神经元中M电流的抑制也不依赖于细胞内钙信号,与已知的两种G蛋白偶联受体激动剂缓激肽与oxo-M的作用机制相比,更接近于oxo-M。因此H1受体在调解M电流的机制方面与已知的M1受体非常一致。这一发现充实并完善了G蛋白偶联受体调节KCNQ/M电流的信号途径,此外组胺对神经元M电流的抑制作用对研究其作为炎症介导因子与神经递质参与并引发神经元兴奋性的机制提供了新的方向。
第三部分抗组胺药物美吡拉敏直接抑制KCNQ/M电流并且引发大鼠颈上神经元去极化
目的:研究第一代H1受体抗组胺药美吡拉敏及苯海拉明对KCNQ/M通道电流的作用及其机制,并研究其对神经元兴奋性的影响,揭示第一代抗组胺药在服用过量下所引发的神经系统不良反应的分子机制。方法:利用电生理膜片钳技术,观察美吡拉敏及苯海拉明对KCNQ/M通道电流的作用,并对通道动力学特征变化进行分析;利用全细胞膜片钳细胞内渗透给药与outside-out膜片钳相结合的实验手段确定药物作用于通道的部位;利用电流钳模式记录药物对细胞膜电位以及细胞兴奋性的影响。
结果:(1)美吡拉敏抑制KCNQ2/Q3通道电流并影响其动力学特征:利用细胞转染的方法,将KCNQ2/Q3基因外源性导入HEK293细胞。第一代H1受体抗组胺药美吡拉敏(100μM)能够强烈抑制KCNQ2/Q3电流,其对KCNQ2/Q3尾电流的抑制率达到92.0±1.7%。这种抑制作用很快,并且能在很大程度上被洗脱。这种作用具有剂量依赖性,其IC50 = 12.5±1.8μM。而H2受体拮抗剂西咪替丁则对KCNQ2/Q3电流没有任何作用。(2)美吡拉敏对通道动力学特征的影响:美吡拉敏可以延长电流激活段时间参数而缩短电流去活段时间参数,并且将通道I-V曲线半数激活电压从-16.9±0.9 mV右移至-7.8±1.4 mV。(3)苯海拉明也具有相似的抑制作用,其对KCNQ2/Q3的抑制率达到66.9±5.9%,并且也可以将通道I-V曲线右移。(4)美吡拉敏对KCNQ通道家族(KCNQ1-5)的作用:除对KCNQ2/Q3电流的抑制作用外,美吡拉敏还能够分别抑制KCNQ1,KCNQ2,KCNQ3,KCNQ4,KCNQ3/5等KCNQ通道成员,并且这种抑制作用均具有剂量依赖性。(5)美吡拉敏从细胞膜外面直接抑制KCNQ2/Q3通道电流:首先我们利用全细胞膜片钳的方法向表达有KCNQ2/Q3的HEK293细胞内部渗透美吡拉敏,研究药物是否通过细胞内部的作用部位发挥对KCNQ通道的抑制作用。在对照情况下,如果利用正常细胞内液进行细胞渗透,经过12分钟后,KCNQ2/Q3电流大小会降低到原来的56.0±9.0%;而使用美吡拉敏进行渗透后,电流会降低到原来的54.8±11.6%,这与对照组相比没有显著差别(p > 0.05)。而与熟知的通过受体激活引发PIP2水解而抑制KCNQ/M通道的M1受体激动剂oxo-M比较后我们发现,oxo-M对电流的抑制作用会受到PLC抑制剂U-73122的影响,而美吡拉敏则不受U-73122的任何影响。通过膜外面向外(outside-out)膜片钳实验我们进一步发现,美吡拉敏可以非常迅速且明显地抑制outside-out膜片钳记录下HEK293细胞的KCNQ电流,抑制率为90.1±2.8%。(6)美吡拉敏抑制大鼠颈上神经元中的M电流:100μM美吡拉敏也同样能够抑制颈上神经元中的M电流,其抑制率达到76.7±2.6%,且这种作用也具有剂量依赖性,IC50 = 25.7±0.7μM。此外美吡拉敏也能够引发颈上神经元明显的去极化,可将细胞膜电位从–51.0±5.6 mV去极化至–38.9±7.3 mV。若使用M通道的特异性抑制剂linopirdine事先作用于细胞后,美吡拉敏便不能在linopirdine持续存在的情况下进一步引发去极化作用。(7)美吡拉敏对神经元兴奋性的影响:我们发现虽然M通道的特异性抑制剂linopirdine的却能够明显引发神经元兴奋性增高,但美吡拉敏却并不能增高兴奋性。
