本文選題：離子通道 + 磷酸化　； 參考：《河北醫(yī)科大學(xué)》2017年博士論文

【摘要】：延遲整流鉀電流(I_K)包括快激活I(lǐng)_Kr和慢激活I(lǐng)_Ks兩種成分,其中KCNQ1和KCNE1分別編碼I_Ks通道的孔區(qū)α亞單位和輔助β亞單位。I_Ks是大動(dòng)物2、3相復(fù)極的重要電流。已有報(bào)道,KCNQ1及KCNE1基因突變引起I_Ks減小或者增大,致動(dòng)作電位復(fù)極延遲或加速,心電圖上分別表現(xiàn)為長(zhǎng)QT綜合征(long QT syndrome,LQT1或者LQT5)和短QT綜合癥(short QT syndrome,SQT2)。在高血壓、冠心病和心肌缺血等引發(fā)的心肌肥厚和心衰以及糖尿病等病理狀態(tài)下,均伴有I_Ks減小,而這是造成獲得性LQT綜合征的重要原因。LQT和SQT均以高發(fā)室性心律失常為特征。因此,研究KCNQ1/KCNE1通道的功能調(diào)節(jié)具有重要意義。據(jù)報(bào)道,磷酸化是通過(guò)調(diào)節(jié)通道功能從而影響心肌電生理功能的有效方式之一,其中PKC磷酸化對(duì)離子通道的調(diào)控備受關(guān)注。早期實(shí)驗(yàn)結(jié)果顯示,PKC對(duì)克隆的小鼠、大鼠心臟的I_Ks表現(xiàn)明顯的抑制作用,而對(duì)豚鼠心臟的I_Ks表現(xiàn)為增大作用,對(duì)此種屬依賴性產(chǎn)生的原因解釋為,PKC使KCNE1的Ser102位點(diǎn)磷酸化而抑制電流,而豚鼠缺乏此位點(diǎn),故表現(xiàn)為增大電流。最新研究表明,PKC磷酸化KCNE1-S102后,通過(guò)加快膜蛋白內(nèi)吞而下調(diào)通道功能。然而,激活PKC可增大表達(dá)人源的KCNQ1/KCNE1通道電流(KCNE1上存在S102),故KCNQ1上存在可上調(diào)通道功能的磷酸化位點(diǎn)。我實(shí)驗(yàn)研究結(jié)果發(fā)現(xiàn),在豚鼠心室肌細(xì)胞(KCNE1上缺乏S102),通過(guò)激活PKC通路抑制I_Ks。綜上,激活PKC對(duì)I_Ks的調(diào)控作用表現(xiàn)為增大電流和抑制電流兩種結(jié)果。由于不同PKC亞型對(duì)通道功能可能具有相反的作用,因此我們推測(cè)PKC對(duì)I_Ks不一致的調(diào)控結(jié)果可能由不同的PKC亞型所致。有報(bào)道PKCβⅡ、ε亞型選擇性增大I_Ks,而α、β1、δ亞型對(duì)電流沒(méi)有明顯影響;PKCε亞型中介PMA和腎上腺素α受體增大豚鼠心房I_Ks的作用。綜上,PKC可顯著地調(diào)控I_Ks功能,但迄今對(duì)于不同PKC亞型特異性調(diào)控I_Ks的認(rèn)識(shí)非常有限。為此,本研究主要采用膜片鉗電生理技術(shù)擬解決以下問(wèn)題(1)激活不同PKC亞型對(duì)I_Ks的調(diào)節(jié)作用。(2)確定介導(dǎo)抑制、增大I_Ks兩種相反作用的PKC亞型。(3)PKC亞型特異性調(diào)控I_Ks的分子機(jī)制。第一部分不同PKC亞型對(duì)I_Ks的調(diào)節(jié)作用目的:激活不同PKC亞型對(duì)I_Ks的影響。方法:構(gòu)建穩(wěn)定表達(dá)KCNQ1和KCNE1的HEK293細(xì)胞線,觀察PMA、cPKC激動(dòng)肽和PKCε激動(dòng)肽對(duì)通道電流的影響。為避免電流的衰減,實(shí)驗(yàn)采用穿孔膜片全細(xì)胞模式記錄I_Ks。結(jié)果:在穩(wěn)定轉(zhuǎn)染I_Ks的HEK293細(xì)胞,外液中加入PMA(100 nM)后,去極化外向電流及復(fù)極化的尾電流明顯增加。在+50mV電壓下,尾電流密度由37.20±6.98 pA/pF 增加到 45.39±7.01 pA/pF(P0.05)。PMA對(duì)I_Ks的增大作用5min出現(xiàn),10-15 min左右達(dá)到穩(wěn)態(tài),沖洗后不能完全恢復(fù)。由激活曲線得出給藥前V_1/2和斜率因子分別為20.04±1.31 mV和15.69±0.76,應(yīng)用PMA后的V_1/2和斜率因子分別為14.48±2.21 mV和16.99±1.94,PMA使I_Ks激活曲線發(fā)生左移。在電壓+10 mV～+50 mV范圍內(nèi),PMA可明顯降低通道的激活時(shí)間常數(shù),在+50 mV電壓下,激活時(shí)間常數(shù)由926.14±128.01 ms減小到756.57±115.23 ms(P0.05)。在電極內(nèi)液中加入cPKC激動(dòng)肽和PKCε激動(dòng)肽及其各自亂碼對(duì)照肽(所有肽都連接穿孔肽),結(jié)果如下:與cPKC對(duì)照肽相比,cPKC激動(dòng)肽使I_Ks電流密度增加,當(dāng)電壓去極至+50 mV時(shí),尾電流密度由26.26±4.46 pA/pF(cPKC 對(duì)照肽)增加到 37.13±4.72 pA/pF(cPKC激動(dòng)肽)(P0.05)。應(yīng)用cPKC對(duì)照肽激活曲線的V_1/2和斜率因子分別為18.03±2.2 mV和17.8±1.5,而應(yīng)用cPKC激動(dòng)肽激活曲線的V1/2和斜率因子分別為8.9±2.7mV和15.6±0.9,曲線明顯發(fā)生左移。在電壓+10 mV～+50 mV范圍內(nèi),cPKC激動(dòng)肽可明顯減小通道的激活時(shí)間常數(shù),在+50 mV,激活時(shí)間常數(shù)由1142.09±168.85 ms降低到608.71±99.38 ms(P0.05)。與PKCε對(duì)照肽相比,PKCε激動(dòng)肽使I_Ks電流密度下降,當(dāng)電壓去極至+50 mV時(shí),尾電流密度由40.81±6.78 pA/pF(PKCε 對(duì)照肽)下降到 16.68±2.26 pA/pF(PKCε 激動(dòng)肽)(P0.01),而激活曲線V1/2和斜率因子沒(méi)有發(fā)生改變,激活時(shí)間常數(shù)也沒(méi)有發(fā)生改變。