EGF对延迟整流性钾离子通道功能调节的研究
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摘要
延迟整流性钾离子通道在心肌动作电位的复极化过程中发挥重要的作用,它包含两种不同成分:快激活延迟整流钾通道电流(IKr)和缓慢激活延迟整流钾通道电流(IKs)。IKs通道是由KCNQ1编码的α亚基和KCNE1编码的β亚基组成的异源多聚体。而IKr通道蛋白的α亚基由hERG基因编码,KCNQ1、hERG编码的α亚基具有6个跨膜结构域和一个具有离子选择性的P环,4个相同α亚基的P环组成一个离子滤过性孔道,孔区的结构高度保守,决定了通道对钾离子的高度选择性,第4个跨膜区(S4)上含有4个带正电荷的氨基酸残基,为电压敏感区,能够感受膜电位的变化,调节孔道开放与关闭。N-末端和C-末端都位于胞内。KCNE1编码的蛋白为单跨膜多肽链,作为辅助亚基调节KCNQ1通道的生物物理特性。KCNQ1、KCNE1和hERG基因突变均可引起先天性长QT综合症。延迟整流性钾离子通道功能异常或数目上调、下调均可导致心律失常的发生,因此,该通道对于维持正常的动作电位以及产生病理性的心律失常都具有重要意义,是多种调节途径作用的重要靶点。
表皮生长因子受体(EGFR)是I型跨膜酪氨酸激酶受体,定位于细胞膜,编码分子量170KDa的跨膜糖蛋白,由三个部分组成:即胞外配体结合区;疏水性的跨膜区;具有酪氨酸蛋白激酶活性的胞内区。与配体EGF结合后EGFR构象发生变化,引起受体在膜上迁移、聚合、形成寡聚体,同时被激活,磷酸化自身的酪氨酸残基,使EGFR激酶活性进一步提高,并对其它底物蛋白进行磷酸化。EGFR受体的酪氨酸激酶发挥两方面作用:一方面磷酸化自身酪氨酸残基,为募集其它信号分子提供平台;另一方面将募集的靶蛋白的酪氨酸残基磷酸化,引起下游信号转导。EGFR在调控细胞的生长、分化、增殖、迁移、凋亡等生理功能中发挥重要作用。
离子通道受细胞内许多信号途径包括蛋白磷酸化和去磷酸化的调节,广谱酪氨酸激酶抑制剂tyrphostin A23、tyrphostin A25和genistein可抑制豚鼠心室肌细胞的基础IKs,酪氨酸磷酸酶抑制剂orthovanadate可反转酪氨酸激酶抑制剂引起的IKs电流的降低,因此认为:基础IKs依赖于IKs通道或通道调节蛋白的酪氨酸磷酸化水平。
本研究以非洲爪蟾卵母细胞作为表达系统,观察EGFR激活后对KCNQ1电流、KCNQ1/KCNE1电流和hERG通道电流的影响,并深入探讨其作用机制,并在豚鼠乳头肌组织上观察了EGF对动作电位的影响,以证实EGFR对IKs和IKr的调节作用是否具有生理学意义。
一、KCNQ1及KCNQ1/KCNE1钾离子通道电流特征
目的:在非洲爪蟾卵母细胞中表达KCNQ1、KCNQ1/KCNE1钾离子通道,观察KCNQ1和KCNQ1/KCNE1钾离子通道的电流特性。
方法:(1)cRNA的制备:KCNQ1、KCNE1的cDNA分别克隆于pGEMHE质粒载体中,提取质粒DNA,经NheⅠ限制性核酸内切酶消化及纯化后,作为模板,用T7体外转录试剂盒(Promega),合成KCNQ1,KCNE1 cRNA,纯化后进行RNA的定量。(2)爪蟾卵母细胞的分离:在爪蟾冰冻麻醉状态下,于下腹部切开一小口,取出卵母细胞,放入含2 g·L-1胶原酶的OR2溶液(mmol·L-1: NaCl 82.5,KCl 2,MgCl2 1,HEPES 5, pH 7.4)中翻摇消化1.5~2hr,制成单个卵母细胞,挑选Ⅴ-Ⅵ级卵母细胞放入ND96培养液中(mmol·L-1:NaCl 96, KCl 1, CaCl2 1.8, MgCl2 1, HEPES 5, pH 7.4)。(3)将5 ng KCNQ1、1 ng KCNE1混合后注射入卵母细胞中,将卵母细胞放入含2.5mM丙酮酸钠和50mg.L-1庆大霉素的ND96培养液中,置于18℃培养箱中,于注射后2~6d,采用双电极电压钳技术观察KCNQ1和KCNQ1/KCNE1钾离子通道的电流特性。
结果:(1)KCNQ1和KCNE1质粒DNA经NheⅠ限制性核酸内切酶消化后,电泳迁移速度明显减慢,与DNA Marker比对,分子量大小约为5Kb和3.4Kb。KCNQ1和KCNE1 cRNA由琼脂糖凝胶电泳证实。(2)KCNE1可明显增大KCNQ1电流。当电压去极至40mV再复极至-50mV时,KCNQ1及KCNQ1/KCNE1尾电流分别为(0.09±0.01)μA和(0.39±0.03)μA(P<0.01,n=6),即KCNE1使KCNQ1通道电流增大4倍多。(3)KCNE1对KCNQ1通道电流动力学特征的影响:膜电压钳制在-80mV,从-60mV开始以阶跃电压10mV的方式逐步去极化至50mV,维持4s,然后电压恢复至-50mV。用Boltzmann方程拟合标准化的尾电流-电压关系曲线,得到半数激活电压(V1/2),KCNQ1通道电流和KCNQ1/KCNE1通道电流的V1/2分别为(-22.8±1.7)mV,(30.9±0.8)mV,因此,KCNE1使KCNQ1的激活电压明显右移。用单指数方程拟合去极至30mV时的激活曲线和从去极化电压30mV复极至-50mV时的尾电流曲线,得到激活时间常数和去活时间常数。KCNQ1/KCNE1电流的激活时间常数(3.84±0.52s),与KCNQ1(0.89±0.05s)相比,明显延长(P<0.01),说明辅助亚基KCNE1明显减慢KCNQ1电流的激活速度。KCNQ1/KCNE1通道电流的去活时间常数(1.01±0.02s)与KCNQ1(2.29±0.39s)相比,明显减小(P<0.05),表明KCNE1可加速KCNQ1电流的去活速度。KCNQ1尾电流的起始部呈弯钩状,是从失活态快速恢复的结果,说明KCNQ1电流存在一定程度的失活,KCNE1可去除KCNQ1电流的失活。(4)KCNE1与KCNQ1共表达后,可增加对IKs特异性阻断剂Chromanol 293B的敏感性,50μmol.L-1Chromanol 293B对KCNQ1和KCNQ1/KCNE1通道电流的抑制率分别为(65.5±5.8)%和(79.9±0.6)%。
结论:KCNQ1通道电流具有快激活、慢去活、部分失活的特性,KCNE1明显改变KCNQ1的门控特性,减慢其激活速度,消除失活,使激活电压向正电位转移。KCNQ1/KCNE1电流幅度比KCNQ1电流大4倍多,此外,KCNE1可提高KCNQ1电流对Chromanol 293B的敏感性。
二、EGF对KCNQ1/KCNE1钾通道电流的影响及其机制研究
目的:观察EGFR激活对爪蟾卵母细胞表达的KCNQ1和KCNQ1/KCNE1电流的影响,并探讨其作用机制;观察EGF对豚鼠乳头肌动作电位的影响。
方法:KCNQ1、KCNE1和EGFR的cDNA分别克隆于pGEMHE质粒载体中,NheⅠ限制性核酸内切酶将质粒DNA线性化,作为模板,应用RibomaxTM large scale RNA production systems-T7 kit,体外合成KCNQ1、KCNE1和EGFR cRNA,纯化后,cRNA的大小和浓度经琼脂糖凝胶电泳,与RNA ladder比对后确定。将5ng KCNQ1、5ng EGFR和1ng KCNE1 cRNA或5ng KCNQ1、5ng EGFR混合后注射入卵母细胞中,放入含2.5mM丙酮酸钠和50mg.L-1庆大霉素的ND96培养液中,置于18℃培养箱,于注射后2~6天,采用双电极电压钳,室温下记录电流。采用细胞内记录观察EGF对豚鼠乳头肌动作电位的影响,应用定向点突变和免疫共沉淀的方法探讨其可能的作用机制。
结果:(1)EGF剂量依赖性地抑制KCNQ1/KCNE1电流,EGF 3,10,30,100,300ng/ml对KCNQ1/KCNE1电流的相对抑制率分别为(6.0±2.1)%, (20.8±3.4)%, (38.9±2.7)%, (57.3±1.5)%和(65.9±2.8)%,半数抑制浓度(IC50)为(24.1±1.8)ng/ml。(2)100ng/ml EGF明显增大KCNQ1电流,灌流EGF前、后的KCNQ1电流大小分别为(0.27±0.02)μA和(0.42±0.04)μA。(3)EGF抑制KCNQ1/KCNE1电流在0～90mV之间呈电压依赖性。随着电压的增大,EGF抑制KCNQ1/KCNE1电流的作用逐渐减小。(4)EGF改变KCNQ1/KCNE1电流的动力学特征:使半数激活电压右移约7mV;对照组KCNQ电流激活时间常数(10mV)为(4.8±0.6)s,给予100ng/ml EGF后,激活时间常数增大至(7.1±0.7)s,说明EGF减慢KCNQ1/KCNE1电流的激活速度;与对照组(1063±102)ms相比,100ng/ml EGF可增加慢去活时间常数(1251±138)ms,与对照组(339±34)ms相比,100ng/ml EGF显著增加快去活时间常数(429±28)ms,表明EGF减慢KCNQ1/KCNE1电流的去活速度。(5)分析与EGF影响KCNQ1/KCNE1电流有关的信号转导途径。Src激酶的抑制剂PP2(200nM)和Ca2+螯合剂EGTA(5μM)不影响EGF对KCNQ1/KCNE1电流的抑制作用;与溶剂对照DMSO组(43.8±4.2%)相比,酪氨酸激酶抑制剂Genistein(200μM)可明显降低EGF对KCNQ1/KCNE1电流的抑制率(4.8±2.9%)(P<0.01,n=6),提示其作用与通道或通道调节蛋白的酪氨酸磷酸化有关。(6)分析EGF引起KCNQ1电流增加的机制。EGTA(5μM)和PLC抑制剂U73122(3μM)降低EGF对KCNQ1电流的增加率(分别由66±2.0%到25±1.8%、76±10%到26±9%),提示其作用与细胞内PLC-PIP2-IP3-Ca2+信号转导途径有关。