结论:(1)第一代H1受体抗组胺药美吡拉敏及苯海拉明能够明显抑制KCNQ/M电流,这种作用是由药物在细胞膜外侧直接阻断通道而引起。(2)美吡拉敏可以改变KCNQ通道激活与去活动力学特征,并引起通道I-V曲线右移。(3)美吡拉敏剂量依赖地抑制所有KCNQ家族成员(KCNQ1-5)。(4)美吡拉敏同样抑制大鼠颈上神经元M电流,并通过这种作用引发神经元明显去极化,但美吡拉敏却并不引起神经元兴奋性改变。第一代H1受体抗组胺药美吡拉敏和苯海拉明在大剂量情况下抑制KCNQ/M电流,并且引发神经元明显去极化的作用,很可能是导致其在中毒剂量引发病人抽搐、痉挛、癫痫等神经系统毒性作用的分子机制之一。
第四部分抗组胺药物选择性抑制Kir通道
目的:研究第一、二、三代H1受体抗组胺药对内向整流钾离子通道(Kir)的作用,试图揭示第一代H1受体抗组胺药在大剂量情况下所引起的神经系统毒性反应的分子机制。
方法:利用分子克隆、RNA体外转录以及细胞显微注射等技术,在非洲爪蟾卵母细胞中,外源性表达Kir2.1,2.3,3.4及Kir2.3突变体通道蛋白,再利用双电极电压钳记录卵母细胞全细胞电流。观察大剂量情况下各种H1受体抗组胺药对通道电流的作用。
结果:(1)抗组胺药对表达于卵母细胞Kir2.3通道电流的作用:在高钾外液孵育下的Kir2.3通道呈现明显的内向整流特性,其反转电位位于0 mV附近。外液给予第一代H1受体抗组胺药美吡拉敏(100μM)或苯海拉明(100μM)后,Kir2.3通道电流均受到明显抑制,其抑制率分别为25.0±2.9%和17.3±0.7%。而第二代及第三代H1受体抗组胺药以及H2受体阻断药阿司咪唑,地氯雷他定及西咪替丁对Kir2.3通道没有任何作用。通过对通道I-V曲线进行分析,美吡拉敏对Kir2.3的抑制作用无电压依赖性。美吡拉敏及苯海拉明能剂量依赖地抑制Kir2.3电流,其IC50分别为306.4μM和689.2μM。(2)抗组胺药对表达于卵母细胞Kir2.1通道电流的作用:第一代H1受体抗组胺药美吡拉敏(100μM)及苯海拉明(100μM)对Kir2.1电流没有明显影响。同样如果给予其他类型药物,包括阿司咪唑,地氯雷他定及西咪替丁,均对Kir2.1电流没有影响。(3)抗组胺药对Kir电流的选择性作用与通道―PIP2相互作用程度有关:如果利用分子生物学方法进行点突变,将Kir2.3位于213位的异亮氨酸置换为亮氨酸(即Kir2.3(I213L)),而增强通道与细胞膜磷脂PIP2的亲和力后,我们发现美吡拉敏对通道电流的抑制作用会被明显削弱。此外通过利用另一种与PIP2结合力较弱的Kir3.4进行实验后我们发现,美吡拉敏能明显抑制其电流,抑制率为30.3±4.6%。因此美吡拉敏对Kir电流抑制率大小顺序为Kir3.4 > Kir2.3 > Kir2.3 (I213L) > Kir2.1,而这与以往关于关于Kir通道与PIP2亲和力所报道的顺序一致。(4)第一代H1受体抗组胺药通过抑制Kir2.3电流引发卵母细胞膜电位去极化:在表达了Kir2.3通道后,卵母细胞膜电位为-95.2±2.3 mV。外液给予美吡拉敏后,可以引起细胞膜电位逐渐去极化,达稳态后,膜电位去极化至-57.1±6.1 mV。苯海拉明也具有相似效应,可将卵母细胞膜电位去极化至-70.0±3.4 mV。
结论:(1)第一代H1受体抗组胺药美吡拉敏和苯海拉明能够明显抑制Kir2.3通道电流,且作用具有剂量依赖性,而第二、三代H1受体抗组胺药以及H2受体拮抗剂阿司咪唑,地氯雷他定及西咪替丁对Kir2.3没有任何作用。(2) Kir2.1通道对任何抗组胺药均不敏感。(3)美吡拉敏对Kir2.1与Kir2.3通道之间作用的明显差别很可能源于通道与PIP2的亲和力大小。美吡拉敏也同样能抑制与PIP2亲和力较弱的Kir3.4通道电流。(4)通过抑制表达于爪蟾卵母细胞的Kir2.3通道电流,美吡拉敏可以明显引发卵母细胞去极化。因此第一代H1受体抗组胺药对Kir2.3通道的抑制作用很可能也是其服用过量或中毒情况下引发神经系统毒性作用的分子机制之一。
总结
1缓激肽能够剂量依赖地抑制存在于小背根神经元中的M电流,缓激肽通过作用于其B2受体,能够激活PLC,导致由IP3介导的内Ca2+释放。由缓激肽所引发的细胞内Ca2+信号能够同时引发M电流的抑制与Ca2+激活Cl-通道的开放。这两种通道的共同作用足以引发背根神经元的去极化并导致其兴奋性的增高。在动物行为学实验中,M通道的特异性增强剂能够显著缓解由缓激肽所引发的疼痛效应。上述实验结果揭示了缓激肽引发炎症性疼痛的详细分子机制,并为炎症性疼痛的治疗提供了崭新方向。