小結(jié):PMA和cPKC激動(dòng)肽增大I_Ks,使通道激活曲線發(fā)生左移,降低激活時(shí)間常數(shù);PKCε激動(dòng)肽抑制I_Ks,但不影響激活曲線和激活時(shí)間常數(shù)。第二部分確定中介I_Ks不同調(diào)節(jié)的PKC亞型目的:前期報(bào)道血管緊張素Ⅱ(Ang Ⅱ)通過(guò)PKC信號(hào)通路抑制豚鼠心室肌I_Ks,第一部分實(shí)驗(yàn)發(fā)現(xiàn)PMA增大I_Ks,為此本部分實(shí)驗(yàn)確定中介抑制、增大I_Ks兩種相反作用的PKC亞型。方法:在穩(wěn)定轉(zhuǎn)染I_KsHEK293細(xì)胞上,瞬時(shí)轉(zhuǎn)染人的Ang Ⅱ受體AT1cDNA,觀察AngⅡ?qū)_Ks的影響。進(jìn)一步采用siRNA技術(shù)分別敲低PKCα PKCβ和PKCε亞型,觀察AngⅡ和PMA對(duì)以上細(xì)胞的調(diào)節(jié)作用,從而確定中介抑制、增大I_Ks兩種相反作用的PKC亞型。結(jié)果:首先在表達(dá)系統(tǒng)進(jìn)一步確定Ang Ⅱ?qū)_Ks的影響。在穩(wěn)定轉(zhuǎn)染I_Ks的HEK293細(xì)胞上,共轉(zhuǎn)染人AT1 cDNA,外液中加入Ang Ⅱ(100 nM)后2-3 min去極化外向電流及復(fù)極化的尾電流均明顯減小,10-15 min左右達(dá)到穩(wěn)態(tài),在+50 mV,尾電流密度由55.40±11.03 pA/pF減小到42.29±8.89pA/pF(P0.05),抑制作用沖洗后不能完全恢復(fù)。其中約400%細(xì)胞在給藥后1 min出現(xiàn)一過(guò)性微弱增大(約7%)。在0.1-1000 nM范圍內(nèi),Ang Ⅱ以濃度依賴性方式抑制I_Ks,以I_Ks尾電流抑制率為縱坐標(biāo)做Ang Ⅱ抑制I_Ks的量效曲線,經(jīng)Hill方程擬合后得到IC50=7.5 nM。Ang Ⅱ?qū)_Ks的半數(shù)激活電壓及激活時(shí)間常數(shù)都沒(méi)有影響。轉(zhuǎn)染siRNA特異性敲低PKC亞型的表達(dá),觀察Ang Ⅱ(K100 nM)和PMA(100 nM)對(duì)抑制、增大I_Ks的影響。結(jié)果發(fā)現(xiàn),轉(zhuǎn)染PKCα+PKCβ siRNA后,Ang Ⅱ?qū)_Ks的抑制作用與轉(zhuǎn)染對(duì)照siRNA無(wú)明顯差別;而轉(zhuǎn)染PKCε siRNA特異性敲低PKCε表達(dá)后,Ang Ⅱ?qū)ξ搽娏鞯囊种?+50 mV下)顯著弱于對(duì)照(10.95%vs 26.06%,P0.05),表明Ang Ⅱ?qū)﹄娏鞯囊种朴蒔KCε中介。轉(zhuǎn)染(亂碼)對(duì)照PKC siRNA后,PMA使I_Ks(+50 mV下)增加31.84%,分別敲低PKCα、PKCβ的表達(dá),PMA對(duì)尾電流的增加幅度較對(duì)照顯著減弱,分別為16.82%(P0.05)、10.58%(P0.01);共同敲低 PKCα 和 PKCβ后,電流增強(qiáng)作用幾乎消失;敲低PKCε的表達(dá)不影響PMA的作用(28.87%vs 31.84%,P0.05)。表明PMA對(duì)電流的增強(qiáng)作用由cPKC的PKCα和PKCβ中介。小結(jié):以上的實(shí)驗(yàn)表明,AngⅡ?qū)寺∪说腎_Ks呈現(xiàn)抑制作用,此抑制作用由PKCε中介,而PMA對(duì)I_Ks的增強(qiáng)作用由PKCα和PKCβ中介。第三部分PKC亞型特異性調(diào)控I_Ks的分子機(jī)制目的:分析不同PKC亞型對(duì)I_Ks調(diào)節(jié)的分子機(jī)制。方法:分別突變KCNQ1、KCNE1上PKC的潛在磷酸化位點(diǎn),觀察Ang Ⅱ和PMA抑制、增強(qiáng)電流作用的改變,分析不同PKC亞型對(duì)通道調(diào)控的分子機(jī)制。結(jié)果:KCNQ1上N端有2個(gè)、C端有4個(gè)評(píng)分較高的PKC潛在磷酸化位點(diǎn),分別是S95、T96和S409、S464、T513、S577,而KCNE1上只有一個(gè)潛在PKC磷酸化位點(diǎn),即S102。將上述KCNQ1上6個(gè)和KCNE1上的1個(gè)潛在磷酸化位點(diǎn)分別突變?yōu)楸彼?另外同時(shí)將KCNQ1 上N端2個(gè)或C端4個(gè)潛在磷酸化位點(diǎn)突變,分別為 KCNQ1-2M(含 N 端 S95A 和 T96A)和 KCNQ1-4M(含 C 端 S409A、S464A、T513A、S577A)。經(jīng)檢測(cè),上述所有突變通道的動(dòng)力學(xué)特征與野生型一致。我們發(fā)現(xiàn),Ang Ⅱ(100 nM)對(duì) KCNQ1/KCNE1-S102A突變通道尾電流的抑制程度明顯小于對(duì)野生型通道的抑制(10.84%,vs 30.59%,P0.05),以上結(jié)果提示KCNE1上的S102是PKC抑制通道功能的磷酸化位點(diǎn)。同時(shí)我們注意到,KCNE1上S102突變后AngⅡ?qū)νǖ赖囊种谱饔貌](méi)有完全消失,推測(cè)除S102外,KCNQ1上也存在抑制通道功能的磷酸化位點(diǎn)。進(jìn)一步的實(shí)驗(yàn)表明,Ang Ⅱ?qū)CNQ1N端突變通道(KCNQ1-2M)尾電流的抑制程度為11.71%,顯著弱于對(duì)野生型通道的作用(30.59%,P0.01),而KCNQ1的C端突變(KCNQ1-4M)和并不影響Ang Ⅱ的抑制作用(30.62%,P0.05)。以上結(jié)果說(shuō)明,KCNQ1的N端參與PKC對(duì)通道功能的抑制。分別突變N端的S95和T96均可減弱Ang Ⅱ?qū)νǖ赖囊种谱饔?對(duì)S95A和T96A通道的抑制分別為15%(P0.01)和16.33%(P0.01),說(shuō)明S95和T96都參與PKC抑制通道功能。