(7)KCNE1(S102A)和KCNE1(Y81A)突变体不影响EGF对KCNQ1/KCNE1电流的抑制作用,但KCNQ1/KCNE1(Y81A)对Chromanol 293B(50μM)的敏感性降低,Chromanol 293B对KCNQ1/KCNE1(Y81A)电流的抑制率为(38.3±8.7)%,而对野生型KCNQ1/KCNE1电流的抑制率为(69.9±2.6)%。此外,KCNE1 ( Y81A )改变电流激活动力学特征,使激活电压右移,KCNQ1/KCNE1(Y81A)电流在+40mV以上时才被激活,提示KCNE1第81位的酪氨酸残基对于调节KCNQ1/KCNE1电流幅度和门控机制是非常重要的。(8)100ng/ml EGF可延长豚鼠乳头肌动作电位的复极过程,增加APD50(给药前127±21ms,给EGF后148±24ms)APD90(给药前189±16ms,给EGF后205±13ms),减慢0相最大上升速率(Vmax)(给药前149±28V/s,给EGF后111±30V/s),提示EGF在抑制延迟整流性K+通道的同时,还可能抑制Na+离子通道。(9)免疫共沉淀的结果显示EGF可增加KCNE1亚基酪氨酸磷酸化的水平,对KCNQ1亚基的酪氨酸磷酸化水平无影响,表明EGF可能通过直接磷酸化KCNE1亚基上的酪氨酸残基而影响KCNQ1/KCNE1电流的大小和门控机制。
结论:EGFR激活后以不同的机制调节KCNQ1通道和KCNQ1/KCNE1通道,可能通过PLC-PIP2-IP3-Ca2+信号转导途径增加KCNQ1通道电流;通过磷酸化KCNE1的酪氨酸残基而抑制KCNQ1/KCNE1通道电流。EGF可延长豚鼠乳头肌动作电位的复极化过程,因此,EGF调节延迟整流性K+通道的作用可能具有生理学意义。
三、EGF对爪蟾卵母细胞上表达的hERG通道电流的影响
目的:在爪蟾卵母细胞上表达hERG通道和EGFR,观察EGFR激活后对hERG通道电流的影响。
方法:EGFR cDNA克隆于pGEMHE载体中,以NheⅠ限制性核酸内切酶线性化的质粒DNA作为模板,应用T7体外转录试剂盒,合成EGFR cRNA;hERG cDNA克隆于pSP64载体中,以EcoRⅠ限制性核酸内切酶线性化的质粒DNA作为模板,应用SP6体外转录试剂盒,获得hERG cRNA。将5ng hERG和5ng EGFR cDNA混合后注入卵母细胞中,采用双电极电压钳观察EGFR激活对hERG通道电流的影响。
结果:hERG电流为电压依赖性的外向电流,具有快速激活,快速失活,被IKr特异性阻断剂E-4031阻断的特点。(1)将电压钳制在-80mV,然后去极化至0mV,再复极至-50mV,记录hERG电流。100ng/ml EGF降低hERG尾电流(复极至-50mV的最大电流),给药前(0.44±0.07)μA,给EGF后(0.28±0.04)μA,用ND96冲洗后,电流可完全恢复。因此,EGF可逆性地抑制hERG电流(。2)分析在-70m~38mV之间EGF对hERG电流作用的电压依赖性。结果显示在-34~14mV之间EGF电压依赖性地抑制hERG电流。(3)EGF右移hERG电流的半数激活电压约15mV,给药前V1/2为(-23.9±0.8)mV,给EGF后V1/2为(-8.6±0.8)mV。(4)100ng/ml EGF可增加hERG电流的激活时间常数(给药前433±85ms,给EGF后574±183ms),减慢hERG电流的激活;100ng/ml EGF可降低hERG电流的去活时间常数(从1500±224ms至810±138ms),加快hERG电流的去活速度。
结论:EGFR激活后抑制hERG电流幅度,改变其动力学特征,使激活电压右移,减慢激活速度,加快去活速度。
四、双苯氟嗪对表达于爪蟾卵母细胞上的KCNQ1/KCNE1钾通道电流的影响
目的:研究双苯氟嗪对KCNQ1/KCNE1钾通道电流的影响,以探讨其抗心律失常作用的可能机制。
方法:采用双电极电压钳技术,观察双苯氟嗪对表达于非洲爪蟾卵母细胞上的KCNQ1/KCNE1钾通道电流的影响。
结果:双苯氟嗪浓度依赖性地抑制KCNQ1/KCNE1电流,双苯氟嗪0.3,1,,3,10,30μmol·L-1对KCNQ1/KCNE1电流的抑制率分别为(6.0±1.0)%,(11.6±1.5)%,(25.7±3.5)%,(45.6±3.5)%,(63.5±7.6)%。IC50为(8.9±1.8)μmol·L-1。在-10~90mV范围内双苯氟嗪对KCNQ1/KCNE1电流的抑制作用具有电压依赖性。10μmol·L-1双苯氟嗪使KCNQ1/KCNE1电流的半数激活电压右移约3mV,显著增大激活时间常数(从3.7±0.6s到5.0±0.5 s),减慢KCNQ1/KCNE1电流的激活;降低慢去活时间常数和快去活时间常数(从1135±91ms、368±27ms至879±78ms、313±45ms),加速KCNQ1/KCNE1电流的去活。
结论:双苯氟嗪浓度依赖性地抑制KCNQ1/KCNE1钾通道电流,并改变其动力学特征,提示双苯氟嗪抗心律失常的作用可能与此有关。
小结
1. KCNQ1通道电流具有快激活、慢去活、部分失活的特征,KCNE1明显改变KCNQ1通道的门控特性,减慢其激活,消除失活,使激活电压右移,KCNE1/KCNQ1电流比KCNQ1电流大4倍多,KCNE1增加KCNQ1电流对Chromanol 293B的敏感性。
2. EGFR激活对KCNQ1电流和KCNQ1/KCNE1电流的作用不同。EGF可能通过PLC-PIP2-IP3-Ca2+信号转导途径增加KCNQ1电流;EGF可能通过磷酸化KCNE1的酪氨酸残基抑制KCNQ1/KCNE1电流。EGF可延长豚鼠乳头肌动作电位的复极过程,表明EGF对延迟整流性K+通道的调节作用具有生理学意义。
3. EGFR激活抑制hERG电流幅度和改变其动力学特征,使激活电压右移,减慢hERG电流的激活速度,加快去活速度。EGF可能通过调节KCNQ1/KCNE1和hERG通道影响心肌动作电位的复极化过程。
4.双苯氟嗪浓度依赖性地抑制KCNQ1/KCNE1电流。IC50为8.9±1.8μmol.L-1;在-10~90 mV范围内双苯氟嗪对KCNQ1/KCNE1电流的抑制作用具有电压依赖性;10μmol·L-1双苯氟嗪使KCNQ1/KCNE1电流的半数激活电压右移3mV,明显增加KCNQ1/KCNE1电流的激活时间常数,降低快、慢去活时间常数。双苯氟嗪抑制KCNQ1/KCNE1电流的幅度,改变其激活和去活动力学特征可能与其抗心律失常的作用有关。
The delayed rectifier potassium currents played a key role in myocardial repolarization, which were composed of two different elements: slowly delayed rectifier potassium currents (IKs) and rapidly delayed rectifier potassium currents (IKr).α-subunit encoded by KCNQ1 heteromultimericly coassembled withβ-subunit encoded by KCNE1 to form IKs channels. hERG encodesα-subunit of IKr. Both KCNQ1 and hERG subunit consisted of six putative transmembrane domains (S1~S6) and an ion-selective P-loop. Fourα-subunits formed the outer pore and contain the selectivity filter. KCNE1 protein consisted of a single transmembrane domain, and altered the biophysical properties of the channels. Mutations in either IKs subunits or IKr subunits were associated with variants of the congenital long QT syndrome. Mutant subunits led to reduction of IKs or IKr by a loss-of-function mechanism, often with a dominant-negative effect. In the heart, reduced IKs or IKr led to prolongation of the cardiac action potential, lengthening of the QT interval, and increased risk of arrhythmia. Thus, the delayed rectifier potassium channels were involved in maintaining normal action potential as well as inducing arrhythmia. The delayed rectifier potassium channels were targets of many modulatory mechanisms.