2组胺能够抑制HEK293细胞与大鼠颈上神经元中的KCNQ/M通道电流,这一作用是通过H1受体―PLC信号通路引发的PIP2水解所造成的,而与细胞内钙信号无关。与作用已知的两种G蛋白偶联受体激动剂缓激肽与oxo-M相比,组胺对M通道抑制作用的机制更接近于oxo-M。因此H1受体在调解M电流的机制方面与M1受体非常一致。
3第一代抗组胺药物美吡拉敏与苯海拉明能够剂量依赖性的抑制外源性表达于HEK293细胞中的KCNQ2/Q3通道电流。美吡拉敏影响通道的激活、失活动力学,并使其I-V曲线显著右移。美吡拉敏除对KCNQ2/Q3电流有抑制作用外,还能剂量依赖地抑制所有KCNQ通道家族成员(包括KCNQ1-4和KCNQ3/Q5)。美吡拉敏还能抑制大鼠颈上神经元中的M电流,并由此引发神经元显著去极化。美吡拉敏和苯海拉明对KCNQ/M通道抑制作用的IC50均位于临床上所观察到的中毒血药浓度范围之间。因此第一代抗组胺药物对M通道的抑制及由此引发的神经元去极化效应很可能导致其中毒剂量下病人所出现的神经系统毒性症状。
4第一代抗组胺药物美吡拉敏与苯海拉明能够剂量依赖地抑制外源性表达于卵母细胞中的Kir2.3通道电流,并能由此产生去极化效应;而第二与第三代抗组胺药物阿斯咪唑及地氯雷他定则无作用。上述所有药物则对Kir2.1通道电流无作用。美吡拉敏同样能够抑制Kir3.4通道电流。美吡拉敏对Kir通道的选择性抑制作用很可能与通道和细胞膜磷脂PIP2的亲和力存在一定关系。
M type potassium channel conducts a voltage and time dependent, slowly activating, slowly deactivating and non-inactivating outward current. It is such named since it can be robustly inhibited by muscarinic M1 receptor activation. At present, it is generally accepted that the molecular basis of M channel is constituted by homo- and hetero-multimers of KCNQ (KCNQ1-5) subunits. M channel opens at a threshold below -60 mV and this feature allows for a fraction of these channels to be open at or near the resting membrane potential of a neuron. It is suggested that M current in neurons can serve as an intrinsic“voltage-clamp”mechanism controlling the resting membrane potential and the threshold for action potential firing thus repressing the over excitability of neurons. M channel is extensively distributed in nervous system, including hippocampus, cortical, sensory and some sympathetic neurons. Moreover, growing evidence suggests that functional M channels are expressed in the peripheral sensory fibers and their activity strongly contributes to the fiber excitability in vivo.
