双孔钾通道TREK-1在大鼠心脏和脑星形胶质细胞中的表达及功能研究
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摘要
双孔钾通道(Two-pore-domain potassium channels, K2P通道)自上世纪90年代中期被发现以来,已经引起了广泛的研究兴趣,而TREK-1(TWIK-Related K+channel-1)做为K2P家族中的一员,因其自身独特的通道理化特性以及在体内的广泛分布,在近年来更是成为关注的焦点。TREK-1在大脑以及心脏等重要器官中有着较高水平的表达,由此推测TREK-1在维持正常生理功能中可能起着十分重要的作用。随着各种生物学技术的发展,TREK-1在中枢神经系统的各种功能逐渐被揭示,但由于缺乏针对TREK-1的特异性的阻断剂,其在心肌细胞以及在脑星形胶质细胞中的具体功能还不明确。心肌肥厚引起的心律失常性猝死约占心肌肥厚死亡人数的三分之一,越来越多的实验结果表明,与心肌肥厚慢性阶段相关的最一致的电生理变化就是动作电位时程的延长,而TREK-1做为牵张激活的钾通道和背景钾通道在其中发挥的主要功能还不清楚。星形胶质细胞是中枢神经系统非兴奋性细胞,其对维持神经元活性正常至关重要,星形胶质细胞的一些病理改变如细胞肿胀,自由脂肪酸升高,细胞内酸中毒等都是TREK-1的易感因素,而TREK-1是否存在于星形胶质细胞上还不明确。一些病理改变如心肌肥厚期间TREK-1的改变也还未见报道,TREK-1在星形胶质细胞中的电生理特性也尚未得到确认。因此本研究中我们侧重于正常条件下或是病理改变条件下大鼠心肌细胞和脑星形胶质细胞中与TREK-1相关的表达和功能研究,为进一步阐明TREK-1在心脏和大脑中的具体功能并寻找新的药物作用靶点提供理论基础。
第一部分：大鼠心肌肥厚期间左心室内膜TREK-1蛋白表达和电流的改变
1.异丙肾上腺素诱导的大鼠心肌肥厚模型
采用大鼠连续颈背皮下注射异丙肾上腺素(5mg/kg)7天诱发左心室肥厚模型,结果显示肥厚组大鼠心脏重量比对照组大鼠心脏重量增加44.9%；肥厚组大鼠心脏重量与体重比比对照组增加45.2%;肥厚组大鼠左心室壁厚度比对照组增加34.9%。以上结果表明大鼠连续颈背皮下注射异丙肾上腺素(5mg/kg)7天可成功诱发出左心室肥厚。
2大鼠,心肌肥厚时左心室内膜双孔钾通道TREK-1蛋白表达的变化
采用蛋白免疫印迹杂交(western blot)的方法观察心肌肥厚过程中大鼠左心室内膜TREK-1蛋白表达的变化趋势。结果显示肥厚组大鼠左心室内膜TREK-1的蛋白表达水平比对照组升高26.6%。以上结果提示心肌肥厚可以显著上调大鼠心室肌内膜细胞TREK-1的蛋白表达水平。
3心肌细胞TREK-1电流的确认
3.1机械牵张(负压)激活的大鼠心室肌细胞外向单通道电流
通过抽吸连接到一个压力计的注射器来诱发机械牵张。结果显示,在inside-out记录模式下不给予细胞任何压力刺激时很少能记录到外向单通道电流,但是给予细胞膜片-30cmH2O的负压刺激时,可记录到明显的外向单通道开放事件,而在撤除负压刺激后,开放的单通道电流几乎被完全逆转。-30cmH20激活的外向单通道的平均开放概率约为0.064+0.01。以上结果表明负压(机械牵张)可以激活心肌细胞上外向的单通道电流。
3.2细胞内酸化作用激活的大鼠心室肌细胞外向单通道电流
观察细胞内酸化作用对心肌细胞外向单通道电流的影响,同时与CHO-TREK-1细胞内的酸化作用作用进行对比。结果显示,在inside-out记录模式下当细胞外液的pH值为7.3时,在心肌细胞记录不到明显的通道开放而在CHO-TREK-1细胞上可以记到明显的外向单通道电流。但当细胞外液的pH值为6.8的时候,两种细胞均可明显记录到外向单通道的开放；当细胞外液pH值为6.3的时候,两种细胞上开放的通道数目进一步增加。细胞外液的pH值为6.8和6.3的时候心肌细胞上激活的外向单通道的平均开放开放概率分别为0.037±0.007和0.124±0.009；细胞外液的pH值为7.3、6.8和6.3的时候CHO-TREK-1上激活的外向单通道的平均开放概率分别为0.068±0.002、0.1334-0.017和0.225±0.022。以上结果表明细胞内的酸化作用可以激活心肌细胞上外向的单通道电流,且这一趋势与细胞内酸化作用对CHO-TREK-1的激活作用一致。
3.3花生四烯酸激活的大鼠心室肌细胞外向单通道电流
观察花生四烯酸对大鼠心肌细胞外向单通道电流的影响。在inside-out记录模式下10μM花生四烯酸可激活心肌细胞外向单通道电流,而CHO-TREK-1不需要给予花生四烯酸处理即可记录到大量开放的外向单通道电流。以上结果表明可以花生四烯酸可以激活心肌细胞上外向的单通道电流。
3.4心肌细胞TREK样通道的单通道电导
根据上述记录的两种细胞不同电位下单通道平均拟合电流的大小计算出两种通道的平均单通道电导分别为123±7pS (CHO-TREK-1)和113±17pS (cardiomyocyte),两者之间无统计学差异。这里我们记录到的心肌细胞TREK-1的单通道电导与文献报道的TREK-1单通道电导范围基本吻合
综合上述四部分的实验结果,我们记录的外向单通道电流可以被负压(机械牵张),细胞内酸化作用以及花生四烯酸激活,而且单通道电导与CHO-TREK-1的单通道电导比较接近,由此可以证明我们在心肌细胞上所记录到的外向单通道电流为心肌细胞TREK-1。
4.L-NBP对CHO-TREK-1、正常心肌细胞以及肥厚心肌细胞TREK-1单通道开放概率的影响
应用inside-out记录技术观察L-NBP对TREK-1的抑制作用。在inside-out记录模式下CHO-TREK-1细胞中不需要给予任何处理即可记录到大量开放的TREK-1通道,给予10μM L-NBP作用5分钟后,通道活性被明显抑制但不能完全被逆转。在正常心肌细胞和肥厚心肌细胞中,0.18mM的氯仿可激活爆发式的TREK-1通道丌放,给予10μM L-NBP处理5分钟后通道丌放事件明显减少,但仍不能完全被抑制。10μM L-NBP对CHO-TREK-1,正常心肌细胞TREK-1和肥厚心肌细胞TREK-1的抑制率分别为48.5+8%,54.3+3%和55.5+4%,三者之间没有统计学差异。上述结果表明,0.18mM氯仿可以显著激活心肌细胞TREK-1;10μM L-NBP对TREK-1有显著的抑制作用,而且10μM L-NBP对三种细胞上TREK-1的抑制作用基本上一致。