The epidermal growth factor receptor (EGFR) belonged to subclass I of the superfamily of the receptor tyrosine kinases. It was composed of an extracellular ligand binding domain, a single transmembrane domain, and an intracellular domain possessing PTK activity. Binding of epidermal growth factor resulted in EGFR dimerization and subsequent activation of the intrinsic tyrosine kinase activity. These phosphorylated tyrosines functioned as docking sites for a variety of signaling molecules that regulated membrane-proximal steps of signal transduction cascades that ultimately brought about cellular responses to EGFR ligands. The EGFR controled a wide variety of biological processes such as cell proliferation, differentiation, and migration and modulation of apoptosis.
Ion channels were targets of many intracellular signaling pathways, including protein phosphorylation and dephosphorylation. Constitutive IKs recorded from guinea-pig ventricular myocytes was suppressed by broad-spectrum tyrosine kinase (TK) inhibitors tyrphostin A23, tyrphostin A25 and genistein. The phosphotyrosyl phosphatase inhibitor orthovanadate almost completely reversed the suppression of IKs induced by broad-spectrum TK inhibitors. Basal IKs was strongly dependent on tyrosine phosphorylation of IKs channel (or channel-regulatory) protein.
In the present study, we used Xenopus oocytes as expression system to observe effect of activation of EGFR on KCNQ1 alone, or KCNQ1/KCNE1 as well as hERG currents, and the mechanism of these effects. Besides this, we studied the effect of EGF on action potential of guinea pig papillary muscles to study the physiological significance of EGFR-mediated modulation of IKs and IKr.
1. The characteristics of KCNQ1 and KCNQ1/KCNE1 currents
Aim: To observe the characteristics of KCNQ1 current and KCNQ1/KCNE1 currents expressed in Xenopus oocyte.
Methods: (1) Preparation of cRNA: KCNQ1 and KCNE1 cDNA were subcloned respectively into the pGEMHE plasmid vector. The sequences of all constructs were confirmed by sequencing. The plasmid DNA was amplified and extracted from E coli transformed with constructs. The plasmid was lineared by NheⅠrestriction endonuclease and purified with DNA fragment purification kit (TakaRa). All cRNAs were synthesized in vitro using RNA production systems-T7 kit (Promega), and were quantified after purification with RNA clean kit (Tiangen). (2) Preparation of Xenopus oocytes: Oocytes were surgically removed under iced anesthesia and placed into OR2 solution (mmol·L-1: NaCl 82.5,KCl 2,MgCl2 1,HEPES 5, pH7.4)containing 2g.L-1 collagenase and were rocked for 1.5 to 2 hours. (3) StageⅤ-Ⅵoocytes were selected and injected with cRNA containing 5ng KCNQ1 with or without 1ng KCNE1 and were incubated at 18℃in ND96 solution (mmol·L-1:NaCl 96, KCl 1, CaCl2 1.8, MgCl2 1, HEPES 5, pH 7.4) plus 2.5 mM pyruvic acid and 50mg.L-1 gentamicin. Currents were recorded at room temperature 2-6d after injection using two-microelectrode voltage-clamp technique.
Results: (1) The lineared plasmid DNA ran slower than the circular plasmid in agarose electrophoresis. Compared with DNA marker, KCNQ1 and KCNE1 plasmid DNA were 5Kb and 3.4Kb, respectively. KCNQ1 and KCNE1 cRNA were verified by agarose electrophoresis. (2) KCNE1 increased KCNQ1 current amplitude. The tail current of KCNQ1 at -50mV following by repolarization to 40mV was (0.09±0.01)μA, whereas KCNQ1/KCNE1 currents at same voltage was (0.39±0.03)μA (P<0.01, n=6). Thus, KCNE1 increased KCNQ1 current over fourfold. (3) KCNE1 altered kinetics characteristics of KCNQ1 current. From a holding potential of -80mV, oocytes were depolarized for 4 s to test potentials between -60mV to 50mV in 10mV steps followed by repolarization to -50mV. Normalized tail current-voltage curves were fitted with Boltzmann equation, and the half activation voltage (V1/2) of KCNQ1 and KCNQ1/KCNE1 currents were (-22.8±1.7) mV and (30.9±0.8) mV, respectively. Thus, KCNE1 shifted KCNQ1 activation voltage to more positive potentials. The activated current trace at +30mV and the tail current trace at -50mV following depolarization to +30mV were fitted with monoexponential function to acquire the time constants of activation and deactivation, respectively. The activation time constants of KCNQ1/KCNE1 was increased significantly to (3.84±0.52s), from (0.89±0.05s) of KCNQ1 alone (P<0.01), suggesting KCNE1 slowed KCNQ1 current activation. The deactivated time constants of KCNQ1/KCNE1 (1.01±0.02s) was lowered significantly, compared with that of (2.29±0.39s) of KCNQ1 alone, suggesting KCNE1 accelerated KCNQ1 current deactivation. KCNQ1 tail currents display a“hook”, indicating that KCNQ1 inactivates to some extent. KCNE1 eliminated inactivation of KCNQ1 current. (4) Coexpression of KCNE1 increased sensitivity of KCNQ1 current to Chromanol 293B, a selective blocker of IKs. The KCNQ1 current inhibition rates with or without KCNE1 by Chromanol 293B were (79.9±0.6)% and (65.5±5.8)%.
Conclusion: KCNQ1 current exhibited a rapidly activating, slow deactivating, partly inactivating characteristics. KCNE1 dramatically modulated KCNQ1 gating, slowing activation, removing inactivation, and shifting the voltage dependence of activation to more positive potentials. KCNE1 plus KCNQ1 had fourfold greater current amplitudes than KCNQ1 alone. KCNE1 increased sensitivity of KCNQ1 current to Chromanol 293B. 2. Effect of EGF on KCNQ1 and KCNQ1/KCNE1 currents expressed in Xenopus oocytes.
Aim: To investigate the effect of activation of EGFR on KCNQ1 and KCNQ1/KCNE1 currents expressed in Xenopus oocytes, and to explore the underlying mechanism. To observe the effect of EGF on action potentials of guinea pig papillary muscles.
Methods: A full-length KCNQ1, KCNE1, EGFR cDNA were subcloned into the pGEMHE plasmid vector, respectively. Complementary RNAs (cRNAs) from all contructs were prepared in vitro from NheⅠ-lineared DNA templates using RibomaxTM large scale RNA production systems-T7 kit. cRNAs size and concentration were estimated by comparison with RNA ladder in agarose gel electrophoresis following purification of cRNAs. StageⅤ-Ⅵoocytes were injected with cRNA containing 5ng KCNQ1, 5ng EGFR with or without 1ng KCNE1 and incubated at 18℃in ND96 solution plus 2.5 mM pyruvic acid and 50mg.L-1 gentamicin. Currents were recorded at room temperature 2-6d after injection using two-microelectrode voltage-clamp technique. The effect of EGF on action potentials from guinea pig right ventricular papillary muscle was observed with a conventional intracellular recording technique. Site-directed mutagenesis and immunoprecipitation technique were used to explore mechanism underlying effect of activation of EGFR on KCNQ1/KCNE1 currents.
