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

【摘要】：延遲整流鉀電流(I_K)包括快激活I_Kr和慢激活I_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是大動物2、3相復極的重要電流。已有報道,KCNQ1及KCNE1基因突變引起I_Ks減小或者增大,致動作電位復極延遲或加速,心電圖上分別表現為長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通道的功能調節(jié)具有重要意義。據報道,磷酸化是通過調節(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磷酸化對離子通道的調控備受關注。早期實驗結果顯示,PKC對克隆的小鼠、大鼠心臟的I_Ks表現明顯的抑制作用,而對豚鼠心臟的I_Ks表現為增大作用,對此種屬依賴性產生的原因解釋為,PKC使KCNE1的Ser102位點磷酸化而抑制電流,而豚鼠缺乏此位點,故表現為增大電流。最新研究表明,PKC磷酸化KCNE1-S102后,通過加快膜蛋白內吞而下調通道功能。然而,激活PKC可增大表達人源的KCNQ1/KCNE1通道電流(KCNE1上存在S102),故KCNQ1上存在可上調通道功能的磷酸化位點。我實驗研究結果發(fā)現,在豚鼠心室肌細胞(KCNE1上缺乏S102),通過激活PKC通路抑制I_Ks。綜上,激活PKC對I_Ks的調控作用表現為增大電流和抑制電流兩種結果。由于不同PKC亞型對通道功能可能具有相反的作用,因此我們推測PKC對I_Ks不一致的調控結果可能由不同的PKC亞型所致。有報道PKCβⅡ、ε亞型選擇性增大I_Ks,而α、β1、δ亞型對電流沒有明顯影響;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可顯著地調控I_Ks功能,但迄今對于不同PKC亞型特異性調控I_Ks的認識非常有限。為此,本研究主要采用膜片鉗電生理技術擬解決以下問題(1)激活不同PKC亞型對I_Ks的調節(jié)作用。(2)確定介導抑制、增大I_Ks兩種相反作用的PKC亞型。(3)PKC亞型特異性調控I_Ks的分子機制。第一部分不同PKC亞型對I_Ks的調節(jié)作用目的:激活不同PKC亞型對I_Ks的影響。方法:構建穩(wěn)定表達KCNQ1和KCNE1的HEK293細胞線,觀察PMA、cPKC激動肽和PKCε激動肽對通道電流的影響。為避免電流的衰減,實驗采用穿孔膜片全細胞模式記錄I_Ks。結果:在穩(wěn)定轉染I_Ks的HEK293細胞,外液中加入PMA(100 nM)后,去極化外向電流及復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I_Ks的增大作用5min出現,10-15 min左右達到穩(wěn)態(tài),沖洗后不能完全恢復。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,PMA可明顯降低通道的激活時間常數,在+50 mV電壓下,激活時間常數由926.14±128.01 ms減小到756.57±115.23 ms(P0.05)。在電極內液中加入cPKC激動肽和PKCε激動肽及其各自亂碼對照肽(所有肽都連接穿孔肽),結果如下:與cPKC對照肽相比,cPKC激動肽使I_Ks電流密度增加,當電壓去極至+50 mV時,尾電流密度由26.26±4.46 pA/pF(cPKC 對照肽)增加到 37.13±4.72 pA/pF(cPKC激動肽)(P0.05)。應用cPKC對照肽激活曲線的V_1/2和斜率因子分別為18.03±2.2 mV和17.8±1.5,而應用cPKC激動肽激活曲線的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,cPKC激動肽可明顯減小通道的激活時間常數,在+50 mV,激活時間常數由1142.09±168.85 ms降低到608.71±99.38 ms(P0.05)。與PKCε對照肽相比,PKCε激動肽使I_Ks電流密度下降,當電壓去極至+50 mV時,尾電流密度由40.81±6.78 pA/pF(PKCε 對照肽)下降到 16.68±2.26 pA/pF(PKCε 激動肽)(P0.01),而激活曲線V1/2和斜率因子沒有發(fā)生改變,激活時間常數也沒有發(fā)生改變。小結:PMA和cPKC激動肽增大I_Ks,使通道激活曲線發(fā)生左移,降低激活時間常數;PKCε激動肽抑制I_Ks,但不影響激活曲線和激活時間常數。第二部分確定中介I_Ks不同調節(jié)的PKC亞型目的:前期報道血管緊張素Ⅱ(Ang Ⅱ)通過PKC信號通路抑制豚鼠心室肌I_Ks,第一部分實驗發(fā)現PMA增大I_Ks,為此本部分實驗確定中介抑制、增大I_Ks兩種相反作用的PKC亞型。方法:在穩(wěn)定轉染I_KsHEK293細胞上,瞬時轉染人的Ang Ⅱ受體AT1cDNA,觀察AngⅡ對I_Ks的影響。進一步采用siRNA技術分別敲低PKCα PKCβ和PKCε亞型,觀察AngⅡ和PMA對以上細胞的調節(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。結果:首先在表達系統(tǒng)進一步確定Ang Ⅱ對I_Ks的影響。