蛋白激酶C亚型特异性调控慢激活延迟整流钾电流及其分子机制

发布时间：2018-07-05 10:06

本文选题：离子通道 + 磷酸化　；参考：《河北医科大学》2017年博士论文

【摘要】：延迟整流钾电流(I_K)包括快激活I_Kr和慢激活I_Ks两种成分,其中KCNQ1和KCNE1分别编码I_Ks通道的孔区α亚单位和辅助β亚单位。I_Ks是大动物2、3相复极的重要电流。已有报道,KCNQ1及KCNE1基因突变引起I_Ks减小或者增大,致动作电位复极延迟或加速,心电图上分别表现为长QT综合征(long QT syndrome,LQT1或者LQT5)和短QT综合症(short QT syndrome,SQT2)。在高血压、冠心病和心肌缺血等引发的心肌肥厚和心衰以及糖尿病等病理状态下,均伴有I_Ks减小,而这是造成获得性LQT综合征的重要原因。LQT和SQT均以高发室性心律失常为特征。因此,研究KCNQ1/KCNE1通道的功能调节具有重要意义。据报道,磷酸化是通过调节通道功能从而影响心肌电生理功能的有效方式之一,其中PKC磷酸化对离子通道的调控备受关注。早期实验结果显示,PKC对克隆的小鼠、大鼠心脏的I_Ks表现明显的抑制作用,而对豚鼠心脏的I_Ks表现为增大作用,对此种属依赖性产生的原因解释为,PKC使KCNE1的Ser102位点磷酸化而抑制电流,而豚鼠缺乏此位点,故表现为增大电流。最新研究表明,PKC磷酸化KCNE1-S102后,通过加快膜蛋白内吞而下调通道功能。然而,激活PKC可增大表达人源的KCNQ1/KCNE1通道电流(KCNE1上存在S102),故KCNQ1上存在可上调通道功能的磷酸化位点。我实验研究结果发现,在豚鼠心室肌细胞(KCNE1上缺乏S102),通过激活PKC通路抑制I_Ks。综上,激活PKC对I_Ks的调控作用表现为增大电流和抑制电流两种结果。由于不同PKC亚型对通道功能可能具有相反的作用,因此我们推测PKC对I_Ks不一致的调控结果可能由不同的PKC亚型所致。有报道PKCβⅡ、ε亚型选择性增大I_Ks,而α、β1、δ亚型对电流没有明显影响;PKCε亚型中介PMA和肾上腺素α受体增大豚鼠心房I_Ks的作用。综上,PKC可显著地调控I_Ks功能,但迄今对于不同PKC亚型特异性调控I_Ks的认识非常有限。为此,本研究主要采用膜片钳电生理技术拟解决以下问题(1)激活不同PKC亚型对I_Ks的调节作用。(2)确定介导抑制、增大I_Ks两种相反作用的PKC亚型。(3)PKC亚型特异性调控I_Ks的分子机制。第一部分不同PKC亚型对I_Ks的调节作用目的:激活不同PKC亚型对I_Ks的影响。方法:构建稳定表达KCNQ1和KCNE1的HEK293细胞线,观察PMA、cPKC激动肽和PKCε激动肽对通道电流的影响。为避免电流的衰减,实验采用穿孔膜片全细胞模式记录I_Ks。结果:在稳定转染I_Ks的HEK293细胞,外液中加入PMA(100 nM)后,去极化外向电流及复极化的尾电流明显增加。在+50mV电压下,尾电流密度由37.20±6.98 pA/pF 增加到 45.39±7.01 pA/pF(P0.05)。PMA对I_Ks的增大作用5min出现,10-15 min左右达到稳态,冲洗后不能完全恢复。由激活曲线得出给药前V_1/2和斜率因子分别为20.04±1.31 mV和15.69±0.76,应用PMA后的V_1/2和斜率因子分别为14.48±2.21 mV和16.99±1.94,PMA使I_Ks激活曲线发生左移。在电压+10 mV～+50 mV范围内,PMA可明显降低通道的激活时间常数,在+50 mV电压下,激活时间常数由926.14±128.01 ms减小到756.57±115.23 ms(P0.05)。