Results: (1) EGF suppressed KCNQ1/KCNE1 currents amplitudes in a dose-dependent manner. EGF at 3,10,30,100,300ng/ml inhibited KCNQ1/KCNE1 currents by (6.0±2.1)%, (20.8±3.4)%, (38.9±2.7)%, (57.3±1.5)%, and (65.9±2.8)%, respectively; The concentration for half maximal inhibition (IC50) was (24.1±1.8)ng/ml. (2) EGF at 100ng/ml increased KCNQ1 current. KCNQ1 current before and after EGF perfusion were (0.27±0.02)μA and (0.42±0.04)μA, respectively. (3) EGF voltage-dependently suppressed KCNQ1/KCNE1 currents amplitudes at membrane potentials between 0 and +90mV, with increased voltages, inhibition decreased. (4) EGF altered kinetics characteristic of KCNQ1/KCNE1 currents. EGF at 100ng/ml shifted V1/2 of KCNQ1/KCNE1 currents activation by 7mV toward more positive potentials, and significantly increased the activating time constant at 10mV (from 4.8±0.6s to 7.1±0.7s), increased slow deactivation time constant (from 1063±102ms to 1251±138ms), and fast deactivation time constant (from 339±34ms to 429±28ms), indicating EGF slowed activation and deactivation of KCNQ1/KCNE1 currents. (5) The signaling associated with effect of EGF on KCNQ1/KCNE1 currents was analyzed. PP2(200nM), a blocker of Src kinase, and Ca2+ chelator EGTA(5μM) did not affect EGF-induced inhibition of KCNQ1/KCNE1 currents. Genistein(200μM), an inhibitor of tyrosine kinase, eliminated the effect of EGF on KCNQ1/KCNE1 currents(from 43.8±4.2% to 4.8±2.9%), suggesting tyrosine phosphorylation of channel or channel modulator was involved. (6) The mechanism of EGF-induced activation of KCNQ1 current was analyzed. EGTA(5μM) or PLC inhibitor U73122(3μM) reduced EGF-induced activation of KCNQ1 current (from 66±2.0% to 25±1.8%, from 76±10% to 26±9%, respectively), indicating involvement of PLC-PIP2-IP3-Ca2+ signaling. (7) Mutation of KCNE1 (Y81A) or (S102A) did not affect EGF-induced modulation of KCNQ1/KCNE1 currents. However KCNQ1/KCNE1(Y81A) became insensitive to Chromanol 293B inhibition. KCNQ1/KCNE1(Y81A) currents were only inhibited by 38.3±8.7% by Chromanol 293B, as compared with a 69.9±2.6% inhibition for wild type KCNQ1/KCNE1. KCNE1(Y81A) also altered activation kinetics, shifting activation potentials to more positive direction. KCNQ1/KCNE1(Y81A) currents did not activate until a membrane depolarization to +40mV. These results suggested tyrosine in KCNE1 81 was important in modulating KCNQ1/KCNE1 currents amplitude and gating. (8) EGF lengthened the action potentials repolarization of guinea pig papillary. EGF at 100ng/ml increased APD50 (from 127±21ms to 148±24ms) and APD90 (from 189±16ms to 205±13ms), slowed the maximal rate of rise of AP (from 149±28V/s to 111±30V/s), suggesting EGF inhibited both delayed rectifier K+ channels and Na+ channel. (9) Results of immunoprecipitation showed EGF increased tyrosine phosphorylation of KCNE1, but did not affect tyrosine phosphorylation of KCNQ1, suggesting KCNQ1/KCNE1 currents amplitude and gating were modulated through tyrosine phosphorylation of KCNE1
Conclusion: Activation of EGFR had different effects on KCNQ1 current and KCNQ1/KCNE1 currents. EGF-induced activation of KCNQ1 current was possibly through PLC-PIP2-IP3-Ca2+ signaling pathway. EGF-induced inhibition of KCNQ1/KCNE1 currents was possibly mediated through tyrosine phosphorylation of KCNE1. EGF lengthened action potential repolarizion in guinea pig papillary. This would indicate modulation of delayed rectifier K+ channels by EGF may have physiological significance. 3. Effect of EGF on hERG channel current expressed in Xenopus oocytes
Aim: To investigate the effect of activation of EGFR on hERG current expressed in Xenopus oocytes.
Methods: EGFR cDNA was subcloned into the pGEMHE plasmid vector. EGFR cRNA was prepared in vitro from NheⅠ-lineared DNA template using RibomaxTM large scale RNA production systems-T7 kit. hERG cDNA was subcloned into pSP64 vector. hERG cRNA was synthesized with RibomaxTM large scale RNA production systems-SP6 kit from EcoRⅠ-lineared DNA template. Mixture of 5ng EGFR cRNA and 5ng hERG cRNA were injected into oocytes. Effect of activation of EGFR on hERG current was analyzed using two electrode voltage-clamp technique.
Results: hERG current possessed characteristic of voltage-dependent fast activating and fast inactivating outward current sensitive to IKr blocker E-4031. (1) hERG current was elicited using a protocol in which the potential was first held at -80mV and then depolarized to 0mV followed by repolarization to -50mV. Tail current was measured upon repolarization to -50mV. EGF at 100ng/ml decreased hERG tailed current from(0.44±0.07)μA to (0.28±0.04)μA. hERG tailed current recovered completely after washout of EGF. Thus, EGF inhibited reversibly hERG current. (2) Voltage-dependency of EGF action on hERG was tested in voltages from -70mV to 38mV. The results showed EGF suppressed hERG current in a voltage-dependent manner from -34mV to 14mV. (3) EGF shifted V1/2 of hERG current about 15 mV towards more positive potentials (from -8.0±0.6 mV to 6.7±0.8 mV). (4) EGF lengthened activation time constant of hERG current from (433±85)ms to (574±183)ms, suggesting EGF slowed activation of hERG current. EGF decreased deactivation time constant from (1500±224)ms to (810±138)ms, suggesting EGF promoted deactivation hERG current.
Conclusion: Activation of EGFR inhibited hERG current amplitude and altered kinetics characteristics, shifting the curve of voltage-dependent activation to the right and slowing the activation and promoting the deactivation.
4. Effect of dipfluzine on KCNQ1/KCNE1 potassium currents expressed in Xenopus oocytes
Aim: To investigate the effect of dipfluzine on KCNQ1/KCNE1 potassium currents expressed heterologously in Xenopus oocytes.
Methods: Using Xenopus oocytes expression system, the current amplitude and kinetic characteristics of KCNQ1/KCNE1 were measured with the two electrode voltage-clamp technique before and after dipfluzine application.
Results: Dipfluzine concentration-dependently inhibited KCNQ1/KCNE1 currents. Dipfluzine at 0.3, 1, 3, 10, 30μmol·L-1 inhibited KCNQ1/KCNE1 currents by 6.0±0.9%, 11.6±0.8% , 25.7±2.9%, 45.6±2.5%, 63.5±1.6%, respectively. IC50 was 8.9±1.8μmol·L-1. The solvent (DMSO, 0.1% ) did not affect KCNQ1/KCNE1 currents. Dipfluzine-induced inhibition of KCNQ1/KCNE1 currents was voltage dependent at membrane potentials between -10 and 90 mV. Dipfluzine at 10μmol·L-1 shifted V1/2 of KCNQ1/KCNE1 currents activation by 3mV toward more positive potentials, and significantly increased the activating time constant (from 3.7±0.6 s to 5.0±0.5 s), slowed KCNQ1/KCNE1 currents activation. Dipfluzine at 10μmol·L-1 significantly decreased the slow and fast deactivating time constants(from 1135±91 ms to 879±78 ms and 368±27ms to 313±45ms, respectively), enhanced KCNQ1/KCNE1 currents deactivation.
Conclusion: Dipfluzine concentration-dependently and voltage- dependently inhibited KCNQ1/KCNE1 currents and modified kinetic characteristics of KCNQ1/KCNE1 activation and deactivation, which might be correlated with its antiarrhythmic effect.
SUMMARY
1. KCNQ1 current exhibited a rapidly activating, slow deactivating, partly inactivating characteristics. KCNE1 dramatically modulated KCNQ1 gating, slowing activation, removing inactivation, and shifting the voltage dependence of activation to more positive potentials. KCNE1 plus KCNQ1 had fourfold greater current amplitudes than KCNQ1 alone. KCNE1 increased sensitivity of KCNQ1 current to Chromanol 293B.
2. Activation of EGFR had different effects on KCNQ1 current and KCNQ1/KCNE1 currents. EGF-induced activation of KCNQ1 current was possibly through PLC-PIP2-IP3-Ca2+ signaling pathway. EGF-induced inhibition of KCNQ1/KCNE1 currents was possibly mediated through tyrosine phosphorylation of KCNE1. EGF lengthened action potential repolarizion in guinea pig papillary. This would indicate modulation of delayed rectifier K+ channels by EGF may have physiological significance.
3. Activation of EGFR inhibited hERG current amplitude and altered kinetics characteristics, shifting the curve of voltage-dependent activation to the right and slowing the activation and promoting the deactivation. Effect of EGF on action potential repolarization may involve modulation of hERG channel besides KCNQ1/KCNE1 channel.