5.L-NBP对正常心肌和肥厚心肌外向电流的影响
应用全细胞记录技术观察TREK-1相对特异性阻断剂L-NBP对正常心肌和肥厚心肌外向电流的影响。以10μM L-NBP抑制的外向电流大小作为一个参数来衡量正常心肌和心肌肥厚TREK-1的差别。在阻断心肌细胞几种主要类型的钾电流的前提下,在全细胞记录模式下正常心肌细胞中10μM L-NBP作用5分钟可将外向全细胞电流从10.21±3.02pA/pF抑制为8.99±2.40pA/pF,而在肥厚心肌细胞中10μM L-NBP可将外向全细胞电流从7.43±1.72pA/pF抑制为5.73±1.75pA/pF。肥厚心肌细胞10μM L-NBP抑制的外向电流大小1.70±0.4pA/pF比正常心肌细胞10μML-NBP抑制的电流外向大小1.22±0.36pA/pF显著升高。上述结果提示L-NBP对肥厚心肌细胞TREK-1电流的抑制率明显高于正常心肌细胞。
6.氯仿对正常心肌和肥厚心肌外向电流的作用
应用全细胞记录技术观察TREK-1特异性激活剂氯仿对正常心肌和肥厚心肌外向电流的影响。以0.18mM的氯仿激活的外向电流大小作为一个与10μM L-NBP抑制的外向电流大小完全相反的参数来衡量正常心肌和心肌肥厚TREK-1的差别。在阻断心肌细胞几种主要类型的钾电流的前提下,在全细胞记录模式下正常心肌细胞中0.18mM氯仿作用5分钟可将外向全细胞电流从6.47±2.13pA/pF增加为8.89±2.38pA/pF,而在肥厚心肌细胞中0.18mM氯仿可将外向全细胞电流从6.04±1.57pA/pF增加为9.47±2.16pA/pF。肥厚心肌0.18mM氯仿诱发的外向电流大小3.43±0.7pA/pF比正常心肌0.18mM氯仿诱发的电流大小2.42±0.5pA/pF显著升高。上述结果提示氯仿对肥厚心肌细胞TREK-1电流的激活作用明显高于正常心肌细胞。
上面两部分实验结果均显示肥厚心肌TREK-1电流与正常心肌TREK-1电流相比有增高的趋势,这个结果与前面的TREK-1的蛋白表达结果一致。
第二部分：大鼠脑星形胶质细胞TREK样通道的电生理确认
1.花生四烯酸和氯仿激活的TREK样外向单通道
观察花生四烯酸和氯仿对星形胶质细胞外向单通道电流的影响。结果显示,在inside-out记录模式下10μM花生四烯酸和0.2M氯仿可显著激活星形胶质细胞外向单通道电流。10μM花生四烯酸和0.2Me1氯仿激活的星形胶质细胞外向单通道的平均开放概率分别为0.173+0.01和0.255±0.02。
根据不同电位下记录的花生四烯酸或氯仿激活的单通道平均拟合电流大小计算出该通道的平均单通道电导为107±16pS,与文献报道的TREK-1单通道电导范围基本吻合。上述结果表明星形胶质细胞上存在可被花生四烯酸和氯仿激活的外向钾通道,且该通道的电导与TREK-1比较接近。
2.机械牵张的影响
通过抽吸连接到一个压力计的注射器来诱发机械牵张。结果显示,在inside-out记录模式下不给予细胞任何压力刺激时很少能记录到外向的单通道电流,但是给予细胞膜片-30cmH2O的负压刺激时,可记录到明显的外向单通道开放事件,而在撤除负压刺激后,开放的单通道电流几乎被完全逆转。-30cmH2O激活的外向单通道的平均开放概率约为0.142±0.02。以上结果表明负压(机械牵张)可以激活星形胶质细胞上外向的单通道电流。
3.细胞内酸化作用的影响
观察细胞内酸化作用对星形胶质细胞外向单通道电流的影响。结果显示,在inside-out记录模式下当细胞外液的pH值为6.8的时候,可明显记录到外向单通道的丌放；当细胞外液pH值为6.3的时候,星形胶质细胞上开放的通道数目进一步增加。细胞内的酸化作用可以呈pH值依赖性的增加外向通道的平均开放概率。在+60mV下细胞外液的pH值为6.8和6.3的时候星形胶质细胞上激活的外向单通道的平均丌放开放概率分别为0.064±0.01和0.176-0.01。以上结果表明细胞内的酸化作用可以激活星形胶质细胞上外向的单通道电流。
综合上面三部分的实验结果,星形胶质细胞上的外向单通道可以被花生四烯酸和氯仿激活,可以被机械牵张(负压)激活,同时也能被细胞内的酸化作用激活,而且该外向通道的单通道电导与TREK-1的单通道电导比较接近,由此可以证明星形胶质细胞上存在野生型的TREK-1通道。
Since K2P channels were found in the middle-1990s, it attracted considerable research interest. TREK-1, as one member of K2P family, has become the focus of attention in recent years because of their own unique channel characteristics. There was extensive evidence that TREK-1was highly expressed in some vital organs such as brain and heart, so it can be speculated that TREK-1plays a very important role in the maintenance of normal physiological function. With the development of a variety of biological technology, the function of TREK-1in the central nervous system and cardiovascular system has been revealed gradually. But because no specific blocker of TREK-1was available, its role in cardiomyocytes and astrocytes still remained unclear. Hypertrophy also presented substrates for lethal ventricular arrhythmias, resulting in sudden arrhythmic deaths that account for about one third of deaths in cardiac