奎尼丁對(duì)HEK293細(xì)胞hERG電流和hERG蛋白的影響

霍建華,郭雪艷,強(qiáng) 華,劉 平,白 玲,馬愛群

(1西安交通大學(xué)醫(yī)學(xué)院第一附屬醫(yī)院心內(nèi)科,西安 710061;2陜西省人民醫(yī)院消化科;*通訊作者,E-mail:huojianhua2005@126.com)

?

奎尼丁對(duì)HEK293細(xì)胞hERG電流和hERG蛋白的影響

霍建華1*,郭雪艷2,強(qiáng) 華1,劉 平1,白 玲1,馬愛群1

(1西安交通大學(xué)醫(yī)學(xué)院第一附屬醫(yī)院心內(nèi)科,西安 710061;2陜西省人民醫(yī)院消化科;*通訊作者,E-mail:huojianhua2005@126.com)

目的 觀察奎尼丁對(duì)hERG電流和hERG通道蛋白表達(dá)的影響,進(jìn)一步明確其導(dǎo)致長(zhǎng)QT間期綜合征的機(jī)制。 方法 用脂質(zhì)體轉(zhuǎn)染法將含有hERG基因的質(zhì)粒轉(zhuǎn)入HEK293細(xì)胞,全細(xì)胞膜片鉗技術(shù)記錄電流和Western blot技術(shù)觀察hERG通道蛋白表達(dá)的影響。實(shí)驗(yàn)分組:①HEK293細(xì)胞轉(zhuǎn)染進(jìn)質(zhì)粒48 h時(shí)加入不同濃度(0，1，3，10 μmol/L)的奎尼丁,并進(jìn)行膜片鉗實(shí)驗(yàn)觀察奎尼丁的瞬時(shí)作用。②HEK293細(xì)胞轉(zhuǎn)染進(jìn)質(zhì)粒24 h時(shí)往培養(yǎng)基加入不同濃度(1，3，10 μmol/L)的奎尼丁,繼續(xù)培養(yǎng)24 h洗脫奎尼丁后立即進(jìn)行膜片鉗及Western blot實(shí)驗(yàn)觀察奎尼丁的慢性作用。 結(jié)果 ①1 μmol/L、3 μmol/L及10 μmol/L組的最大尾電流分別為(55.9±3.8)pA/pF、(35.7±3.2)pA/pF及(15.5±1.4)pA/pF,均明顯小于對(duì)照(0 μmol/L)組(均P<0.05)。②不同濃度的奎尼丁孵育HEK293細(xì)胞后hERG電流無(wú)明顯變化。Western blot觀察到不同濃度奎尼丁作用下hERG蛋白表達(dá)無(wú)明顯變化。 結(jié)論 奎尼丁對(duì)hERG電流有瞬時(shí)直接抑制作用,但并非影響hERG通道蛋白的表達(dá)來(lái)改變hERG電流?？岫?duì)hERG通道的直接抑制作用可能為其引起長(zhǎng)QT間期綜合征的根本機(jī)制。

長(zhǎng)QT間期綜合征； 奎尼丁； hERG； HEK293

人ether-a-go-go相關(guān)基因(human-ether-a-go-go-related gene,hERG)定位于染色體7q35-36,在心肌組織中高度表達(dá),編碼心肌細(xì)胞電壓門控鉀通道的α亞單位,α亞單位與MiRP1基因編碼的β亞單位結(jié)合后產(chǎn)生快速激活延遲整流鉀電流(rapidly activating component of delayed rectifier potassium current,IKr)[1,2]。多種藥物(常規(guī)抗心律失常藥物、抗生素、抗組胺藥物以及抗精神病藥物等)可阻斷hERG通道引起藥物性長(zhǎng)QT綜合征,心電圖上表現(xiàn)為QT間期延長(zhǎng),T波異常,常引起尖端扭轉(zhuǎn)型室性心動(dòng)過(guò)速,臨床上表現(xiàn)為反復(fù)發(fā)作性暈厥,甚至猝死。其主要機(jī)制是藥物對(duì)hERG 鉀通道產(chǎn)生直接抑制作用[3]。近年來(lái)發(fā)現(xiàn),某些藥物對(duì)hERG鉀通道蛋白的轉(zhuǎn)運(yùn)有抑制作用,導(dǎo)致細(xì)胞膜hERG通道表達(dá)降低,從而發(fā)揮阻滯效應(yīng),引起長(zhǎng)QT綜合征[4]?？岫∈且环N非選擇性鈉離子通道阻滯劑,為Ⅰa類抗心律失常藥,由于其致心律失常作用嚴(yán)重影響了其在臨床的引用。已有研究顯示奎尼丁對(duì)hERG通道電流的直接抑制作用為其致心律失常作用的主要機(jī)制[5]。但其奎尼丁是否對(duì)hERG鉀通道蛋白的表達(dá)轉(zhuǎn)運(yùn)有影響還未見報(bào)道。本研究利用分子生物學(xué)及膜片鉗技術(shù)觀察其對(duì)hERG鉀通道的影響。