聯(lián)合突變KCNQ1的N端與 KCNE1 的 S102 后,Ang Ⅱ 對(duì)通道(KCNQ1-2M/KCNE1-S102A)的抑制作用幾乎消失(2.76%,P0.01)。以上結(jié)果說(shuō)明PKC在I_Ks上有三個(gè)抑制位點(diǎn),分別是KCNQ1上的S95、T96和KCNE1上的S102。KCNE1上S102突變并不影響PMA增大I_Ks的作用,我們推斷KCNQ1亞基有增強(qiáng)通道功能的位點(diǎn)。PMA對(duì)KCNQ1-2M/KCNE1和KCNQ1-4M/KCNE1通道的電流增加分別為31.74%和3.18%,N端突變與野生型通道作用無(wú)明顯區(qū)別(P0.05),而C端突變幾乎取消了其增強(qiáng)作用。結(jié)果說(shuō)明KCNQ1亞基C端參與PKC對(duì)通道作用的增強(qiáng)性調(diào)控。進(jìn)一步分別突變C端四個(gè)位點(diǎn),結(jié)果顯示,PMA對(duì)S409A、S464A、T513A、S577A突變通道電流分別增加12.98%、8.56%、6.37%以及11.71%,均較野生型顯著降低。結(jié)果說(shuō)明KCNQ1亞基C端的S409、S464、T513以及S577是PKC增強(qiáng)通道功能的磷酸化位點(diǎn)。我們進(jìn)一步在HEK293細(xì)胞上表達(dá)克隆的豚鼠KCNQ1/KCNE1通道,將KCNQ1上N端唯一的潛在磷酸化位點(diǎn)S96突變?yōu)楸彼岷?Ang Ⅱ?qū)ν蛔兺ǖ赖囊种谱饔脦缀跸?進(jìn)一步表明KCNQ1上N端磷酸化抑制通道功能。小結(jié):KCNQ1亞基存在PKC抑制、增強(qiáng)通道功能的位點(diǎn),N端磷酸化抑制通道功能,而C端磷酸化增強(qiáng)通道功能;KCNE1上的S102磷酸化則抑制通道功能。
[Abstract]:Delayed rectifier potassium current (I_K) consists of two components, fast activated I_Kr and slow activated I_Ks, in which KCNQ1 and KCNE1 encode the pore region alpha subunit of the I_Ks channel and the auxiliary beta subunit.I_Ks, an important current for the 2,3 phase repolarization of large animals. It has been reported that the KCNQ1 and KCNE1 gene mutations cause I_Ks to decrease or increase, causing action potential repolarization delay or QT syndrome (long QT syndrome, LQT1 or LQT5) and short QT syndrome (short QT syndrome, SQT2) were accelerated respectively. In the pathological conditions, such as hypertension, coronary heart disease, and myocardial ischemia, such as cardiac hypertrophy, heart failure and diabetes, all of them were associated with I_Ks reduction, which was an important cause of acquired syndrome. LQT and SQT are characterized by high incidence of ventricular arrhythmia. Therefore, the study of the functional regulation of KCNQ1/KCNE1 channels is of great significance. It is reported that phosphorylation is one of the effective ways to affect the electrophysiological function of the myocardium by regulating the function of the channel, and the regulation of the phosphorylation of PKC has attracted much attention. Early experimental results show that PKC The I_Ks expression in the heart of the cloned mice is obviously inhibited, and the I_Ks expression of the guinea pig's heart is increased. The reason for this dependence is that PKC makes the Ser102 site of KCNE1 phosphorylation and inhibits the current, while the guinea pig lacks this site, so it is present to increase the current. The latest research shows that PKC phosphorylation of KCNE1-S1 is the most recent study. After 02, the channel function was downregulated by accelerating the membrane protein endocytosis. However, activation of PKC could increase the KCNQ1/KCNE1 channel