在穩(wěn)定轉染I_Ks的HEK293細胞上,共轉染人AT1 cDNA,外液中加入Ang Ⅱ(100 nM)后2-3 min去極化外向電流及復極化的尾電流均明顯減小,10-15 min左右達到穩(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。其中約400%細胞在給藥后1 min出現一過性微弱增大(約7%)。在0.1-1000 nM范圍內,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做Ang Ⅱ抑制I_Ks的量效曲線,經Hill方程擬合后得到IC50=7.5 nM。Ang Ⅱ對I_Ks的半數激活電壓及激活時間常數都沒有影響。轉染siRNA特異性敲低PKC亞型的表達,觀察Ang Ⅱ(K100 nM)和PMA(100 nM)對抑制、增大I_Ks的影響。結果發(fā)現,轉染PKCα+PKCβ siRNA后,Ang Ⅱ對I_Ks的抑制作用與轉染對照siRNA無明顯差別;而轉染PKCε siRNA特異性敲低PKCε表達后,Ang Ⅱ對尾電流的抑制(+50 mV下)顯著弱于對照(10.95%vs 26.06%,P0.05),表明Ang Ⅱ對電流的抑制由PKCε中介。轉染(亂碼)對照PKC siRNA后,PMA使I_Ks(+50 mV下)增加31.84%,分別敲低PKCα、PKCβ的表達,PMA對尾電流的增加幅度較對照顯著減弱,分別為16.82%(P0.05)、10.58%(P0.01);共同敲低 PKCα 和 PKCβ后,電流增強作用幾乎消失;敲低PKCε的表達不影響PMA的作用(28.87%vs 31.84%,P0.05)。表明PMA對電流的增強作用由cPKC的PKCα和PKCβ中介。小結:以上的實驗表明,AngⅡ對克隆人的I_Ks呈現抑制作用,此抑制作用由PKCε中介,而PMA對I_Ks的增強作用由PKCα和PKCβ中介。第三部分PKC亞型特異性調控I_Ks的分子機制目的:分析不同PKC亞型對I_Ks調節(jié)的分子機制。方法:分別突變KCNQ1、KCNE1上PKC的潛在磷酸化位點,觀察Ang Ⅱ和PMA抑制、增強電流作用的改變,分析不同PKC亞型對通道調控的分子機制。結果:KCNQ1上N端有2個、C端有4個評分較高的PKC潛在磷酸化位點,分別是S95、T96和S409、S464、T513、S577,而KCNE1上只有一個潛在PKC磷酸化位點,即S102。將上述KCNQ1上6個和KCNE1上的1個潛在磷酸化位點分別突變?yōu)楸彼?另外同時將KCNQ1 上N端2個或C端4個潛在磷酸化位點突變,分別為 KCNQ1-2M(含 N 端 S95A 和 T96A)和 KCNQ1-4M(含 C 端 S409A、S464A、T513A、S577A)。經檢測,上述所有突變通道的動力學特征與野生型一致。我們發(fā)現,Ang Ⅱ(100 nM)對 KCNQ1/KCNE1-S102A突變通道尾電流的抑制程度明顯小于對野生型通道的抑制(10.84%,vs 30.59%,P0.05),以上結果提示KCNE1上的S102是PKC抑制通道功能的磷酸化位點。同時我們注意到,KCNE1上S102突變后AngⅡ對通道的抑制作用并沒有完全消失,推測除S102外,KCNQ1上也存在抑制通道功能的磷酸化位點。進一步的實驗表明,Ang Ⅱ對KCNQ1N端突變通道(KCNQ1-2M)尾電流的抑制程度為11.71%,顯著弱于對野生型通道的作用(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)。以上結果說明,KCNQ1的N端參與PKC對通道功能的抑制。分別突變N端的S95和T96均可減弱Ang Ⅱ對通道的抑制作用,對S95A和T96A通道的抑制分別為15%(P0.01)和16.33%(P0.01),說明S95和T96都參與PKC抑制通道功能。聯合突變KCNQ1的N端與 KCNE1 的 S102 后,Ang Ⅱ 對通道(KCNQ1-2M/KCNE1-S102A)的抑制作用幾乎消失(2.76%,P0.01)。以上結果說明PKC在I_Ks上有三個抑制位點,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亞基有增強通道功能的位點。PMA對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明顯區(qū)別(P0.05),而C端突變幾乎取消了其增強作用。結果說明KCNQ1亞基C端參與PKC對通道作用的增強性調控。進一步分別突變C端四個位點,結果顯示,PMA對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。結果說明KCNQ1亞基C端的S409、S464、T513以及S577是PKC增強通道功能的磷酸化位點。我們進一步在HEK293細胞上表達克隆的豚鼠KCNQ1/KCNE1通道,將KCNQ1上N端唯一的潛在磷酸化位點S96突變?yōu)楸彼岷?Ang Ⅱ對突變通道的抑制作用幾乎消失,進一步表明KCNQ1上N端磷酸化抑制通道功能。小結:KCNQ1亞基存在PKC抑制、增強通道功能的位點,N端磷酸化抑制通道功能,而C端磷酸化增強通道功能;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.
【學位授予單位】：河北醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：R54

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