4. Dipfluzine concentration-dependently inhibited KCNQ1/KCNE1 currents. IC50 was (8.9±1.8)μmol·L-1. Dipfluzine-induced inhibition of KCNQ1/KCNE1 currents was voltage dependent at membrane potentials between -10 and 90 mV. Dipfluzine at 10μmol·L-1 shifted V1/2 of KCNQ1/KCNE1 currents activation by 3mV toward more positive potentials, and significantly increased the activating time constant, and decreased the slow and fast deactivating time constant. Dipfluzine inhibited KCNQ1/KCNE1 currents amplitudes and modifies kinetic characteristics of KCNQ1/KCNE1 activation and deactivation, which might be correlated with its antiarrhythmic effect.
表皮生长因子受体(EGFR)是I型跨膜酪氨酸激酶受体,定位于细胞膜,编码分子量170KDa的跨膜糖蛋白,由三个部分组成:即胞外配体结合区;疏水性的跨膜区;具有酪氨酸蛋白激酶活性的胞内区。与配体EGF结合后EGFR构象发生变化,引起受体在膜上迁移、聚合、形成寡聚体,同时被激活,磷酸化自身的酪氨酸残基,使EGFR激酶活性进一步提高,并对其它底物蛋白进行磷酸化。EGFR受体的酪氨酸激酶发挥两方面作用:一方面磷酸化自身酪氨酸残基,为募集其它信号分子提供平台;另一方面将募集的靶蛋白的酪氨酸残基磷酸化,引起下游信号转导。EGFR在调控细胞的生长、分化、增殖、迁移、凋亡等生理功能中发挥重要作用。
离子通道受细胞内许多信号途径包括蛋白磷酸化和去磷酸化的调节,广谱酪氨酸激酶抑制剂tyrphostin A23、tyrphostin A25和genistein可抑制豚鼠心室肌细胞的基础IKs,酪氨酸磷酸酶抑制剂orthovanadate可反转酪氨酸激酶抑制剂引起的IKs电流的降低,因此认为:基础IKs依赖于IKs通道或通道调节蛋白的酪氨酸磷酸化水平。
本研究以非洲爪蟾卵母细胞作为表达系统,观察EGFR激活后对KCNQ1电流、KCNQ1/KCNE1电流和hERG通道电流的影响,并深入探讨其作用机制,并在豚鼠乳头肌组织上观察了EGF对动作电位的影响,以证实EGFR对IKs和IKr的调节作用是否具有生理学意义。
一、KCNQ1及KCNQ1/KCNE1钾离子通道电流特征
目的:在非洲爪蟾卵母细胞中表达KCNQ1、KCNQ1/KCNE1钾离子通道,观察KCNQ1和KCNQ1/KCNE1钾离子通道的电流特性。
方法:(1)cRNA的制备:KCNQ1、KCNE1的cDNA分别克隆于pGEMHE质粒载体中,提取质粒DNA,经NheⅠ限制性核酸内切酶消化及纯化后,作为模板,用T7体外转录试剂盒(Promega),合成KCNQ1,KCNE1 cRNA,纯化后进行RNA的定量。(2)爪蟾卵母细胞的分离:在爪蟾冰冻麻醉状态下,于下腹部切开一小口,取出卵母细胞,放入含2 g·L-1胶原酶的OR2溶液(mmol·L-1: NaCl 82.5,KCl 2,MgCl2 1,HEPES 5, pH 7.4)中翻摇消化1.5~2hr,制成单个卵母细胞,挑选Ⅴ-Ⅵ级卵母细胞放入ND96培养液中(mmol·L-1:NaCl 96, KCl 1, CaCl2 1.8, MgCl2 1, HEPES 5, pH 7.4)。(3)将5 ng KCNQ1、1 ng KCNE1混合后注射入卵母细胞中,将卵母细胞放入含2.5mM丙酮酸钠和50mg.L-1庆大霉素的ND96培养液中,置于18℃培养箱中,于注射后2~6d,采用双电极电压钳技术观察KCNQ1和KCNQ1/KCNE1钾离子通道的电流特性。
结果:(1)KCNQ1和KCNE1质粒DNA经NheⅠ限制性核酸内切酶消化后,电泳迁移速度明显减慢,与DNA Marker比对,分子量大小约为5Kb和3.4Kb。KCNQ1和KCNE1 cRNA由琼脂糖凝胶电泳证实。(2)KCNE1可明显增大KCNQ1电流。当电压去极至40mV再复极至-50mV时,KCNQ1及KCNQ1/KCNE1尾电流分别为(0.09±0.01)μA和(0.39±0.03)μA(P<0.01,n=6),即KCNE1使KCNQ1通道电流增大4倍多。(3)KCNE1对KCNQ1通道电流动力学特征的影响:膜电压钳制在-80mV,从-60mV开始以阶跃电压10mV的方式逐步去极化至50mV,维持4s,然后电压恢复至-50mV。用Boltzmann方程拟合标准化的尾电流-电压关系曲线,得到半数激活电压(V1/2),KCNQ1通道电流和KCNQ1/KCNE1通道电流的V1/2分别为(-22.8±1.7)mV,(30.9±0.8)mV,因此,KCNE1使KCNQ1的激活电压明显右移。用单指数方程拟合去极至30mV时的激活曲线和从去极化电压30mV复极至-50mV时的尾电流曲线,得到激活时间常数和去活时间常数。KCNQ1/KCNE1电流的激活时间常数(3.84±0.52s),与KCNQ1(0.89±0.05s)相比,明显延长(P<0.01),说明辅助亚基KCNE1明显减慢KCNQ1电流的激活速度。KCNQ1/KCNE1通道电流的去活时间常数(1.01±0.02s)与KCNQ1(2.29±0.39s)相比,明显减小(P<0.05),表明KCNE1可加速KCNQ1电流的去活速度。KCNQ1尾电流的起始部呈弯钩状,是从失活态快速恢复的结果,说明KCNQ1电流存在一定程度的失活,KCNE1可去除KCNQ1电流的失活。(4)KCNE1与KCNQ1共表达后,可增加对IKs特异性阻断剂Chromanol 293B的敏感性,50μmol.L-1Chromanol 293B对KCNQ1和KCNQ1/KCNE1通道电流的抑制率分别为(65.5±5.8)%和(79.9±0.6)%。
结论:KCNQ1通道电流具有快激活、慢去活、部分失活的特性,KCNE1明显改变KCNQ1的门控特性,减慢其激活速度,消除失活,使激活电压向正电位转移。KCNQ1/KCNE1电流幅度比KCNQ1电流大4倍多,此外,KCNE1可提高KCNQ1电流对Chromanol 293B的敏感性。
二、EGF对KCNQ1/KCNE1钾通道电流的影响及其机制研究
目的:观察EGFR激活对爪蟾卵母细胞表达的KCNQ1和KCNQ1/KCNE1电流的影响,并探讨其作用机制;观察EGF对豚鼠乳头肌动作电位的影响。
方法:KCNQ1、KCNE1和EGFR的cDNA分别克隆于pGEMHE质粒载体中,NheⅠ限制性核酸内切酶将质粒DNA线性化,作为模板,应用RibomaxTM large scale RNA production systems-T7 kit,体外合成KCNQ1、KCNE1和EGFR cRNA,纯化后,cRNA的大小和浓度经琼脂糖凝胶电泳,与RNA ladder比对后确定。将5ng KCNQ1、5ng EGFR和1ng KCNE1 cRNA或5ng KCNQ1、5ng EGFR混合后注射入卵母细胞中,放入含2.5mM丙酮酸钠和50mg.L-1庆大霉素的ND96培养液中,置于18℃培养箱,于注射后2~6天,采用双电极电压钳,室温下记录电流。采用细胞内记录观察EGF对豚鼠乳头肌动作电位的影响,应用定向点突变和免疫共沉淀的方法探讨其可能的作用机制。
结果:(1)EGF剂量依赖性地抑制KCNQ1/KCNE1电流,EGF 3,10,30,100,300ng/ml对KCNQ1/KCNE1电流的相对抑制率分别为(6.0±2.1)%, (20.8±3.4)%, (38.9±2.7)%, (57.3±1.5)%和(65.9±2.8)%,半数抑制浓度(IC50)为(24.1±1.8)ng/ml。(2)100ng/ml EGF明显增大KCNQ1电流,灌流EGF前、后的KCNQ1电流大小分别为(0.27±0.02)μA和(0.42±0.04)μA。(3)EGF抑制KCNQ1/KCNE1电流在0～90mV之间呈电压依赖性。随着电压的增大,EGF抑制KCNQ1/KCNE1电流的作用逐渐减小。(4)EGF改变KCNQ1/KCNE1电流的动力学特征:使半数激活电压右移约7mV;对照组KCNQ电流激活时间常数(10mV)为(4.8±0.6)s,给予100ng/ml EGF后,激活时间常数增大至(7.1±0.7)s,说明EGF减慢KCNQ1/KCNE1电流的激活速度;与对照组(1063±102)ms相比,100ng/ml EGF可增加慢去活时间常数(1251±138)ms,与对照组(339±34)ms相比,100ng/ml EGF显著增加快去活时间常数(429±28)ms,表明EGF减慢KCNQ1/KCNE1电流的去活速度。(5)分析与EGF影响KCNQ1/KCNE1电流有关的信号转导途径。Src激酶的抑制剂PP2(200nM)和Ca2+螯合剂EGTA(5μM)不影响EGF对KCNQ1/KCNE1电流的抑制作用;与溶剂对照DMSO组(43.8±4.2%)相比,酪氨酸激酶抑制剂Genistein(200μM)可明显降低EGF对KCNQ1/KCNE1电流的抑制率(4.8±2.9%)(P<0.01,n=6),提示其作用与通道或通道调节蛋白的酪氨酸磷酸化有关。(6)分析EGF引起KCNQ1电流增加的机制。