current of the human source (S102 on KCNE1). Therefore, there is a phosphorylation site on KCNQ1 that can increase the function of the channel. In S. synthesis, the activation of PKC to I_Ks shows two results of increasing current and inhibiting current. Since different PKC subtypes may have the opposite effect on channel function, we speculate that the result of PKC on I_Ks inconsistencies may be caused by different PKC subtypes. It is reported that PKC beta II, epsilon subtype is selective increasing I_Ks, and alpha, beta 1, Delta. Subtypes have no obvious effects on the current; PKC - epsilon PMA and adrenaline receptors increase the role of I_Ks in the guinea pig atrium. To sum up, PKC can significantly regulate the function of I_Ks, but so far, the understanding of the specific regulation of I_Ks in different PKC subtypes is very limited. Therefore, this study mainly uses the patch clamp electrophysiological technique to solve the following problems (1) The regulating effect of different PKC subtypes on I_Ks. (2) determine the mediating inhibition and increase the PKC subtypes of the opposite action of I_Ks. (3) the molecular mechanism of the PKC subtype specific regulation of I_Ks. The first part of the regulation of I_Ks by different PKC subtypes: activation of the effect of different PKC subtypes on I_Ks. Method: to construct a stable expression of KCNQ1 and KCNE1 cells. Line, observe the effect of PMA, cPKC agonist and PKC epsilon on channel current. In order to avoid current attenuation, the experiment uses perforated diaphragm whole cell mode to record the result of I_Ks.: after adding PMA (100 nM) to I_Ks HEK293 cells, the depolarization extrovert current and repolarization current increase obviously. The flow density increased from 37.20 + 6.98 pA/pF to 45.39 + 7.01 pA/pF (P0.05).PMA to I_Ks, 5min appeared, and 10-15 min reached steady state. After washing, it could not be completely recovered. The V_1/2 and slope factors were 20.04 + 1.31 mV and 15.69 + 0.76 respectively before the injection, and V_1/2 and slope factors after PMA were 14.48 + 2.2 respectively. 1 mV and 16.99 + 1.94, PMA makes the I_Ks activation curve move left. Within the range of +10 mV to +50 mV, PMA can obviously reduce the activation time constant of the channel. At the +50 mV voltage, the activation time constant decreases from 926.14 + 128.01 MS to 756.57 + 115.23 MS. The results were as follows: compared with cPKC control peptide, cPKC agonist increased the current density of I_Ks. When the voltage went to +50 mV, the tail current density increased from 26.26 + 4.46 pA/pF (cPKC control peptide) to 37.13 + 4.72 pA/pF (cPKC agonist) (P0.05). The V_1/2 and slope factor of the cPKC control peptide activation