1 材料與方法

1.1 質(zhì)粒及細(xì)胞株

質(zhì)粒pcDNA3-WT-hERG、GFP-pRK5及HEK293細(xì)胞株由西安交通大學(xué)環(huán)境與疾病相關(guān)基因?qū)嶒?yàn)室保存。

1.2 主要試劑及儀器

脂質(zhì)體2000TM試劑盒購(gòu)自美國(guó)Invitrogen公司?？筯ERG抗體購(gòu)自以色列Alomone公司。辣根過(guò)氧物酶標(biāo)記的羊抗兔IgG抗體購(gòu)自美國(guó)Sigma-Aldrich公司?？岫≠?gòu)自美國(guó)Sigma-Aldrich公司。Axon-700B膜片鉗系統(tǒng)、DigiData-1322A數(shù)模轉(zhuǎn)換裝置及pCLAMP9.2膜片鉗數(shù)據(jù)處理軟件均為美國(guó)Axon公司產(chǎn)品。Syngene Chemi-Genius成像系統(tǒng)購(gòu)自英國(guó)SynGene公司。

1.3 HEK293細(xì)胞的培養(yǎng)、轉(zhuǎn)染及處理

HEK293細(xì)胞培養(yǎng)在含有10%胎牛血清的高糖DMEM中。按照脂質(zhì)體2000TM試劑說(shuō)明進(jìn)行操作,將pcDNA3-WT-hERG及GFP-pRK5(綠色熒光蛋白表達(dá)質(zhì)粒,可作為pcDNA3-WT-hERG成功轉(zhuǎn)染進(jìn)細(xì)胞的指示劑)共轉(zhuǎn)染進(jìn)HEK293細(xì)胞。轉(zhuǎn)染質(zhì)粒48 h后立刻進(jìn)行實(shí)驗(yàn)。實(shí)驗(yàn)分組:①觀察奎尼丁的瞬時(shí)作用:HEK293細(xì)胞轉(zhuǎn)染進(jìn)質(zhì)粒48 h時(shí)加入不同濃度(1 μmol/L、3 μmol/L及10 μmol/L)的奎尼丁進(jìn)行膜片鉗實(shí)驗(yàn);②觀察奎尼丁的慢性作用:HEK293細(xì)胞轉(zhuǎn)染進(jìn)質(zhì)粒24 h時(shí)往培養(yǎng)基加入不同濃度(1 μmol/L、3 μmol/L及10 μmol/L)的奎尼丁,繼續(xù)培養(yǎng)24 h洗脫奎尼丁后立即進(jìn)行膜片鉗及Western blot實(shí)驗(yàn)。

1.4 Western blot檢測(cè)hERG蛋白表達(dá)

HEK293細(xì)胞培養(yǎng)在100 mm培養(yǎng)皿中,將真核表達(dá)載體pcDNA3-WT-hERG轉(zhuǎn)染入細(xì)胞后第48小時(shí)時(shí)用PBS液洗滌兩次,NP-40裂解液(20 mmol//L Tris-HCl at pH 8,137 mmol/L NaCl,10% glycerol,1% nonidet P-40 and 2 mmol/L EDTA)和蛋白酶抑制劑進(jìn)行細(xì)胞裂解。Bradford 法測(cè)量細(xì)胞總蛋白濃度。蛋白樣品用8% SDS-聚丙烯酰胺凝膠電泳后電轉(zhuǎn)移至硝酸纖維素膜,5%脫脂奶粉室溫封閉2 h,一抗兔抗人HERG單克隆抗體4 ℃孵育12 h,二抗辣根過(guò)氧物酶標(biāo)記的羊抗兔IgG抗體室溫孵育2 h,ECL發(fā)光液反應(yīng)后利用Syngene Chemi-Genius成像系統(tǒng)照相。