在电极内液中加入cPKC激动肽和PKCε激动肽及其各自乱码对照肽(所有肽都连接穿孔肽),结果如下:与cPKC对照肽相比,cPKC激动肽使I_Ks电流密度增加,当电压去极至+50 mV时,尾电流密度由26.26±4.46 pA/pF(cPKC 对照肽)增加到 37.13±4.72 pA/pF(cPKC激动肽)(P0.05)。应用cPKC对照肽激活曲线的V_1/2和斜率因子分别为18.03±2.2 mV和17.8±1.5,而应用cPKC激动肽激活曲线的V1/2和斜率因子分别为8.9±2.7mV和15.6±0.9,曲线明显发生左移。在电压+10 mV～+50 mV范围内,cPKC激动肽可明显减小通道的激活时间常数,在+50 mV,激活时间常数由1142.09±168.85 ms降低到608.71±99.38 ms(P0.05)。与PKCε对照肽相比,PKCε激动肽使I_Ks电流密度下降,当电压去极至+50 mV时,尾电流密度由40.81±6.78 pA/pF(PKCε 对照肽)下降到 16.68±2.26 pA/pF(PKCε 激动肽)(P0.01),而激活曲线V1/2和斜率因子没有发生改变,激活时间常数也没有发生改变。小结:PMA和cPKC激动肽增大I_Ks,使通道激活曲线发生左移,降低激活时间常数;PKCε激动肽抑制I_Ks,但不影响激活曲线和激活时间常数。第二部分确定中介I_Ks不同调节的PKC亚型目的:前期报道血管紧张素Ⅱ(Ang Ⅱ)通过PKC信号通路抑制豚鼠心室肌I_Ks,第一部分实验发现PMA增大I_Ks,为此本部分实验确定中介抑制、增大I_Ks两种相反作用的PKC亚型。方法:在稳定转染I_KsHEK293细胞上,瞬时转染人的Ang Ⅱ受体AT1cDNA,观察AngⅡ对I_Ks的影响。进一步采用siRNA技术分别敲低PKCα PKCβ和PKCε亚型,观察AngⅡ和PMA对以上细胞的调节作用,从而确定中介抑制、增大I_Ks两种相反作用的PKC亚型。结果:首先在表达系统进一步确定Ang Ⅱ对I_Ks的影响。在稳定转染I_Ks的HEK293细胞上,共转染人AT1 cDNA,外液中加入Ang Ⅱ(100 nM)后2-3 min去极化外向电流及复极化的尾电流均明显减小,10-15 min左右达到稳态,在+50 mV,尾电流密度由55.40±11.03 pA/pF减小到42.29±8.89pA/pF(P0.05),抑制作用冲洗后不能完全恢复。其中约400%细胞在给药后1 min出现一过性微弱增大(约7%)。在0.1-1000 nM范围内,Ang Ⅱ以浓度依赖性方式抑制I_Ks,以I_Ks尾电流抑制率为纵坐标做Ang Ⅱ抑制I_Ks的量效曲线,经Hill方程拟合后得到IC50=7.5 nM。Ang Ⅱ对I_Ks的半数激活电压及激活时间常数都没有影响。转染siRNA特异性敲低PKC亚型的表达,观察Ang Ⅱ(K100 nM)和PMA(100 nM)对抑制、增大I_Ks的影响。结果发现,转染PKCα+PKCβ siRNA后,Ang Ⅱ对I_Ks的抑制作用与转染对照siRNA无明显差别;而转染PKCε siRNA特异性敲低PKCε表达后,Ang Ⅱ对尾电流的抑制(+50 mV下)显著弱于对照(10.95%vs 26.06%,P0.05),表明Ang Ⅱ对电流的抑制由PKCε中介。转染(乱码)对照PKC siRNA后,PMA使I_Ks(+50 mV下)增加31.84%,分别敲低PKCα、PKCβ的表达,PMA对尾电流的增加幅度较对照显著减弱,分别为16.82%(P0.05)、10.58%(P0.01);共同敲低 PKCα 和 PKCβ后,电流增强作用几乎消失;敲低PKCε的表达不影响PMA的作用(28.87%vs 31.84%,P0.05)。表明PMA对电流的增强作用由cPKC的PKCα和PKCβ中介。小结:以上的实验表明,AngⅡ对克隆人的I_Ks呈现抑制作用,此抑制作用由PKCε中介,而PMA对I_Ks的增强作用由PKCα和PKCβ中介。