curve was applied. The V1/2 and slope factors were 18.03 + 2.2 mV and 17.8 + 1.5 respectively, and the curves of cPKC agonist activation curves were 8.9 + 2.7mV and 15.6 + 0.9 respectively. In the range of voltage +10 mV to +50 mV, the cPKC agonist peptide could obviously reduce the activation time constant of the channel, and the activation time constant was reduced from 1142.09 + 168.85 MS to 608 at +50 mV. .71 + 99.38 MS (P0.05). Compared with PKC e control peptide, PKC e agonist decreased the current density of I_Ks. When the voltage went to +50 mV, the tail current density decreased from 40.81 + 6.78 pA/pF (PKC epsilon) to 16.68 + 2.26 pA/pF (PKC epsilon), but the activation curve and the slope factor did not change, and the activation time constant was not Changes. Summary: PMA and cPKC agonists increase I_Ks, make the activation curve of channel left shift and decrease activation time constant; PKC epsilon peptide inhibits I_Ks, but does not affect activation curve and activation time constant. The second part determines the PKC subtype of mediator I_Ks with different regulation: the anterior report of angiotensin II (Ang II) through PKC signaling pathway Inhibition of I_Ks in the guinea pig ventricular muscle, the first part of the experiment found that PMA increased I_Ks, so this part of the experiment determined the intermediary inhibition and increased the PKC subtype of the I_Ks two opposite effects. Method: transfection of Ang II receptor AT1cDNA on the stable transfection of I_KsHEK293 cells, observe the effect of Ang II on I_Ks, and further adopt siRNA technology to knock low PKC alpha respectively. PKC beta and PKC epsilon subtypes were used to observe the regulatory effect of Ang II and PMA on the above cells, thus determining the mediating inhibition and increasing the PKC subtypes of the two opposite acts of I_Ks. Results: first, the effect of Ang II on I_Ks was further determined in the expression system. The AT1 cDNA was transferred on the HEK293 cells that stably transfected with I_Ks, and 2-3 of the external fluids were added to the I_Ks. The tail current of in depolarization extrovert current and repolarization decreased obviously, and reached steady state at about 10-15 min. At +50 mV, the tail current density decreased from 55.40 + 11.03 pA/pF to 42.29 + 8.89pA/pF (P0.05). The inhibition effect could not be completely recovered after the inhibition. About 400% cells were slightly enlarged (about 7%) in 1 min after the drug delivery. In 0.1-1000 nM norm. In the circumference, Ang II inhibited I_Ks in a concentration dependent manner, taking the I_Ks tail current inhibition rate as the longitudinal coordinate to do Ang II inhibition of I_Ks. After the Hill equation, it