hypertrophy. Accumulated experimental data suggested that the most consistent electrical change which has been described in association with the chronic stage of cardiac hypertrophy is prolongation of action potential duration (APD).Considering TREK-1was a stretch-activated potassium channel and meanwhile a background potassium channel, its role in regulating the cardiac action potential was anticipating. Astrocytes are non-excitable cells of the central nervous system and provide many important functions that are critical for the normal activity of neurons. TREK-1was sensitive to some pathological conditions in astrocytes such as cell swelling, a rise in free fatty acid level and intracellular acidification, its role in astrocytes also remained unknown. Pathological changes of TREK-1in cardiomyocytes and electrophysiological identification of TREK-1in astrocytes were uncertain. So in this study we focused on the expressional and functional studies in normal or pathological conditions of caidiomyocytes or astrocytes, and provided theoretical basis for further clarifying the concise function of TREK-1and seachering for new drug targets.
Part I:Changes of protein expression and current of TREK-1in endocardial myocytes during left ventricular hypertrophy
1. Hypertrophy in rat induced by isoproterenol Left ventricular hypertrophy was evoked by repeated subcutaneous administration of isoproterenol at a dosage of5mg/kg for7days. The results showed that compared to normal rats, the heart weight, heart weight/body weight and left ventricular wall thickness of hypertrophic rats were increased by44.9%,45.2%and34.9%, respectively. It meant isoproterenol-induced hypertrophy was successful.
2. Changes of TREK-1protein expression in endocardial myocytes during left ventricular hypertrophy
Western blot was used to observe changes of TREK-1protein expression in endocardial myocytes during left ventricular hypertrophy. The result showed compared to control group, TREK-1protein expression was enhanced by26.6%in hypertrophy group. It meant hypertrophy significantly up-regulated TREK-1protein expression in endocardial myocytes.
3Identification of cardiac TREK-1currents
3.1Outward single channel currents activated by stretch (negative pressure) in cardiomyocytes
Stretch was elicited via suction of a syringe connected to a manometer. The results showed in inside-out recordings little outward current could be recorded when no stretch was applied to the excised patches. When a-30cmH2O negative pressure was applied, obvious outward openings with an open probability of0.064±0.01could be detected. After the negative pressure was removed, the openings were almost reversed. It meant stretch could activate an outward single channel in cardiomyocytes.