EGTA(5μM)和PLC抑制剂U73122(3μM)降低EGF对KCNQ1电流的增加率(分别由66±2.0%到25±1.8%、76±10%到26±9%),提示其作用与细胞内PLC-PIP2-IP3-Ca2+信号转导途径有关。(7)KCNE1(S102A)和KCNE1(Y81A)突变体不影响EGF对KCNQ1/KCNE1电流的抑制作用,但KCNQ1/KCNE1(Y81A)对Chromanol 293B(50μM)的敏感性降低,Chromanol 293B对KCNQ1/KCNE1(Y81A)电流的抑制率为(38.3±8.7)%,而对野生型KCNQ1/KCNE1电流的抑制率为(69.9±2.6)%。此外,KCNE1 ( Y81A )改变电流激活动力学特征,使激活电压右移,KCNQ1/KCNE1(Y81A)电流在+40mV以上时才被激活,提示KCNE1第81位的酪氨酸残基对于调节KCNQ1/KCNE1电流幅度和门控机制是非常重要的。(8)100ng/ml EGF可延长豚鼠乳头肌动作电位的复极过程,增加APD50(给药前127±21ms,给EGF后148±24ms)APD90(给药前189±16ms,给EGF后205±13ms),减慢0相最大上升速率(Vmax)(给药前149±28V/s,给EGF后111±30V/s),提示EGF在抑制延迟整流性K+通道的同时,还可能抑制Na+离子通道。(9)免疫共沉淀的结果显示EGF可增加KCNE1亚基酪氨酸磷酸化的水平,对KCNQ1亚基的酪氨酸磷酸化水平无影响,表明EGF可能通过直接磷酸化KCNE1亚基上的酪氨酸残基而影响KCNQ1/KCNE1电流的大小和门控机制。
结论:EGFR激活后以不同的机制调节KCNQ1通道和KCNQ1/KCNE1通道,可能通过PLC-PIP2-IP3-Ca2+信号转导途径增加KCNQ1通道电流;通过磷酸化KCNE1的酪氨酸残基而抑制KCNQ1/KCNE1通道电流。EGF可延长豚鼠乳头肌动作电位的复极化过程,因此,EGF调节延迟整流性K+通道的作用可能具有生理学意义。
三、EGF对爪蟾卵母细胞上表达的hERG通道电流的影响
目的:在爪蟾卵母细胞上表达hERG通道和EGFR,观察EGFR激活后对hERG通道电流的影响。
方法:EGFR cDNA克隆于pGEMHE载体中,以NheⅠ限制性核酸内切酶线性化的质粒DNA作为模板,应用T7体外转录试剂盒,合成EGFR cRNA;hERG cDNA克隆于pSP64载体中,以EcoRⅠ限制性核酸内切酶线性化的质粒DNA作为模板,应用SP6体外转录试剂盒,获得hERG cRNA。将5ng hERG和5ng EGFR cDNA混合后注入卵母细胞中,采用双电极电压钳观察EGFR激活对hERG通道电流的影响。
结果:hERG电流为电压依赖性的外向电流,具有快速激活,快速失活,被IKr特异性阻断剂E-4031阻断的特点。(1)将电压钳制在-80mV,然后去极化至0mV,再复极至-50mV,记录hERG电流。100ng/ml EGF降低hERG尾电流(复极至-50mV的最大电流),给药前(0.44±0.07)μA,给EGF后(0.28±0.04)μA,用ND96冲洗后,电流可完全恢复。因此,EGF可逆性地抑制hERG电流(。2)分析在-70m~38mV之间EGF对hERG电流作用的电压依赖性。结果显示在-34~14mV之间EGF电压依赖性地抑制hERG电流。(3)EGF右移hERG电流的半数激活电压约15mV,给药前V1/2为(-23.9±0.8)mV,给EGF后V1/2为(-8.6±0.8)mV。(4)100ng/ml EGF可增加hERG电流的激活时间常数(给药前433±85ms,给EGF后574±183ms),减慢hERG电流的激活;100ng/ml EGF可降低hERG电流的去活时间常数(从1500±224ms至810±138ms),加快hERG电流的去活速度。
结论:EGFR激活后抑制hERG电流幅度,改变其动力学特征,使激活电压右移,减慢激活速度,加快去活速度。
四、双苯氟嗪对表达于爪蟾卵母细胞上的KCNQ1/KCNE1钾通道电流的影响
目的:研究双苯氟嗪对KCNQ1/KCNE1钾通道电流的影响,以探讨其抗心律失常作用的可能机制。
方法:采用双电极电压钳技术,观察双苯氟嗪对表达于非洲爪蟾卵母细胞上的KCNQ1/KCNE1钾通道电流的影响。
结果:双苯氟嗪浓度依赖性地抑制KCNQ1/KCNE1电流,双苯氟嗪0.3,1,,3,10,30μmol·L-1对KCNQ1/KCNE1电流的抑制率分别为(6.0±1.0)%,(11.6±1.5)%,(25.7±3.5)%,(45.6±3.5)%,(63.5±7.6)%。IC50为(8.9±1.8)μmol·L-1。在-10~90mV范围内双苯氟嗪对KCNQ1/KCNE1电流的抑制作用具有电压依赖性。10μmol·L-1双苯氟嗪使KCNQ1/KCNE1电流的半数激活电压右移约3mV,显著增大激活时间常数(从3.7±0.6s到5.0±0.5 s),减慢KCNQ1/KCNE1电流的激活;降低慢去活时间常数和快去活时间常数(从1135±91ms、368±27ms至879±78ms、313±45ms),加速KCNQ1/KCNE1电流的去活。
结论:双苯氟嗪浓度依赖性地抑制KCNQ1/KCNE1钾通道电流,并改变其动力学特征,提示双苯氟嗪抗心律失常的作用可能与此有关。
小结
1. KCNQ1通道电流具有快激活、慢去活、部分失活的特征,KCNE1明显改变KCNQ1通道的门控特性,减慢其激活,消除失活,使激活电压右移,KCNE1/KCNQ1电流比KCNQ1电流大4倍多,KCNE1增加KCNQ1电流对Chromanol 293B的敏感性。
2. EGFR激活对KCNQ1电流和KCNQ1/KCNE1电流的作用不同。EGF可能通过PLC-PIP2-IP3-Ca2+信号转导途径增加KCNQ1电流;EGF可能通过磷酸化KCNE1的酪氨酸残基抑制KCNQ1/KCNE1电流。EGF可延长豚鼠乳头肌动作电位的复极过程,表明EGF对延迟整流性K+通道的调节作用具有生理学意义。
3. EGFR激活抑制hERG电流幅度和改变其动力学特征,使激活电压右移,减慢hERG电流的激活速度,加快去活速度。EGF可能通过调节KCNQ1/KCNE1和hERG通道影响心肌动作电位的复极化过程。
4.双苯氟嗪浓度依赖性地抑制KCNQ1/KCNE1电流。IC50为8.9±1.8μmol.L-1;在-10~90 mV范围内双苯氟嗪对KCNQ1/KCNE1电流的抑制作用具有电压依赖性;10μmol·L-1双苯氟嗪使KCNQ1/KCNE1电流的半数激活电压右移3mV,明显增加KCNQ1/KCNE1电流的激活时间常数,降低快、慢去活时间常数。双苯氟嗪抑制KCNQ1/KCNE1电流的幅度,改变其激活和去活动力学特征可能与其抗心律失常的作用有关。
The delayed rectifier potassium currents played a key role in myocardial repolarization, which were composed of two different elements: slowly delayed rectifier potassium currents (IKs) and rapidly delayed rectifier potassium currents (IKr).α-subunit encoded by KCNQ1 heteromultimericly coassembled withβ-subunit encoded by KCNE1 to form IKs channels. hERG encodesα-subunit of IKr. Both KCNQ1 and hERG subunit consisted of six putative transmembrane domains (S1~S6) and an ion-selective P-loop. Fourα-subunits formed the outer pore and contain the selectivity filter. KCNE1 protein consisted of a single transmembrane domain, and altered the biophysical properties of the channels. Mutations in either IKs subunits or IKr subunits were associated with variants of the congenital long QT syndrome. Mutant subunits led to reduction of IKs or IKr by a loss-of-function mechanism, often with a dominant-negative effect. In the heart, reduced IKs or IKr led to prolongation of the cardiac action potential, lengthening of the QT interval, and increased risk of arrhythmia. Thus, the delayed rectifier potassium channels were involved in maintaining normal action potential as well as inducing arrhythmia. The delayed rectifier potassium channels were targets of many modulatory mechanisms.