Ca2+ activated Cl- channels (CACC) are a prominent group of ion channels robustly expressed in many mammalian cell types including neurons, smooth muscles and epithelia. In the nervous system, functional CaCCs are best characterized in neurons mediating different sensory inputs, such as olfactory neurons, photosensitive rods and cons, taste calls and DRG neurons. CaCCs play a role in depolarization or amplification of a depolarizing input produced by other channels in the nervous system. The molecular identity of CaCC remained controversial until very recently when three groups independently identified a member of the transmembrane protein 16A (TMEM16A) protein family as a CaCC subunit. This important finding makes it possible to alter CaCCs in native cells via genetic manipulations
Inwardly rectifying potassium (Kir) channel family contains at least 7 subfamilies (Kir1-7). These channels are extensively distributed in various tissues and serve a role in maintaining K+ equilibrium and keeping membrane potential. These channels play an important role in the repolarization phase of action potential and regulation of cell excitability. Furthermore, recent studies indicate PIP2 may play an important role in modulation of Kir2.0 channel function by several modulators.
Based upon the above mentioned channels, in my thesis, we use techniques such as patch clamp, calcium imaging, siRNA, native neuron transfection, TEVC, LSCM and animal behavioral assay, etc. to systematically study the following subjects: (1) Ionic mechanisms for the acute nociceptive signals induced by inflammatory mediator bradykinin (BK); (2) Mechanism underlying inflammatory mediator histamine regulation of KCNQ/M channels; (3) Mechanism underlying the first-generation antihistamine induced severe neuronal adverse effects.
Part 1 Ionic mechanisms for the acute nociceptive signals induced by bradykinin
Objective: We aim to investigate the mechanisms of acute nociceptive signaling induced by bradykinin. We characterized major membrane currents directly affected by BK in nociceptive sensory neurons, investigated their molecular identities, signaling cascades coupling their regulation to BK receptors and the contribution of these currents to the excitability of nociceptive neurons and to the BK-induced nocifensive behaviour in rats.
Methods: Perforated patch clamp (voltage clamp) was used to monitor the effects of BK on ion channels in DRG neurons. Current clamp was used to record the neuron excitability. Calcium photometry was used to measure the changes of intracellular Ca2+ concentration. Membrane PIP2 hydrolysis was monitored by confocal microscope. Whole animal behavioral study was carried out to monitor the nocifensive behavior of rats upon BK application.
Results: (1) BK inhibits M current in small DRG neurons: BK caused a prominent concentration-dependent inhibition of M current in small DRG neurons, with an EC50 of 60.0±16.3 nM. The inhibitory effect was independent of non-neuronal cells since purified neuron culture did not affect BK’s response. (2) Signaling pathway of BK-induced M current inhibition: BK produced significant phospholipase C (PLC) activation since PLCδ-PH-GFP, a fluorescent probe that binds PIP2 and IP3, translocated from the membrane to the cytosol after BK application. However, membrane PIP2 concentration did not drop significantly since a more specific PIP2 probe YFP-tubby failed to translocate to the cytosol following BK application. Ca2+ imaging experiments showed that BK induced significant intracellular Ca2+ rises in DRG neurons. Patch clamp studies further revealed that the inhibitory effect of BK was significantly attenuated by B2R antagonist Hoe-140, PLC inhibitor edelfosine, IP3 receptor antagonist Xe-C, SERCA inhibitor thapsigargin and high BAPTA internal solution, but was not affected by PKC inhibitor bisindolylmaleimide and PLA2 inhibitor quinacrine. (3) BK activates TMEM16A-dependent Cl- channels: BK can induce a prominent inward current accompanied by M current inhibition. This inward current was not due to TRPV1 or HCN channel opening since ruthenium red and ZD7288 did not affect it. However, this inward current was abolished by Cl- channel blocker DIDS and low Cl- internal solution. Further experiments showed that the inward current was also abolished by B2R antagonist Hoe-140, PLC inhibitor edelfosine, IP3 receptor antagonist Xe-C, SERCA inhibitor thapsigargin and high BAPTA internal solution. The above results suggest that the inward current was conducted by Ca2+ activated Cl- channels (CACC). The gene encoding essential CaCC subunit has recently been identified as Tmem 16a. Experiments using siRNA against Tmem 16a significantly attenuated BK induced inward current, revealing that Tmem 16a is a molecular correlate of the BK-induced Cl- current in DRG neurons. (4) M-current inhibition and CaCC activation contribute to and can account for the excitatory effect of BK: BK and XE991 could both significantly increase DRG neuron excitability and cause depolarization. In low Cl- intracellular solution which abolished BK-induced inward current, co-application of BK + XE991 failed to produce further excitation upon XE991 application. However, XE991 could further excite the neuron in the presence of BK. (5) M channel openers can avert nocifensive behaviour induced by BK: Intraplantar injection of BK (10 nM/site) into the rat hind paw produced strong nocifensive behaviour, which was significantly alleviated by specific M channel openers retigabine or zinc pyrithione. However, the Cl- channel blocker DIDS had no clear effect on the BK-induced nocifensive behaviour and co-application of retigabine and DIDS had only a marginal additivity.