3.2Outward single channel currents induced by intracellular acidification in cardiomyocytes
Observe the effects of intracellular acidification on cardiac outward single channel currents and CHO-TREK-1. The results showed in inside-out recordings when the pH of the external solution was7.3, no obvious openings could be recorded in cardiomyocytes but flittering openings could be detected in CHO-TREK-1cells. When the pH was6.8and6.3, both in Cardiomyocytes and CHO-TREK-1cells visible opening could be seen.PH6.8and6.3solution elicited an outward single channel in cardiomyocytes with an open probability of0.037±0.007and0.124±0.009, respectively. pH7.3,6.8and6.3solution elicited an outward single channel in CHO-TREK-1cells with open probabilities of0.068±0.002,0.133±0.017and0.225±0.022, respectively. These results indicated that intracellular acidification could activate an outward current in cardiomyocytes.
3.3Outward single channel currents induced by arachidonic acid in cardiomyocytes
Observe the effects of arachidonic acid on cardiac outward single channel currents. The results showed in inside-out recordings10μM arachidonic acid could obviously induce an outward single channel current in cardiomyocytes while evident openings also could be detected in CHO-TREK-1cells without any treatment. It suggested arachidonic acid could activated an activated an outward current in cardiomyocytes.
3.4Conductance of cardiac TREK-like channels
Single channel conductances of two cell types calculated according to the fitted current amplitude of the above-recorded currents were123±7pS (CHO-TREK-1) and113±17pS (cardiomyocyte) with no statistical significance. The conductance of our recorded currents induced by arachidonic acid in cardiomyocyts was close to the previously reported conductance of TREK-1. It meant the conductance of our recorded currents induced by arachidonic acid in cardiomyocyts was consistent with the conductance of CHO-TREK-1.
According to the above results, the outward channels we recorded in cardiomyocyes could be activated by stretch, intracellular acidification and AA and had a similar conductance to CHO-TREK-1, so it could be proved that the current we recorded in cardiomyocytes was native TREK-1.
4. Effects of L-NBP on the open probability of TREK-1in CHO-TREK-1cells, normal and hypertrophic cardiomyocytes
Inside-out recordings were used to observe the inhibitive effects of L-NBP on TREK-1. In inside-out recording, obvious channel openings could be recorded without any treatment in CHO-TREK-1cells, while after application of10μM L-NBP for5min, the channel activity could be apparently inhibited but could not be reversed totally. In normal and hypertrophic cardiomyocytes,0.18chloroform could induce burst TREK-1openings, while after application of10μM L-NBP for5min, the openings were significantly decreased but could not be inhibited completely. The inhibition rate of10μM L-NBP on TREK-1in CHO-TREK-1cells, normal and hypertrophic cardiomyocytes were48.5±8%,54.3±3%and55.5±4%, no statistical significance existed among the three groups. These results indicated that chloroform could easily elicit TREK-1in cardiomyocytes and10μM L-NBP had significant inhibitory effects on TREK-1in three cell types.
5. Effects of L-NBP on outward currents in normal and hypertrophic cardiomyocytes
Whole cell recordings were used to observe the effects of TREK-1blocker L-BNP on outward currents in normal and hypertrophic cardiomyocytes.10μM L-NBP-inhibited outward current was considered as an index to evaluate the difference of TREK-1between normal and hypertrophic cardiomyocytes. On the base of blocking other major potassium channels, application of10μM L-NBP for5min could reduce the outward current from10.21±3.02pA/pF to8.99±2.40pA/pF in normal cardiomyocytes and from7.43±1.72pA/pF to5.73±1.75pA/pF in hypertrophic cardiomyocytes.10μM L-NBP-inhibited outward current in hypertrophic cardiomyocytes (1.70±0.4pA/pF) was significantly larger than in normal cardiomyocytes (1.22±0.36pA/pF). The results suggested the background current of TREK-1in hypertrophic cardiomyocytes was larger than in normal cardiomyocytes.
6. Effects of chloroform on outward currents in normal and hypertrophic cardiomyocytes
Whole cell recordings were used to observe the effects of TREK-1selective agonist chloroform on outward currents in normal and hypertrophic cardiomyocytes.0.18mM chloroform-activated outward current was considered as an index to evaluate the difference of TREK-1between normal and hypertrophic cardiomyocytes. On the base of blocking other major potassium channels, application of0.18mM chloroform for5min could enhance the outward current from6.47±2.13pA/pF to8.89±2.38pA/pF in normal cardiomyocytes and from6.04±1.57pA/pF to9.47±2.16pA/pF in hypertrophic cardiomyocytes.0.18mM chloroform-activated outward current in hypertrophic cardiomyocytes (3.43±0.7pA/pF) was obviously larger than in normal cardiomyocytes (2.42±0.5pA/pF). The results suggested the background current of TREK-1in hypertrophic cardiomyocytes was larger than in normal cardiomyocytes.
The above results meant that there was an increasing tendency of TREK-1in hypertrophic cardiomyocytes compared to normal cardiomyocytes, which was consistent with our western blot data.