The epidermal growth factor receptor (EGFR) belonged to subclass I of the superfamily of the receptor tyrosine kinases. It was composed of an extracellular ligand binding domain, a single transmembrane domain, and an intracellular domain possessing PTK activity. Binding of epidermal growth factor resulted in EGFR dimerization and subsequent activation of the intrinsic tyrosine kinase activity. These phosphorylated tyrosines functioned as docking sites for a variety of signaling molecules that regulated membrane-proximal steps of signal transduction cascades that ultimately brought about cellular responses to EGFR ligands. The EGFR controled a wide variety of biological processes such as cell proliferation, differentiation, and migration and modulation of apoptosis.
Ion channels were targets of many intracellular signaling pathways, including protein phosphorylation and dephosphorylation. Constitutive IKs recorded from guinea-pig ventricular myocytes was suppressed by broad-spectrum tyrosine kinase (TK) inhibitors tyrphostin A23, tyrphostin A25 and genistein. The phosphotyrosyl phosphatase inhibitor orthovanadate almost completely reversed the suppression of IKs induced by broad-spectrum TK inhibitors. Basal IKs was strongly dependent on tyrosine phosphorylation of IKs channel (or channel-regulatory) protein.
In the present study, we used Xenopus oocytes as expression system to observe effect of activation of EGFR on KCNQ1 alone, or KCNQ1/KCNE1 as well as hERG currents, and the mechanism of these effects. Besides this, we studied the effect of EGF on action potential of guinea pig papillary muscles to study the physiological significance of EGFR-mediated modulation of IKs and IKr.
1. The characteristics of KCNQ1 and KCNQ1/KCNE1 currents
Aim: To observe the characteristics of KCNQ1 current and KCNQ1/KCNE1 currents expressed in Xenopus oocyte.
Methods: (1) Preparation of cRNA: KCNQ1 and KCNE1 cDNA were subcloned respectively into the pGEMHE plasmid vector. The sequences of all constructs were confirmed by sequencing. The plasmid DNA was amplified and extracted from E coli transformed with constructs. The plasmid was lineared by NheⅠrestriction endonuclease and purified with DNA fragment purification kit (TakaRa). All cRNAs were synthesized in vitro using RNA production systems-T7 kit (Promega), and were quantified after purification with RNA clean kit (Tiangen). (2) Preparation of Xenopus oocytes: Oocytes were surgically removed under iced anesthesia and placed into OR2 solution (mmol·L-1: NaCl 82.5,KCl 2,MgCl2 1,HEPES 5, pH7.4)containing 2g.L-1 collagenase and were rocked for 1.5 to 2 hours. (3) StageⅤ-Ⅵoocytes were selected and injected with cRNA containing 5ng KCNQ1 with or without 1ng KCNE1 and were incubated at 18℃in ND96 solution (mmol·L-1:NaCl 96, KCl 1, CaCl2 1.8, MgCl2 1, HEPES 5, pH 7.4) plus 2.5 mM pyruvic acid and 50mg.L-1 gentamicin. Currents were recorded at room temperature 2-6d after injection using two-microelectrode voltage-clamp technique.
Results: (1) The lineared plasmid DNA ran slower than the circular plasmid in agarose electrophoresis. Compared with DNA marker, KCNQ1 and KCNE1 plasmid DNA were 5Kb and 3.4Kb, respectively. KCNQ1 and KCNE1 cRNA were verified by agarose electrophoresis. (2) KCNE1 increased KCNQ1 current amplitude. The tail current of KCNQ1 at -50mV following by repolarization to 40mV was (0.09±0.01)μA, whereas KCNQ1/KCNE1 currents at same voltage was (0.39±0.03)μA (P<0.01, n=6). Thus, KCNE1 increased KCNQ1 current over fourfold. (3) KCNE1 altered kinetics characteristics of KCNQ1 current. From a holding potential of -80mV, oocytes were depolarized for 4 s to test potentials between -60mV to 50mV in 10mV steps followed by repolarization to -50mV. Normalized tail current-voltage curves were fitted with Boltzmann equation, and the half activation voltage (V1/2) of KCNQ1 and KCNQ1/KCNE1 currents were (-22.8±1.7) mV and (30.9±0.8) mV, respectively. Thus, KCNE1 shifted KCNQ1 activation voltage to more positive potentials. The activated current trace at +30mV and the tail current trace at -50mV following depolarization to +30mV were fitted with monoexponential function to acquire the time constants of activation and deactivation, respectively. The activation time constants of KCNQ1/KCNE1 was increased significantly to (3.84±0.52s), from (0.89±0.05s) of KCNQ1 alone (P<0.01), suggesting KCNE1 slowed KCNQ1 current activation. The deactivated time constants of KCNQ1/KCNE1 (1.01±0.02s) was lowered significantly, compared with that of (2.29±0.39s) of KCNQ1 alone, suggesting KCNE1 accelerated KCNQ1 current deactivation. KCNQ1 tail currents display a“hook”, indicating that KCNQ1 inactivates to some extent. KCNE1 eliminated inactivation of KCNQ1 current. (4) Coexpression of KCNE1 increased sensitivity of KCNQ1 current to Chromanol 293B, a selective blocker of IKs. The KCNQ1 current inhibition rates with or without KCNE1 by Chromanol 293B were (79.9±0.6)% and (65.5±5.8)%.
Conclusion: KCNQ1 current exhibited a rapidly activating, slow deactivating, partly inactivating characteristics. KCNE1 dramatically modulated KCNQ1 gating, slowing activation, removing inactivation, and shifting the voltage dependence of activation to more positive potentials. KCNE1 plus KCNQ1 had fourfold greater current amplitudes than KCNQ1 alone. KCNE1 increased sensitivity of KCNQ1 current to Chromanol 293B. 2. Effect of EGF on KCNQ1 and KCNQ1/KCNE1 currents expressed in Xenopus oocytes.
Aim: To investigate the effect of activation of EGFR on KCNQ1 and KCNQ1/KCNE1 currents expressed in Xenopus oocytes, and to explore the underlying mechanism. To observe the effect of EGF on action potentials of guinea pig papillary muscles.
Methods: A full-length KCNQ1, KCNE1, EGFR cDNA were subcloned into the pGEMHE plasmid vector, respectively. Complementary RNAs (cRNAs) from all contructs were prepared in vitro from NheⅠ-lineared DNA templates using RibomaxTM large scale RNA production systems-T7 kit. cRNAs size and concentration were estimated by comparison with RNA ladder in agarose gel electrophoresis following purification of cRNAs. StageⅤ-Ⅵoocytes were injected with cRNA containing 5ng KCNQ1, 5ng EGFR with or without 1ng KCNE1 and incubated at 18℃in ND96 solution plus 2.5 mM pyruvic acid and 50mg.L-1 gentamicin. Currents were recorded at room temperature 2-6d after injection using two-microelectrode voltage-clamp technique. The effect of EGF on action potentials from guinea pig right ventricular papillary muscle was observed with a conventional intracellular recording technique. Site-directed mutagenesis and immunoprecipitation technique were used to explore mechanism underlying effect of activation of EGFR on KCNQ1/KCNE1 currents.