第三部分PKC亚型特异性调控I_Ks的分子机制目的:分析不同PKC亚型对I_Ks调节的分子机制。方法:分别突变KCNQ1、KCNE1上PKC的潜在磷酸化位点,观察Ang Ⅱ和PMA抑制、增强电流作用的改变,分析不同PKC亚型对通道调控的分子机制。结果:KCNQ1上N端有2个、C端有4个评分较高的PKC潜在磷酸化位点,分别是S95、T96和S409、S464、T513、S577,而KCNE1上只有一个潜在PKC磷酸化位点,即S102。将上述KCNQ1上6个和KCNE1上的1个潜在磷酸化位点分别突变为丙氨酸,另外同时将KCNQ1 上N端2个或C端4个潜在磷酸化位点突变,分别为 KCNQ1-2M(含 N 端 S95A 和 T96A)和 KCNQ1-4M(含 C 端 S409A、S464A、T513A、S577A)。经检测,上述所有突变通道的动力学特征与野生型一致。我们发现,Ang Ⅱ(100 nM)对 KCNQ1/KCNE1-S102A突变通道尾电流的抑制程度明显小于对野生型通道的抑制(10.84%,vs 30.59%,P0.05),以上结果提示KCNE1上的S102是PKC抑制通道功能的磷酸化位点。同时我们注意到,KCNE1上S102突变后AngⅡ对通道的抑制作用并没有完全消失,推测除S102外,KCNQ1上也存在抑制通道功能的磷酸化位点。进一步的实验表明,Ang Ⅱ对KCNQ1N端突变通道(KCNQ1-2M)尾电流的抑制程度为11.71%,显著弱于对野生型通道的作用(30.59%,P0.01),而KCNQ1的C端突变(KCNQ1-4M)和并不影响Ang Ⅱ的抑制作用(30.62%,P0.05)。以上结果说明,KCNQ1的N端参与PKC对通道功能的抑制。分别突变N端的S95和T96均可减弱Ang Ⅱ对通道的抑制作用,对S95A和T96A通道的抑制分别为15%(P0.01)和16.33%(P0.01),说明S95和T96都参与PKC抑制通道功能。联合突变KCNQ1的N端与 KCNE1 的 S102 后,Ang Ⅱ 对通道(KCNQ1-2M/KCNE1-S102A)的抑制作用几乎消失(2.76%,P0.01)。以上结果说明PKC在I_Ks上有三个抑制位点,分别是KCNQ1上的S95、T96和KCNE1上的S102。KCNE1上S102突变并不影响PMA增大I_Ks的作用,我们推断KCNQ1亚基有增强通道功能的位点。PMA对KCNQ1-2M/KCNE1和KCNQ1-4M/KCNE1通道的电流增加分别为31.74%和3.18%,N端突变与野生型通道作用无明显区别(P0.05),而C端突变几乎取消了其增强作用。结果说明KCNQ1亚基C端参与PKC对通道作用的增强性调控。进一步分别突变C端四个位点,结果显示,PMA对S409A、S464A、T513A、S577A突变通道电流分别增加12.98%、8.56%、6.37%以及11.71%,均较野生型显著降低。结果说明KCNQ1亚基C端的S409、S464、T513以及S577是PKC增强通道功能的磷酸化位点。我们进一步在HEK293细胞上表达克隆的豚鼠KCNQ1/KCNE1通道,将KCNQ1上N端唯一的潜在磷酸化位点S96突变为丙氨酸后,Ang Ⅱ对突变通道的抑制作用几乎消失,进一步表明KCNQ1上N端磷酸化抑制通道功能。小结:KCNQ1亚基存在PKC抑制、增强通道功能的位点,N端磷酸化抑制通道功能,而C端磷酸化增强通道功能;KCNE1上的S102磷酸化则抑制通道功能。
[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.
【学位授予单位】：河北医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R54

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