Part II:Electrophysiological Identification of a native TREK-like channel in rat brain astrocytes
1. TREK-like outward channels activated by arachidonic acid and chloroform
Observe the effects of arachidonic acid and chloroform on outward single channel currents in astrocytes. In inside-out recordings,10μM arachidonic acid and0.2mM chloroform could obviously induce outward openings in cultured astrocytes. The open probabilities of10μM arachidonic acid-activated and0.2mM chloroform-activated outward channels were0.173±0.01and0.255±0.02. The calculated single channel conductance of the recorded channels was107±16pS, which was close to previous literature. These results indicated that arachidonic acid and chloroform could activate outward channel openings with a similar conductance to TREK-1in astrocytes.
2Outward single channel currents activated by stretch (negative pressure) in astrocytes
Stretch was elicited via suction of a syringe connected to a manometer. The results showed in inside-out recordings little outward current could be recorded when no stretch was applied to the excised patches. When a-30cmH2O negative pressure was applied, obvious outward openings with an open probability of0.142±0.02could be detected. After the negative pressure was removed, the openings were almost reversed. It meant stretch could activate an outward single channel in cardiomyocytes.
3.2Outward single channel currents induced by intracellular acidification in astrocytes
Observe the effects of intracellular acidification on outward single channel currents in cultured astrocytes. The results showed in inside-out recordings when the pH of the external solution was7.3, no obvious openings could be recorded in cardiomyocytes. When the pH was6.8and6.3, visible opening could be seen. Outward channel openings could be increased pH-dependently. PH6.8and6.3solutions elicited an outward single channel in astrocytes with open probabilities of0.064±0.01and0.176±0.01, respectively. These results indicated that intracellular acidification could activate an outward current in cardiomyocytes.
According to the above results, the outward channels we recorded in astrocytes could be activated by stretch, intracellular acidification, AA and chloroform and had a similar conductance to TREK-1, so it could be proved that the current we recorded in astrocytes was native TREK-1.
第一部分：大鼠心肌肥厚期间左心室内膜TREK-1蛋白表达和电流的改变
1.异丙肾上腺素诱导的大鼠心肌肥厚模型
采用大鼠连续颈背皮下注射异丙肾上腺素(5mg/kg)7天诱发左心室肥厚模型,结果显示肥厚组大鼠心脏重量比对照组大鼠心脏重量增加44.9%；肥厚组大鼠心脏重量与体重比比对照组增加45.2%;肥厚组大鼠左心室壁厚度比对照组增加34.9%。以上结果表明大鼠连续颈背皮下注射异丙肾上腺素(5mg/kg)7天可成功诱发出左心室肥厚。
2大鼠,心肌肥厚时左心室内膜双孔钾通道TREK-1蛋白表达的变化
采用蛋白免疫印迹杂交(western blot)的方法观察心肌肥厚过程中大鼠左心室内膜TREK-1蛋白表达的变化趋势。结果显示肥厚组大鼠左心室内膜TREK-1的蛋白表达水平比对照组升高26.6%。以上结果提示心肌肥厚可以显著上调大鼠心室肌内膜细胞TREK-1的蛋白表达水平。
3心肌细胞TREK-1电流的确认
3.1机械牵张(负压)激活的大鼠心室肌细胞外向单通道电流
通过抽吸连接到一个压力计的注射器来诱发机械牵张。结果显示,在inside-out记录模式下不给予细胞任何压力刺激时很少能记录到外向单通道电流,但是给予细胞膜片-30cmH2O的负压刺激时,可记录到明显的外向单通道开放事件,而在撤除负压刺激后,开放的单通道电流几乎被完全逆转。-30cmH20激活的外向单通道的平均开放概率约为0.064+0.01。以上结果表明负压(机械牵张)可以激活心肌细胞上外向的单通道电流。
3.2细胞内酸化作用激活的大鼠心室肌细胞外向单通道电流
观察细胞内酸化作用对心肌细胞外向单通道电流的影响,同时与CHO-TREK-1细胞内的酸化作用作用进行对比。结果显示,在inside-out记录模式下当细胞外液的pH值为7.3时,在心肌细胞记录不到明显的通道开放而在CHO-TREK-1细胞上可以记到明显的外向单通道电流。但当细胞外液的pH值为6.8的时候,两种细胞均可明显记录到外向单通道的开放；当细胞外液pH值为6.3的时候,两种细胞上开放的通道数目进一步增加。细胞外液的pH值为6.8和6.3的时候心肌细胞上激活的外向单通道的平均开放开放概率分别为0.037±0.007和0.124±0.009；细胞外液的pH值为7.3、6.8和6.3的时候CHO-TREK-1上激活的外向单通道的平均开放概率分别为0.068±0.002、0.1334-0.017和0.225±0.022。以上结果表明细胞内的酸化作用可以激活心肌细胞上外向的单通道电流,且这一趋势与细胞内酸化作用对CHO-TREK-1的激活作用一致。