1.5 全細(xì)胞膜片鉗技術(shù)記錄hERG電流

HEK293細(xì)胞轉(zhuǎn)染進(jìn)pcDNA3-WT-hERG及GFP-pRK5后48 h收獲。實(shí)驗(yàn)前將HEK293細(xì)胞制備成單細(xì)胞懸液并置于浴槽中使其貼壁良好;全細(xì)胞電流利用Axon-700B單探頭膜片鉗放大器、DigiData-1322A數(shù)模轉(zhuǎn)換裝置及pCLAMP9.2膜片鉗數(shù)據(jù)處理軟件進(jìn)行記錄處理。細(xì)胞外液:137 mmol/L NaCl,4 mmol/L KCl,1.8 mmol/L CaCl2,1 mmol/L MgCl2,10 mmol/L Glucose,10 mmol/L HEPES(pH 7.4)。電極內(nèi)液:130 mmol/L KCl,1 mmol/L MgCl2,5 mmol/L EGTA,5 mmol/L MgATP,10 mmol/L HEPES(用KOH 調(diào)節(jié)pH值在7.2)。實(shí)驗(yàn)在室溫(22-24 ℃)下進(jìn)行。

1.6 統(tǒng)計(jì)學(xué)分析

2 結(jié)果

2.1 奎尼丁對(duì)hERG電流的瞬時(shí)作用

HEK293細(xì)胞轉(zhuǎn)染進(jìn)質(zhì)粒后48 h時(shí)加入不同濃度的奎尼丁后記錄hERG電流。細(xì)胞的鉗制電壓是-80 mV,命令電壓是-60 mV到+60 mV,階躍是10 mV,每一個(gè)命令電壓維持時(shí)間是2 s,緊接著細(xì)胞被鉗制在-40 mV并持續(xù)5 s以獲得尾電流[6]。從圖1A中可以看出,不同濃度的奎尼丁作用下hERG電流的形態(tài)相一致。圖1B顯示的是激活電流的I-V曲線,可以看出各組細(xì)胞的最大激活電流的命令電壓都是0 mV,且隨著濃度的增加,奎尼丁對(duì)hERG電流有抑制作用越明顯。圖1C顯示的是尾電流的I-V曲線,1 μmol/L、3 μmol/L及10 μmol/L組的最大尾電流分別為(55.9±3.8)pA/pF、(35.7±3.2)pA/pF及(15.5±1.4)pA/pF,均明顯低于對(duì)照(0 μmol/L)組(P<0.05,各組n=9)。以上結(jié)果說(shuō)明奎尼丁對(duì)hERG電流有明顯的抑制。

圖1 給予不同濃度奎尼丁hERG電流的激活電流和尾電流I-V曲線Figure 1 I-V relationships for amplitudes of activation currents and tail currents recorded during test pulses under different concentrations of quinidine

2.2 奎尼丁對(duì)hERG電流的慢性作用

HEK293細(xì)胞轉(zhuǎn)染進(jìn)質(zhì)粒24 h后往培養(yǎng)基加入不同濃度的奎尼丁,繼續(xù)培養(yǎng)24 h后立即洗脫奎尼丁后記錄hERG電流。從圖2A顯示的是不同濃度的奎尼丁孵育HEK293細(xì)胞后記錄的hERG電流圖形。圖2B顯示的是激活電流的I-V曲線,可以看出各組細(xì)胞的最大激活電流的命令電壓都是0 mV,但各組在不同命令電壓下的激活電流密度無(wú)明顯變化(P>0.05)。圖2C顯示的是尾電流的I-V曲線,各組在不同命令電壓下的尾電流密度無(wú)明顯變化(P>0.05)。

2.3 奎尼丁對(duì)hERG蛋白表達(dá)的影響

HEK293細(xì)胞轉(zhuǎn)染進(jìn)質(zhì)粒后24 h時(shí)往培養(yǎng)基加入不同濃度的奎尼丁,繼續(xù)培養(yǎng)24 h立即洗脫奎尼丁后進(jìn)行Western blot檢測(cè)hERG蛋白。運(yùn)用Western blot技術(shù)發(fā)現(xiàn)hERG通道蛋白表現(xiàn)為兩條帶,135 kD為不完全糖基化的通道蛋白,滯留在胞質(zhì)中;而155 kD為完全糖基化的通道蛋白,為轉(zhuǎn)運(yùn)到胞膜上的hERG通道蛋白。圖3顯示的是不同濃度奎尼丁孵育轉(zhuǎn)染hERG質(zhì)粒的細(xì)胞后檢測(cè)到的hERG蛋白表達(dá),從圖中可看出,不同濃度奎尼丁下hERG蛋白表達(dá)無(wú)明顯變化,說(shuō)明奎尼丁對(duì)hERG蛋白的表達(dá)無(wú)明顯影響。