Conclusions: (1) BK concentration-dependently inhibits M current in small DRG neurons. The action of BK on M current is not mediated by non-neuronal cells but due to a primary effect on DRG neurons. (2) BK activates PLC but does not produce a global depletion of the membrane PIP2. BK can also induce intracellular Ca2+ mobilization. Pharmacological studies reveal that the inhibitory effect of BK on M current is due to B2R-PLC and IP3-mediated intracellular Ca2+ rise. (3) A series of pharmacological experiments show that the inward current induced by BK is conducted by CACC. Furthermore, siRNA experiments reveal that the recently discovered Tmem 16a underlies molecular correlate of CACC in DRG neurons. (4) Current clamp studies show that BK causes hyperexcitability in DRG neurons. By comparing the effects of BK and Xe991 in high and low Cl- containing internal solution, we show that M current and CACC are both involved in BK’s excitatory effect. (5) BK induced nocifensive behavior in rat is significantly attenuated by M current enhancers, suggesting inhibition of M channels in the peripheral sensory terminals can indeed produce firing of action potentials in vivo.
Part 2 Phosphatidylinositol 4, 5-bisphosphate hydrolysis mediates histamine-induced KCNQ/M current inhibition
Objective: We aim to study the effects of histamine on KCNQ/M channel current in HEK293 cells and rat SCG neurons. We further explore the signaling pathway and compare the effects of histamine on SCG neurons with those of the well characterized M1 and B2 receptor agonists, oxo-M and BK. The final goal will be to understand the common mechanisms underlying GPCR modulation of KCNQ/M channels in more detail.
Methods: Patch clamp technique was used to study the effect of histamine on channel currents and calcium photometry was used to measure the changes of intracellular Ca2+ concentration. Pharmacology methods, in combination with LSCM, were used to study the signaling pathway underlying histamine modulation of KCNQ/M channel in detail.
Results: (1) Histamine suppresses KCNQ/M current in HEK293 cells and rat SCG neurons via activation of H1 receptor: Histamine (10μM) strongly inhibited KCNQ2/Q3 channel currents heterologusly expressed in HEK293 cells and the inhibition was 77.9±3.5%. The inhibitory effect was not affected by H2 receptor antagonist cimetidine (p > 0.05 vs. control) but was attenuated by H1 receptor antagonist mepyramine (p < 0.01 vs. control). Histamine could also inhibit M channel current in rat SCG neurons in a concentration dependent manner and this effect was also attenuated by mepyramine (p < 0.01 vs. control). The EC50 deduced from the concentration response relationship was 3.3±1.1μM. Moreover, the existence of H1 receptor in SCG neurons was confirmed by Western blot. (2) Histamine suppresses KCNQ/M current via activation of PLC: The inhibitory effect of histamine was significantly reduced to 7.1±0.6% (p < 0.01 vs. control) by a PLC antagonist U-73122. Meanwhile, the inhibitory effect of histamine on M current in SCG neurons was also significantly abolished by U-73122 (p < 0.01 vs. control). (3) Histamine-induced inhibition of KCNQ2/Q3 currents is the result of membrane PIP2 hydrolysis: Histamine produced an obvious translocation of the PLCδ1-PH-GFP probe from the membrane to the cytosol and the effect was reversible upon histamine washout. The rise of fluorescence signals in the cytosol evoked by application of histamine was 80.4±6.0%. This effect was blocked by mepyramine and U-73122 (p < 0.01 vs. control). Patch clamp studies showed that the recovery of the current inhibited by histamine was significantly reduced to 10.5±3.4% (p < 0.01 vs. control) when treated with PI4-Kinase inhibitor wortmannin, compared with 88.4±6.2% in control conditions. In addition, PAO, another PI4-Kinase inhibitor, also significantly reduced