was obtained that IC50=7.5 nM.Ang II had no effect on the median activation voltage and the activation time constant of I_Ks. The expression of siRNA specific low PKC subtype was observed and Ang II was observed. M) and PMA (100 nM) affect the inhibition and increase the effect of I_Ks. It was found that after transfection of PKC alpha +PKC beta siRNA, the inhibitory effect of Ang II on I_Ks was not significantly different from that of the transfected control siRNA. The suppression of current is mediated by PKC e. After transfection (chaotic code) against PKC siRNA, PMA increases I_Ks (+50 mV) by 31.84%, knocks low PKC a, PKC beta, and PMA increases the tail current significantly less than the control, which is 16.82% (P0.05) and 10.58% (P0.01). The effect of PMA (28.87%vs 31.84%, P0.05). Indicates that the enhancement of PMA to the current is mediated by PKC alpha and PKC beta of cPKC. Conclusion: the above experiments show that Ang II inhibits the I_Ks of human cloning, and this inhibition is mediated by PKC epsilon, while PMA on I_Ks enhancement is mediated by alpha and beta. The third part of the specific modulation of the subtype. The molecular mechanism of controlling I_Ks: analysis the molecular mechanism of different PKC subtypes on I_Ks regulation. Methods: mutation KCNQ1, the potential phosphorylation site of PKC on KCNE1, Ang II and PMA inhibition, enhancement of current action, and analysis of the molecular mechanism of channel regulation by different PKC subtypes. Results: KCNQ1 N ends have 2, and C ends have 4 higher scores. The potential phosphorylation sites of PKC are S95, T96 and S409, S464, T513, S577, and there is only one potential PKC phosphorylation site on KCNE1, namely, S102. mutation of the above 6 and 1 potential phosphorylation sites on KCNE1, respectively, and 4 potential phosphorylation sites on the 2 or 4 terminals. N terminal S95A and T96A) and KCNQ1-4M (including C terminal S409A, S464A, T513A, S577A). The kinetic characteristics of all the mutation channels are in accordance with the wild type. We found that Ang II (100 nM) inhibited the tail current of the KCNQ1/KCNE1-S102A mutation channel significantly less than that of the wild type channel (10.84%, 30.59%,), above The results suggest that S102 on KCNE1 is the phosphorylation site of the function of PKC inhibition channel. At the same time, we notice that the inhibitory effect of Ang II on the channel is not completely disappeared after the S102 mutation on KCNE1. It is speculated that there is also a phosphorylation site that inhibits the channel function except for S102. Further evidence shows that Ang II has a mutation channel (KCNQ1-2M) on the KCNQ1N end. The suppression of the tail current was 