3.3花生四烯酸激活的大鼠心室肌细胞外向单通道电流
观察花生四烯酸对大鼠心肌细胞外向单通道电流的影响。在inside-out记录模式下10μM花生四烯酸可激活心肌细胞外向单通道电流,而CHO-TREK-1不需要给予花生四烯酸处理即可记录到大量开放的外向单通道电流。以上结果表明可以花生四烯酸可以激活心肌细胞上外向的单通道电流。
3.4心肌细胞TREK样通道的单通道电导
根据上述记录的两种细胞不同电位下单通道平均拟合电流的大小计算出两种通道的平均单通道电导分别为123±7pS (CHO-TREK-1)和113±17pS (cardiomyocyte),两者之间无统计学差异。这里我们记录到的心肌细胞TREK-1的单通道电导与文献报道的TREK-1单通道电导范围基本吻合
综合上述四部分的实验结果,我们记录的外向单通道电流可以被负压(机械牵张),细胞内酸化作用以及花生四烯酸激活,而且单通道电导与CHO-TREK-1的单通道电导比较接近,由此可以证明我们在心肌细胞上所记录到的外向单通道电流为心肌细胞TREK-1。
4.L-NBP对CHO-TREK-1、正常心肌细胞以及肥厚心肌细胞TREK-1单通道开放概率的影响
应用inside-out记录技术观察L-NBP对TREK-1的抑制作用。在inside-out记录模式下CHO-TREK-1细胞中不需要给予任何处理即可记录到大量开放的TREK-1通道,给予10μM L-NBP作用5分钟后,通道活性被明显抑制但不能完全被逆转。在正常心肌细胞和肥厚心肌细胞中,0.18mM的氯仿可激活爆发式的TREK-1通道丌放,给予10μM L-NBP处理5分钟后通道丌放事件明显减少,但仍不能完全被抑制。10μM L-NBP对CHO-TREK-1,正常心肌细胞TREK-1和肥厚心肌细胞TREK-1的抑制率分别为48.5+8%,54.3+3%和55.5+4%,三者之间没有统计学差异。上述结果表明,0.18mM氯仿可以显著激活心肌细胞TREK-1;10μM L-NBP对TREK-1有显著的抑制作用,而且10μM L-NBP对三种细胞上TREK-1的抑制作用基本上一致。
5.L-NBP对正常心肌和肥厚心肌外向电流的影响
应用全细胞记录技术观察TREK-1相对特异性阻断剂L-NBP对正常心肌和肥厚心肌外向电流的影响。以10μM L-NBP抑制的外向电流大小作为一个参数来衡量正常心肌和心肌肥厚TREK-1的差别。在阻断心肌细胞几种主要类型的钾电流的前提下,在全细胞记录模式下正常心肌细胞中10μM L-NBP作用5分钟可将外向全细胞电流从10.21±3.02pA/pF抑制为8.99±2.40pA/pF,而在肥厚心肌细胞中10μM L-NBP可将外向全细胞电流从7.43±1.72pA/pF抑制为5.73±1.75pA/pF。肥厚心肌细胞10μM L-NBP抑制的外向电流大小1.70±0.4pA/pF比正常心肌细胞10μML-NBP抑制的电流外向大小1.22±0.36pA/pF显著升高。上述结果提示L-NBP对肥厚心肌细胞TREK-1电流的抑制率明显高于正常心肌细胞。
6.氯仿对正常心肌和肥厚心肌外向电流的作用
应用全细胞记录技术观察TREK-1特异性激活剂氯仿对正常心肌和肥厚心肌外向电流的影响。以0.18mM的氯仿激活的外向电流大小作为一个与10μM L-NBP抑制的外向电流大小完全相反的参数来衡量正常心肌和心肌肥厚TREK-1的差别。在阻断心肌细胞几种主要类型的钾电流的前提下,在全细胞记录模式下正常心肌细胞中0.18mM氯仿作用5分钟可将外向全细胞电流从6.47±2.13pA/pF增加为8.89±2.38pA/pF,而在肥厚心肌细胞中0.18mM氯仿可将外向全细胞电流从6.04±1.57pA/pF增加为9.47±2.16pA/pF。肥厚心肌0.18mM氯仿诱发的外向电流大小3.43±0.7pA/pF比正常心肌0.18mM氯仿诱发的电流大小2.42±0.5pA/pF显著升高。上述结果提示氯仿对肥厚心肌细胞TREK-1电流的激活作用明显高于正常心肌细胞。
上面两部分实验结果均显示肥厚心肌TREK-1电流与正常心肌TREK-1电流相比有增高的趋势,这个结果与前面的TREK-1的蛋白表达结果一致。
第二部分：大鼠脑星形胶质细胞TREK样通道的电生理确认
1.花生四烯酸和氯仿激活的TREK样外向单通道
观察花生四烯酸和氯仿对星形胶质细胞外向单通道电流的影响。结果显示,在inside-out记录模式下10μM花生四烯酸和0.2M氯仿可显著激活星形胶质细胞外向单通道电流。10μM花生四烯酸和0.2Me1氯仿激活的星形胶质细胞外向单通道的平均开放概率分别为0.173+0.01和0.255±0.02。
根据不同电位下记录的花生四烯酸或氯仿激活的单通道平均拟合电流大小计算出该通道的平均单通道电导为107±16pS,与文献报道的TREK-1单通道电导范围基本吻合。上述结果表明星形胶质细胞上存在可被花生四烯酸和氯仿激活的外向钾通道,且该通道的电导与TREK-1比较接近。
2.机械牵张的影响
通过抽吸连接到一个压力计的注射器来诱发机械牵张。结果显示,在inside-out记录模式下不给予细胞任何压力刺激时很少能记录到外向的单通道电流,但是给予细胞膜片-30cmH2O的负压刺激时,可记录到明显的外向单通道开放事件,而在撤除负压刺激后,开放的单通道电流几乎被完全逆转。-30cmH2O激活的外向单通道的平均开放概率约为0.142±0.02。以上结果表明负压(机械牵张)可以激活星形胶质细胞上外向的单通道电流。
3.细胞内酸化作用的影响
观察细胞内酸化作用对星形胶质细胞外向单通道电流的影响。结果显示,在inside-out记录模式下当细胞外液的pH值为6.8的时候,可明显记录到外向单通道的丌放；当细胞外液pH值为6.3的时候,星形胶质细胞上开放的通道数目进一步增加。细胞内的酸化作用可以呈pH值依赖性的增加外向通道的平均开放概率。在+60mV下细胞外液的pH值为6.8和6.3的时候星形胶质细胞上激活的外向单通道的平均丌放开放概率分别为0.064±0.01和0.176-0.01。以上结果表明细胞内的酸化作用可以激活星形胶质细胞上外向的单通道电流。
综合上面三部分的实验结果,星形胶质细胞上的外向单通道可以被花生四烯酸和氯仿激活,可以被机械牵张(负压)激活,同时也能被细胞内的酸化作用激活,而且该外向通道的单通道电导与TREK-1的单通道电导比较接近,由此可以证明星形胶质细胞上存在野生型的TREK-1通道。