the recovery to 6.3±0.9% (p < 0.01 vs. control) compared with 95.4±4.6% in control conditions. (4) Histamine inhibits KCNQ2/Q3 currents in HEK293 cells deprived of [Ca2+]i rises: Histamine (10μM) could induce an obvious [Ca2+]i rise and this effect still existed in Ca2+-free extracellular solution but largely abolished by U-73122. In whole-cell patch clamp configuration, the histamine induced KCNQ current inhibition in HEK293 cells was not altered (81.3±4.0%, p > 0.05 vs. control) when cells were dialyzed with high BAPTA intracellular solution. Besides, thapsigargin, a sarcoplasmic-endoplasmic reticulum Ca2+ pump inhibitor, which can empty the Ca2+ stores, did not affect the effect of histamine, either (88.6±3.9%, p > 0.05 vs. control). (5) H1 receptor stimulation does not evoke [Ca2+]i signal in SCG neurons: Calcium photometry was used to monitor the intracellular Ca2+ changes upon histamine application and the effect of histamine was compared with M1 and BK2 receptor agonist oxo-M and BK. BK could evoke an obvious [Ca2+]i rise, while oxo-M and histamine could not. The rises in the F340/380 ratio produced by histamine, oxo-M and BK were 0.04±0.008, 0.03±0.004 and 0.32±0.05. (6) Histamine does not require [Ca2+]i signal for M current inhibition in SCG neurons: Application of histamine, oxo-M and BK suppressed M currents by 38.3±7.9%, 98.8±0.4% and 85.3±4.4%, respectively, when neurons were dialyzed with low BAPTA internal solution (containing 0.1 mM BAPTA). When neurons were dialyzed with high BAPTA internal solutions, the suppression of M currents by histamine and oxo-M was 32.1±3.1% and 87.3±7.0%, respectively, which was not significantly altered (p > 0.05 vs. control). In contrast, the inhibitory effect of BK was significantly reduced to 20.5±2.6% (p < 0.01 vs. control).
Conclusions: (1) Histamine inhibits KCNQ2/Q3 and M channel currents in heterologusly expressed HEK293 cells and rat SCG neurons by H1 receptor activation and this inhibitory effect shows dose dependence. (2) The inhibitory effect of histamine is closely related to PLC activation. (3) Histamine can induce membrane PIP2 hydrolysis and its inhibitory effect on KCNQ currents may likely be due to the hydrolysis of membrane PIP2. (4) The inhibitory effect of histamine on KCNQ currents in HEK293 cells does not rely on intracellular Ca2+ signals despite the fact that it can mobilize intracellular Ca2+ in these cells. (5) The inhibitory effect of histamine on M currents in SCG neurons is also independent of intracellular Ca2+ signals. This effect is similar with that of the oxo-M. Thus activation of H1 receptor inhibits M currents in rat SCG neurons through a similar mechanism with that of M1 receptor. This finding further enriches our understanding of signaling pathway underlying GPCR modulation of KCNQ/M channel. Moreover, the inhibitory effect of histamine on M current may provide new insights into the understanding of the excitatory effects of histamine on neurons.
Part 3 Antihistamine mepyramine directly inhibits KCNQ/M channel and depolarizes rat superior cervical ganglion neurons Objective: We aim to study the effects of the first-generation
antihistamine mepyramine and diphenhydramine on KCNQ/M channel currents and the underlying mechanisms. We also aim to study their effects on neuron excitability. We try to unravel the molecular mechanism underlying the neuronal toxicity induced by overdose of first-generation antihistamines.
Methods: Patch clamp technique was used to study the effect of mepyramine and diphenhydramine on KCNQ/M channel currents and the changes in channel kinetics were analyzed. Whole-cell patch clamp for intracellular drug application and outside-out patch were used to determine the action side of the drug. Current clamp recording mode was used to monitor the effects of drugs on membrane potentials and neuron excitability.