11.71%, significantly weaker than the effect on the wild type channel (30.59%, P0.01), while the C end mutation (KCNQ1-4M) of KCNQ1 did not affect the inhibitory effect of Ang II (30.62%, P0.05). The above results indicated that the N end of KCNQ1 was involved in the suppression of the channel function by PKC. The S95 and T96 of the N ends could weaken the inhibition of the channel II to the channel. The suppression of the S95A and T96A channels is 15% (P0.01) and 16.33% (P0.01) respectively, indicating that both S95 and T96 are involved in the PKC suppression channel function. The inhibition of the Ang II to the channel is almost disappeared after the N end and KCNE1 S102 of the joint mutation KCNQ1 (2.76%,). The above results indicate that there are three inhibitory sites on the KCNQ1. Point, the S102 mutation on S102.KCNE1 on the KCNQ1 on the S95, T96 and KCNE1 does not affect the effect of PMA on I_Ks. We infer that the increase of the channel function of the KCNQ1 subunit is 31.74% and 3.18% for KCNQ1-2M/KCNE1 and KCNQ1-4M/KCNE1 channels, respectively, but there is no significant difference between the end process and the wild type channel. The C end mutation almost cancelled its enhancement. The results showed that the C end of the KCNQ1 subunit was involved in the enhancement of the effect of PKC on the channel function. Further mutation of four loci of the C terminal respectively. The results showed that PMA increased the current of the mutation channel of S409A, S464A, T513A and S577A respectively by 12.98%, 8.56%, 6.37% and 11.71%, respectively, compared with the wild type. The result indicated KCNQ1. S409, S464, T513 and S577 at the subunit C end are the phosphorylation sites of PKC enhanced channel function. We further express the cloned guinea pig KCNQ1/KCNE1 channel on HEK293 cells, after the only potential phosphorylation site of the N end of KCNQ1 is S96 mutation to alanine, and the inhibition effect of Ang II to the mutant channel almost disappeared. Phosphorylation inhibition channel function. Summary: KCNQ1 subunit has PKC inhibition, enhanced channel function site, N terminal phosphorylation inhibition channel function, while C terminal phosphorylation enhanced channel function, and S102 phosphorylation on KCNE1 inhibited channel function.
【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：R54

【相似文獻(xiàn)】

相關(guān)期刊論文 前3條

1 鄒志強(qiáng);李濤;陳鎮(zhèn)奇;莊文;李廷俊;張錦忠;楊湘輝;;凝血酶誘導(dǎo)人單核細(xì)胞分泌白細(xì)胞介素-6的分析[J];中國(guó)醫(yī)學(xué)創(chuàng)新;2010年29期

2 李廷俊;鄒志強(qiáng);李濤;陳鎮(zhèn)奇;張錦忠;楊湘輝;;凝血酶和PARs激動(dòng)肽誘導(dǎo)單核細(xì)胞分泌IL-6作用的研究[J];臨床輸血與檢驗(yàn);2011年04期

3 ;[J];;年期

相關(guān)博士學(xué)位論文 前1條

1 勾向博;蛋白激酶C亞型特異性調(diào)控慢激活延遲整流鉀電流及其分子機(jī)制[D];河北醫(yī)科大學(xué);2017年

，

本文編號(hào)：2099852