Since K2P channels were found in the middle-1990s, it attracted considerable research interest. TREK-1, as one member of K2P family, has become the focus of attention in recent years because of their own unique channel characteristics. There was extensive evidence that TREK-1was highly expressed in some vital organs such as brain and heart, so it can be speculated that TREK-1plays a very important role in the maintenance of normal physiological function. With the development of a variety of biological technology, the function of TREK-1in the central nervous system and cardiovascular system has been revealed gradually. But because no specific blocker of TREK-1was available, its role in cardiomyocytes and astrocytes still remained unclear. Hypertrophy also presented substrates for lethal ventricular arrhythmias, resulting in sudden arrhythmic deaths that account for about one third of deaths in cardiac hypertrophy. Accumulated experimental data suggested that the most consistent electrical change which has been described in association with the chronic stage of cardiac hypertrophy is prolongation of action potential duration (APD).Considering TREK-1was a stretch-activated potassium channel and meanwhile a background potassium channel, its role in regulating the cardiac action potential was anticipating. Astrocytes are non-excitable cells of the central nervous system and provide many important functions that are critical for the normal activity of neurons. TREK-1was sensitive to some pathological conditions in astrocytes such as cell swelling, a rise in free fatty acid level and intracellular acidification, its role in astrocytes also remained unknown. Pathological changes of TREK-1in cardiomyocytes and electrophysiological identification of TREK-1in astrocytes were uncertain. So in this study we focused on the expressional and functional studies in normal or pathological conditions of caidiomyocytes or astrocytes, and provided theoretical basis for further clarifying the concise function of TREK-1and seachering for new drug targets.
Part I:Changes of protein expression and current of TREK-1in endocardial myocytes during left ventricular hypertrophy
1. Hypertrophy in rat induced by isoproterenol Left ventricular hypertrophy was evoked by repeated subcutaneous administration of isoproterenol at a dosage of5mg/kg for7days. The results showed that compared to normal rats, the heart weight, heart weight/body weight and left ventricular wall thickness of hypertrophic rats were increased by44.9%,45.2%and34.9%, respectively. It meant isoproterenol-induced hypertrophy was successful.
2. Changes of TREK-1protein expression in endocardial myocytes during left ventricular hypertrophy
Western blot was used to observe changes of TREK-1protein expression in endocardial myocytes during left ventricular hypertrophy. The result showed compared to control group, TREK-1protein expression was enhanced by26.6%in hypertrophy group. It meant hypertrophy significantly up-regulated TREK-1protein expression in endocardial myocytes.
3Identification of cardiac TREK-1currents
3.1Outward single channel currents activated by stretch (negative pressure) in cardiomyocytes
Stretch was elicited via suction of a syringe connected to a manometer. The results showed in inside-out recordings little outward current could be recorded when no stretch was applied to the excised patches. When a-30cmH2O negative pressure was applied, obvious outward openings with an open probability of0.064±0.01could be detected. After the negative pressure was removed, the openings were almost reversed. It meant stretch could activate an outward single channel in cardiomyocytes.