Results: (1) Mepyramine inhibits KCNQ2/Q3 channel currents and affects channel gating properties: KCNQ2/Q3 channels were expressed heterologusly in HEK293 cells by means of transfection. The first-generation antihistamine mepyramine could strongly inhibit KCNQ2/Q3 currents and the inhibition rate reached 92.0±1.7%. The inhibition developed rapidly and could be largely washed out. This effect showed concentration dependency and IC50 was 12.5±1.8μM. However, the H2 receptor antagonist cimetidine did not affect KCNQ2/Q3 currents. (2) The effect of mepyramine on channel kinetics: Mepyramine could prolong activation and shorten deactivation kinetics of KCNQ2/Q3 channels and right shift the half-maximal activation (V50) from -16.9±0.9 mV to -7.8±1.4 mV. (3) Diphenhydramine shows similar inhibitory effect. The inhibition produced by 100μM diphenhydramine was 66.9±5.9% and the I-V curve was shifted to the right. (4) Effect of mepyramine on KCNQ families (KCNQ1-5): Mepyramine could inhibit homomeric KCNQ1, KCNQ2, KCNQ3, KCNQ4 and heteromeric KCNQ3/5 channel currents and the inhibitory effects showed concentration dependence. (5) Mepyramine directly inhibits KCNQ2/Q3 channel current from outside of the cell membrane: We included mepyramine in the recording pipette for whole-cell recording to see if direct introduction of mepyramine into the cell would result in KCNQ2/Q3 current inhibition in HEK293 cells. In control and mepyramine treated groups, KCNQ2/Q3 current was decreased to 56.0±9.0% and 54.8±11.6% of its initial level after 12 min dialysis. There was no significant difference between these two groups (p > 0.05). After rundown of KCNQ2/Q3 currents, the inhibitory effect of oxo-M, an M1 receptor agonist that hydrolyzes PIP2 on KCNQ2/Q3 current, was significantly reduced, while effect of mepyramine was not. Outside-out patch clamp studies revealed that mepyramine could quickly and significantly inhibit KCNQ currents (90.1±2.8%) in the membrane excised from HEK293 cells. (6) Mepyramine inhibits M current in rat SCG neurons: Meyramine also inhibited M current in SCG neurons, the inhibition was 76.7±2.6% and showed concentration dependence (IC50 = 25.7±0.7μM). In addition, mepyramine also significantly depolarized the SCG neurons from–51.0±5.6 mV to–38.9±7.3 mV. Mepyramine could not induce depolarization in the continued presence of a specific M channel blocker, linopirdine. (7) Effect of mepyramine on evoked neuronal action potentials: We found that linopirdine could induce remarkable hyperexcitability in SCG neurons. On the contrary, mepyramine did not show any effect on the excitability of neurons.
Conclusions: (1) The first-generation antihistamine mepyramine and diphenhydramine can significantly inhibit KCNQ/M currents and this effect is due to direct drug inhibition from the extracellular side. (2) Mepyramine alters KCNQ channel activation and deactivation kinetics and shifts the I-V curve to the right. (3) Mepyramine concentration-dependently inhibits all members of KCNQ family (KCNQ1-5). (4) Mepyramine also inhibits M current in rat SCG neurons and induces depolarization in neurons. However, mepyramine does not evoke hyperexcitability in neurons. These actions are likely to be involved in the adverse neuroexcitatory effects including seizers, convulsion and epilepsy, observed in patients intoxicated by overdose of first-generation antihistamines.
Part 4 Selective inhibition of Kir currents by antihistamines
Objective: We aim to study the effects of the first, second and the third-generation antihistamines on inwardly rectifying potassium (Kir) channels and try to reveal the molecular mechanisms underlying neurotoxic effects by overdose of H1 receptor antagonists.
Methods: DNA preparation, RNA in vitro transcription and microinjection methods were used to express Kir2.1, 2.3 and 3.4 channels in Xenopus oocyte. Two electrode voltage clamp (TEVC) was used to record the whole cell currents in oocyte and to observe the effects of each type of antihistamines on channel currents.