3 討論

奎尼丁是一種非選擇性鈉離子通道阻滯劑,為Ⅰa類抗心律失常藥,由于其致心律失常作用已嚴(yán)重影響其在臨床上的應(yīng)用。本實(shí)驗(yàn)利用全細(xì)胞膜片鉗及Western blot技術(shù)觀察到:奎尼丁對(duì)hERG電流有明顯的瞬時(shí)抑制作用,并表現(xiàn)為濃度依賴性;慢性作用下,奎尼丁對(duì)hERG電流及hERG通道蛋白無(wú)明顯影響。

目前已經(jīng)發(fā)現(xiàn)多種藥物(常規(guī)抗心律失常藥物、抗生素、抗組胺藥物以及抗精神病藥物等)可阻斷hERG通道引起藥物性長(zhǎng)QT綜合征,其主要機(jī)制之一是藥物對(duì)hERG鉀通道產(chǎn)生直接抑制作用[7,8]?？岫∈禽^早發(fā)現(xiàn)的引起QT間期延長(zhǎng)的藥物,可使2%-6%病人引起暈厥?？岫〔粌H是鈉通道阻斷劑,研究發(fā)現(xiàn)其主要通過(guò)阻斷快速激活延遲整流鉀通道的α亞單位(hERG通道)引起QT間期延長(zhǎng),從而引起尖端扭轉(zhuǎn)性室性心律失常等臨床癥狀。本實(shí)驗(yàn)通過(guò)膜片鉗亦證明了奎尼丁對(duì)hERG電流有明顯的直接抑制作用,并呈現(xiàn)濃度依賴性。

圖2 奎尼丁孵育24 h后hERG電流的激活電流和尾電流I-V曲線Figure 2 I-V relationships for amplitudes of activation currents and tail currents recorded during test pulses after cultured with quinidine for 24 h

圖3 不同濃度奎尼丁孵育后hERG通道蛋白的表達(dá)Figure 3 Western blot analysis of hERG protein after treated with quinidine

hERG通道蛋白在內(nèi)質(zhì)網(wǎng)中初步合成和組裝折疊后,在內(nèi)質(zhì)網(wǎng)中進(jìn)行不完全糖基化。然后不完全糖基化的HERG蛋白被轉(zhuǎn)運(yùn)到高爾基體內(nèi)進(jìn)行完全糖基化。最終完全糖基化和正確折疊的HERG通道蛋白被轉(zhuǎn)運(yùn)到細(xì)胞膜上。運(yùn)用Western blot技術(shù)發(fā)現(xiàn)hERG通道蛋白表現(xiàn)為兩條帶,135 kD為不完全糖基化的通道蛋白,而155 kD為完全糖基化的通道蛋白。近期研究顯示藥物性長(zhǎng)QT綜合征的機(jī)制不僅是藥物對(duì)hERG通道有直接抑制作用,某些藥物還對(duì)hERG通道蛋白的表達(dá)與轉(zhuǎn)運(yùn)有抑制作用[9,10]。三氧化二砷對(duì)hERG電流無(wú)直接抑制作用,而阻礙了hERG蛋白的轉(zhuǎn)運(yùn)表達(dá),當(dāng)三氧化二砷作為治療血液系統(tǒng)疾病時(shí),長(zhǎng)期使用亦可引起長(zhǎng)QT綜合征。西沙比利及普羅布考不僅直接抑制hERG電流,還影響hERG蛋白的表達(dá)轉(zhuǎn)運(yùn)[11,12]。本實(shí)驗(yàn)用不同濃度的奎尼丁孵育表達(dá)hERG蛋白的HEK293細(xì)胞后,運(yùn)用Western blot技術(shù)檢測(cè)到hERG通道蛋白無(wú)明顯變化。說(shuō)明奎尼丁不影響hERG通道蛋白的表達(dá)轉(zhuǎn)運(yùn)。

綜上所述,奎尼丁對(duì)hERG鉀電流有直接抑制作用,但并不影響hERG通道蛋白的表達(dá)轉(zhuǎn)運(yùn)。其對(duì)hERG通道的直接阻斷作用為奎尼丁引起長(zhǎng)QT間期綜合征的根本機(jī)制。

[1] Wymore RS,Gintant GA,Wymore RT,etal. Tissue and species distribution of mRNA for the IKr-like K+channel,erg[J].Circ Res,1997,80(2):261-268.

[2] Trudeau MC,Warmke JW,Ganetzky B,etal.HERG,a human inward rectifier in the voltage-gated potassium channel family[J].Science,1995,269(5220):92-95.

[3] Zhang KP,Yang BF,Li BX.Translational toxicology and rescue strategies of the hERG channel dysfunction:biochemical and molecular mechanistic aspects[J].Acta Pharmacol Sin,2014,35(12):1473-1484.