Results: (1) EGF suppressed KCNQ1/KCNE1 currents amplitudes in a dose-dependent manner. EGF at 3,10,30,100,300ng/ml inhibited KCNQ1/KCNE1 currents by (6.0±2.1)%, (20.8±3.4)%, (38.9±2.7)%, (57.3±1.5)%, and (65.9±2.8)%, respectively; The concentration for half maximal inhibition (IC50) was (24.1±1.8)ng/ml. (2) EGF at 100ng/ml increased KCNQ1 current. KCNQ1 current before and after EGF perfusion were (0.27±0.02)μA and (0.42±0.04)μA, respectively. (3) EGF voltage-dependently suppressed KCNQ1/KCNE1 currents amplitudes at membrane potentials between 0 and +90mV, with increased voltages, inhibition decreased. (4) EGF altered kinetics characteristic of KCNQ1/KCNE1 currents. EGF at 100ng/ml shifted V1/2 of KCNQ1/KCNE1 currents activation by 7mV toward more positive potentials, and significantly increased the activating time constant at 10mV (from 4.8±0.6s to 7.1±0.7s), increased slow deactivation time constant (from 1063±102ms to 1251±138ms), and fast deactivation time constant (from 339±34ms to 429±28ms), indicating EGF slowed activation and deactivation of KCNQ1/KCNE1 currents. (5) The signaling associated with effect of EGF on KCNQ1/KCNE1 currents was analyzed. PP2(200nM), a blocker of Src kinase, and Ca2+ chelator EGTA(5μM) did not affect EGF-induced inhibition of KCNQ1/KCNE1 currents. Genistein(200μM), an inhibitor of tyrosine kinase, eliminated the effect of EGF on KCNQ1/KCNE1 currents(from 43.8±4.2% to 4.8±2.9%), suggesting tyrosine phosphorylation of channel or channel modulator was involved. (6) The mechanism of EGF-induced activation of KCNQ1 current was analyzed. EGTA(5μM) or PLC inhibitor U73122(3μM) reduced EGF-induced activation of KCNQ1 current (from 66±2.0% to 25±1.8%, from 76±10% to 26±9%, respectively), indicating involvement of PLC-PIP2-IP3-Ca2+ signaling. (7) Mutation of KCNE1 (Y81A) or (S102A) did not affect EGF-induced modulation of KCNQ1/KCNE1 currents. However KCNQ1/KCNE1(Y81A) became insensitive to Chromanol 293B inhibition. KCNQ1/KCNE1(Y81A) currents were only inhibited by 38.3±8.7% by Chromanol 293B, as compared with a 69.9±2.6% inhibition for wild type KCNQ1/KCNE1. KCNE1(Y81A) also altered activation kinetics, shifting activation potentials to more positive direction. KCNQ1/KCNE1(Y81A) currents did not activate until a membrane depolarization to +40mV. These results suggested tyrosine in KCNE1 81 was important in modulating KCNQ1/KCNE1 currents amplitude and gating. (8) EGF lengthened the action potentials repolarization of guinea pig papillary. EGF at 100ng/ml increased APD50 (from 127±21ms to 148±24ms) and APD90 (from 189±16ms to 205±13ms), slowed the maximal rate of rise of AP (from 149±28V/s to 111±30V/s), suggesting EGF inhibited both delayed rectifier K+ channels and Na+ channel. (9) Results of immunoprecipitation showed EGF increased tyrosine phosphorylation of KCNE1, but did not affect tyrosine phosphorylation of KCNQ1, suggesting KCNQ1/KCNE1 currents amplitude and gating were modulated through tyrosine phosphorylation of KCNE1
Conclusion: Activation of EGFR had different effects on KCNQ1 current and KCNQ1/KCNE1 currents. EGF-induced activation of KCNQ1 current was possibly through PLC-PIP2-IP3-Ca2+ signaling pathway. EGF-induced inhibition of KCNQ1/KCNE1 currents was possibly mediated through tyrosine phosphorylation of KCNE1. EGF lengthened action potential repolarizion in guinea pig papillary. This would indicate modulation of delayed rectifier K+ channels by EGF may have physiological significance. 3. Effect of EGF on hERG channel current expressed in Xenopus oocytes
Aim: To investigate the effect of activation of EGFR on hERG current expressed in Xenopus oocytes.
Methods: EGFR cDNA was subcloned into the pGEMHE plasmid vector. EGFR cRNA was prepared in vitro from NheⅠ-lineared DNA template using RibomaxTM large scale RNA production systems-T7 kit. hERG cDNA was subcloned into pSP64 vector. hERG cRNA was synthesized with RibomaxTM large scale RNA production systems-SP6 kit from EcoRⅠ-lineared DNA template. Mixture of 5ng EGFR cRNA and 5ng hERG cRNA were injected into oocytes. Effect of activation of EGFR on hERG current was analyzed using two electrode voltage-clamp technique.
Results: hERG current possessed characteristic of voltage-dependent fast activating and fast inactivating outward current sensitive to IKr blocker E-4031. (1) hERG current was elicited using a protocol in which the potential was first held at -80mV and then depolarized to 0mV followed by repolarization to -50mV. Tail current was measured upon repolarization to -50mV. EGF at 100ng/ml decreased hERG tailed current from(0.44±0.07)μA to (0.28±0.04)μA. hERG tailed current recovered completely after washout of EGF. Thus, EGF inhibited reversibly hERG current. (2) Voltage-dependency of EGF action on hERG was tested in voltages from -70mV to 38mV. The results showed EGF suppressed hERG current in a voltage-dependent manner from -34mV to 14mV. (3) EGF shifted V1/2 of hERG current about 15 mV towards more positive potentials (from -8.0±0.6 mV to 6.7±0.8 mV). (4) EGF lengthened activation time constant of hERG current from (433±85)ms to (574±183)ms, suggesting EGF slowed activation of hERG current. EGF decreased deactivation time constant from (1500±224)ms to (810±138)ms, suggesting EGF promoted deactivation hERG current.
Conclusion: Activation of EGFR inhibited hERG current amplitude and altered kinetics characteristics, shifting the curve of voltage-dependent activation to the right and slowing the activation and promoting the deactivation.
4. Effect of dipfluzine on KCNQ1/KCNE1 potassium currents expressed in Xenopus oocytes
Aim: To investigate the effect of dipfluzine on KCNQ1/KCNE1 potassium currents expressed heterologously in Xenopus oocytes.
Methods: Using Xenopus oocytes expression system, the current amplitude and kinetic characteristics of KCNQ1/KCNE1 were measured with the two electrode voltage-clamp technique before and after dipfluzine application.
Results: Dipfluzine concentration-dependently inhibited KCNQ1/KCNE1 currents. Dipfluzine at 0.3, 1, 3, 10, 30μmol·L-1 inhibited KCNQ1/KCNE1 currents by 6.0±0.9%, 11.6±0.8% , 25.7±2.9%, 45.6±2.5%, 63.5±1.6%, respectively. IC50 was 8.9±1.8μmol·L-1. The solvent (DMSO, 0.1% ) did not affect KCNQ1/KCNE1 currents. Dipfluzine-induced inhibition of KCNQ1/KCNE1 currents was voltage dependent at membrane potentials between -10 and 90 mV. Dipfluzine at 10μmol·L-1 shifted V1/2 of KCNQ1/KCNE1 currents activation by 3mV toward more positive potentials, and significantly increased the activating time constant (from 3.7±0.6 s to 5.0±0.5 s), slowed KCNQ1/KCNE1 currents activation. Dipfluzine at 10μmol·L-1 significantly decreased the slow and fast deactivating time constants(from 1135±91 ms to 879±78 ms and 368±27ms to 313±45ms, respectively), enhanced KCNQ1/KCNE1 currents deactivation.
Conclusion: Dipfluzine concentration-dependently and voltage- dependently inhibited KCNQ1/KCNE1 currents and modified kinetic characteristics of KCNQ1/KCNE1 activation and deactivation, which might be correlated with its antiarrhythmic effect.
SUMMARY
1. KCNQ1 current exhibited a rapidly activating, slow deactivating, partly inactivating characteristics. KCNE1 dramatically modulated KCNQ1 gating, slowing activation, removing inactivation, and shifting the voltage dependence of activation to more positive potentials. KCNE1 plus KCNQ1 had fourfold greater current amplitudes than KCNQ1 alone. KCNE1 increased sensitivity of KCNQ1 current to Chromanol 293B.
2. Activation of EGFR had different effects on KCNQ1 current and KCNQ1/KCNE1 currents. EGF-induced activation of KCNQ1 current was possibly through PLC-PIP2-IP3-Ca2+ signaling pathway. EGF-induced inhibition of KCNQ1/KCNE1 currents was possibly mediated through tyrosine phosphorylation of KCNE1. EGF lengthened action potential repolarizion in guinea pig papillary. This would indicate modulation of delayed rectifier K+ channels by EGF may have physiological significance.
3. Activation of EGFR inhibited hERG current amplitude and altered kinetics characteristics, shifting the curve of voltage-dependent activation to the right and slowing the activation and promoting the deactivation. Effect of EGF on action potential repolarization may involve modulation of hERG channel besides KCNQ1/KCNE1 channel.
4. Dipfluzine concentration-dependently inhibited KCNQ1/KCNE1 currents. IC50 was (8.9±1.8)μmol·L-1. Dipfluzine-induced inhibition of KCNQ1/KCNE1 currents was voltage dependent at membrane potentials between -10 and 90 mV. Dipfluzine at 10μmol·L-1 shifted V1/2 of KCNQ1/KCNE1 currents activation by 3mV toward more positive potentials, and significantly increased the activating time constant, and decreased the slow and fast deactivating time constant. Dipfluzine inhibited KCNQ1/KCNE1 currents amplitudes and modifies kinetic characteristics of KCNQ1/KCNE1 activation and deactivation, which might be correlated with its antiarrhythmic effect.
引文
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