Results: (1) Effect of antihistamines on Kir2.3 currents expressed in Xenopus oocyte: Kir2.3 channel showed a substantial inwardly rectifying property in a high extra-cellular K+ bath solution. The reverse potential was near 0 mV. Extra-cellular perfusion with 100μM mepyramine or diphenhydramine, two histamine H1 receptor antagonist of the first-generation antihistamine, both reduced the current amplitude by 25.0±2.9% and 17.3±0.7%, respectively. However, the second and third-generation H1 receptor antihistamines astemizole, desloratadine and H2 receptor antagonist cimetidine had no effect on Kir2.3 channel current. When deduced from the I-V curve, mepyramine-induced inhibition of Kir2.3 currents was not voltage-dependent. The Kir2.3 current inhibitions induced by mepyramine and diphenhydramine showed concentration dependence and IC50 was 306.4μM and 689.2μM, respectively. (2) Effect of antihistamines on Kir2.1 currents expressed in Xenopus oocyte: Mepyramine and diphenhydramine, at concentrations of 100μM, had little or no inhibitory effect on Kir2.1 channel currents. Similar results were also obtained from other drugs. Astemizole, desloratadine and cimetidine had no effect on Kir2.1 currents. (3) Selective inhibition of antihistamines on Kir currents depends on characteristic channel-PIP2 interaction: We found that the effect of mepyramine could be significantly abolished by a point mutation of Kir2.3, Kir2.3 (I213L), which could confer a stronger channel-PIP2 interaction to Kir2.3 channel. In addition, mepyramine could also inhibit Kir3.4, another member of the Kir subfamily, which has also been shown to be a Kir channel interacting weakly with PIP2 as Kir2.3. Our results indicated the rank order for current inhibition among Kir channels as follows: Kir3.4 > Kir2.3 > Kir2.3 (I213L) > Kir2.1. This result coincided with our previous results showing the rank order of the strength of channel-PIP2 interactions among Kir channels. (4) Inhibition of Kir2.3 current by the first-generation antihistamines leads to the depolarization of resting membrane potential of oocytes: After expressing Kir2.3 channels, the oocytes displayed resting membrane potential (Vm) values of -95.2±2.3 mV. Application of mepyramine (300μM) gradually and significantly depolarized Vm to -57.1±6.1 mV. Similar result was also obtained for 300μM diphenhydramine, which reduced the resting Vm value to -70.0±3.4mV.
Conclusions: (1) The first-generation antihistamines mepyramine and diphenhydramine significantly and concentration dependently inhibit Kir2.3 channel current. However, the second and third-generation H1 receptor antihistamine and H2 receptor antagonist astemizole, desloratadine and cimetidine have no effect on Kir2.3 currnet. (2) Kir2.1 channel is insensitive to any antihistamines. (3) The obvious discrepancy of the effect of mepyramine between Kir2.1 and Kir2.3 is due to channel-PIP2 interactions. Mepyramine also inhibits Kir3.4 channel, which shows weak channel-PIP2 interaction. (4) Mepyramine induces a significant depolarization in oocytes by inhibiting Kir2.3 channel. Thus, Kir2.3 channel inhibition may likely be involved in the molecular mechanisms underlying overdose first-generation antihistamine induced neurotoxic effects.
SUMMARY
1 BK inhibits M-type K+ channel and activates Ca2+-activated Cl- channel in small DRG neurons. BK, acting through its B2 receptors, PLC and releasing of Ca2+ ions from intracellular stores, robustly inhibits M-type K+ channels and activates TMEM 16A-dependent Ca2+-activated Cl- channels. Summation of these two effects adequately accounts for the depolarization and increase in action potential firing induced by BK in DRG neurons. Preclusion of BK-induced inhibition of M channels with specific openers strongly attenuates the nociceptive effect of BK in vivo. The above results reveal the detailed molecular mechanism underlying BK-induced inflammatory pain and provide new directions for the treatment of inflammatory pain.
2 Histamine, via activating H1 receptor, PLC and hydrolyzing PIP2, inhibits KCNQ/M channel in HEK293 cells and rat SCG neurons. This process does not involve Ca2+ signal. By comparing the effects of histamine on SCG neurons with those of the well-characterized M1 and B2 receptor agonist, oxo-M, and BK, we suggest that histamine inhibits M current in a similar mechanism with oxo-M.
3 The first-generation antihistamine mepyramne and diphenhydramine concentration dependently inhibit KCNQ2/Q3 channel exogenously expressed in HEK293 cells. Meyramine alters channel activation and deactivation kinetics and shift the I-V curve to the right. In addition, mepyramine concentration dependently inhibits all KCNQ channels, including KCNQ1-4 and KCNQ3/Q5. Moreover, mepyrmaine, by inhibiting M channel current in rat SCG neurons, induces significant membrane potential depolarization in neurons. The IC50 for the inhibitory effect of mepyramine lies in the range of plasma drug concentration of intoxicated patients. Thus, KCNQ/M channel inhibition and the subsequent marked depolarization of neuron membrane potential may participate in adverse effects observed in patients intoxicated by overdose with first-generation antihistamines.
4 The first-generation antihistamine mepyramne and diphenhydramine dose dependently inhibit Kir2.3 channel current expressed in Xenopus oocytes and induce membrane depolarization, while the second and third-generation antihistamine astemizole and desloratadine do not. Kir2.1 current is insensitive to any of these drugs. Mepyrmaine also inhibits Kir3.4 current. We speculate that the specific Kir channel inhibition produced by the first-generation antihistamine is likely to be determined by the strength of channel-PIP2 interactions.
引文
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