[4] Nogawa H,Kawai T.hERG trafficking inhibition in drug-induced lethal cardiac arrhythmia[J].Eur J Pharmacol,2014,741:336-339.

[5] Tsujimae K,Suzuki S,Yamada M,etal.Comparison of kinetic properties of quinidine and dofetilide block of HERG channels[J].Eur J Pharmacol,2004,493(1-3):29-40.

[6] Anson BD,Ackerman MJ,Tester DJ,etal.Molecular and functional characterization of common polymorphisms in HERG(KCNH2)potassium channels. American journal of physiology[J].Am J Physiol Heart Circ Physiol,2004,286(6):H2434-2441.

[7] Vandenberg JI,Perry MD,Perrin MJ,etal.hERG K(+) channels:structure,function,and clinical significance[J].Physiol Rev,2012,92(3):1393-1478.

[8] Witchel HJ.Drug-induced hERG block and long QT syndrome[J].Cardiovasc Ther,2011,29(4):251-259.

[9] Smith JL,Reloj AR,Nataraj PS,etal.Pharmacological correction of long QT-linked mutations in KCNH2(hERG)increases the trafficking of Kv11.1 channels stored in the transitional endoplasmic reticulum[J].Am J Physiol Cell Physiol,2013,305:C919-C930.

[10] Cubeddu LX.Drug-induced inhibition and trafficking disruption of ion channels:pathogenesis of QT abnormalities and drug-induced fatal arrhythmias[J].Curr Cardiol Rev,2016,12(2):141-154.

[11] Staudacher I,Schweizer PA,Katus HA,etal.hERG:protein trafficking and potential for therapy and drug side effects[J].Curr Opin Drug Discov Devel,2010,13(1):23-30.

[12] Dennis A,Wang L,Wan X,etal.hERG channel trafficking:novel targets in drug-induced long QT syndrome[J].Biochem Soc Trans,2007,35(Pt 5):1060-1063.

Effects of quinidine on hERG potassium current and hERG channel protein in HEK293 cell line

HUO Jianhua1*,GUO Xueyan2,QIANG Hua1,LIU Ping1,BAI Ling1,MA Aiqun1

(1DepartmentofCardiovascularMedicine,FirstAffiliatedHospitalofXi’anJiaotongUniversity,Xi’an710061,China;2DepartmentofGastroenterology,ShaanxiProvincialPeople’sHospital;*Correspondingauthor,E-mail:huojianhua2005@126.com)

ObjectiveTo explore the effect of quinidine on ether-a-go-go related gene(hERG)current and hERG channel protein and its possible mechanism of quinidine-induced long QT syndrome.MethodsHEK293 cells were transiently transfected with plasmid containing hERG gene via Lipofectamine.The hERG current was observed by whole-cell patch-clamping and the expression of hERG channel protein was detected by Western blot analysis.①HEK293 cells were transfected with plasmid,and after 48 h the cells were treated with different concentrations(0,1,3,10 μmol/L)of quinidine and patch clamp experiments were operated for instantaneous effect of quinidine.②HEK293 cells were transfected into plasmid for 24 h,then treated with different concentrations of quinidine(1,3,10 μmol/L)for 24 h,and then the patch clamp and Western blot were operated to observe the chronic effect of quinidine.ResultsAfter HEK293 cells were transfected into the plasmid for 48 h,the hERG current was significantly inhibited after treated with different concentrations of quinidine.The maximal density of tail currents were(55.9±3.8)pA/pF,(35.7±3.2)pA/pF and(15.5±1.4)pA/pF in 1 μmol/L,3 μmol/L and 10 μmol/L quinidine groups,respectively(P<0.05vs0 μmol/L).HEK293 cells were transfected into plasmid for 24 h and cultured with different concentrations of quinidine for 24 h,the hERG current showed no significant change.Western blot result showed that there was no significant change in the expression of hERG protein in HEK293 cells cultured with different concentrations of quinidine.ConclusionQuinidine shows an immediate and direct inhibitory effect on hERG potassium current,but it does not affect the expression of hERG channel protein.The direct inhibition of hERG channel is the basic mechanism of long QT syndrome caused by quinidine.

long QT syndrome； quinidine； hERG； HEK293

國(guó)家自然科學(xué)基金資助項(xiàng)目(30801133)；陜西省自然科學(xué)基礎(chǔ)研究計(jì)劃項(xiàng)目(2014JM2-8154)

霍建華,男,1979-03生,博士,主治醫(yī)生,E-mail:huojianhua2005@126.com

2017-01-04

R541.7

A

1007-6611(2017)04-0301-05

10.13753/j.issn.1007-6611.2017.04.001