3.2Outward single channel currents induced by intracellular acidification in cardiomyocytes
Observe the effects of intracellular acidification on cardiac outward single channel currents and CHO-TREK-1. The results showed in inside-out recordings when the pH of the external solution was7.3, no obvious openings could be recorded in cardiomyocytes but flittering openings could be detected in CHO-TREK-1cells. When the pH was6.8and6.3, both in Cardiomyocytes and CHO-TREK-1cells visible opening could be seen.PH6.8and6.3solution elicited an outward single channel in cardiomyocytes with an open probability of0.037±0.007and0.124±0.009, respectively. pH7.3,6.8and6.3solution elicited an outward single channel in CHO-TREK-1cells with open probabilities of0.068±0.002,0.133±0.017and0.225±0.022, respectively. These results indicated that intracellular acidification could activate an outward current in cardiomyocytes.
3.3Outward single channel currents induced by arachidonic acid in cardiomyocytes
Observe the effects of arachidonic acid on cardiac outward single channel currents. The results showed in inside-out recordings10μM arachidonic acid could obviously induce an outward single channel current in cardiomyocytes while evident openings also could be detected in CHO-TREK-1cells without any treatment. It suggested arachidonic acid could activated an activated an outward current in cardiomyocytes.
3.4Conductance of cardiac TREK-like channels
Single channel conductances of two cell types calculated according to the fitted current amplitude of the above-recorded currents were123±7pS (CHO-TREK-1) and113±17pS (cardiomyocyte) with no statistical significance. The conductance of our recorded currents induced by arachidonic acid in cardiomyocyts was close to the previously reported conductance of TREK-1. It meant the conductance of our recorded currents induced by arachidonic acid in cardiomyocyts was consistent with the conductance of CHO-TREK-1.
According to the above results, the outward channels we recorded in cardiomyocyes could be activated by stretch, intracellular acidification and AA and had a similar conductance to CHO-TREK-1, so it could be proved that the current we recorded in cardiomyocytes was native TREK-1.
4. Effects of L-NBP on the open probability of TREK-1in CHO-TREK-1cells, normal and hypertrophic cardiomyocytes
Inside-out recordings were used to observe the inhibitive effects of L-NBP on TREK-1. In inside-out recording, obvious channel openings could be recorded without any treatment in CHO-TREK-1cells, while after application of10μM L-NBP for5min, the channel activity could be apparently inhibited but could not be reversed totally. In normal and hypertrophic cardiomyocytes,0.18chloroform could induce burst TREK-1openings, while after application of10μM L-NBP for5min, the openings were significantly decreased but could not be inhibited completely. The inhibition rate of10μM L-NBP on TREK-1in CHO-TREK-1cells, normal and hypertrophic cardiomyocytes were48.5±8%,54.3±3%and55.5±4%, no statistical significance existed among the three groups. These results indicated that chloroform could easily elicit TREK-1in cardiomyocytes and10μM L-NBP had significant inhibitory effects on TREK-1in three cell types.
5. Effects of L-NBP on outward currents in normal and hypertrophic cardiomyocytes
Whole cell recordings were used to observe the effects of TREK-1blocker L-BNP on outward currents in normal and hypertrophic cardiomyocytes.10μM L-NBP-inhibited outward current was considered as an index to evaluate the difference of TREK-1between normal and hypertrophic cardiomyocytes. On the base of blocking other major potassium channels, application of10μM L-NBP for5min could reduce the outward current from10.21±3.02pA/pF to8.99±2.40pA/pF in normal cardiomyocytes and from7.43±1.72pA/pF to5.73±1.75pA/pF in hypertrophic cardiomyocytes.10μM L-NBP-inhibited outward current in hypertrophic cardiomyocytes (1.70±0.4pA/pF) was significantly larger than in normal cardiomyocytes (1.22±0.36pA/pF). The results suggested the background current of TREK-1in hypertrophic cardiomyocytes was larger than in normal cardiomyocytes.
6. Effects of chloroform on outward currents in normal and hypertrophic cardiomyocytes
Whole cell recordings were used to observe the effects of TREK-1selective agonist chloroform on outward currents in normal and hypertrophic cardiomyocytes.0.18mM chloroform-activated outward current was considered as an index to evaluate the difference of TREK-1between normal and hypertrophic cardiomyocytes. On the base of blocking other major potassium channels, application of0.18mM chloroform for5min could enhance the outward current from6.47±2.13pA/pF to8.89±2.38pA/pF in normal cardiomyocytes and from6.04±1.57pA/pF to9.47±2.16pA/pF in hypertrophic cardiomyocytes.0.18mM chloroform-activated outward current in hypertrophic cardiomyocytes (3.43±0.7pA/pF) was obviously larger than in normal cardiomyocytes (2.42±0.5pA/pF). The results suggested the background current of TREK-1in hypertrophic cardiomyocytes was larger than in normal cardiomyocytes.
The above results meant that there was an increasing tendency of TREK-1in hypertrophic cardiomyocytes compared to normal cardiomyocytes, which was consistent with our western blot data.
Part II:Electrophysiological Identification of a native TREK-like channel in rat brain astrocytes
1. TREK-like outward channels activated by arachidonic acid and chloroform
Observe the effects of arachidonic acid and chloroform on outward single channel currents in astrocytes. In inside-out recordings,10μM arachidonic acid and0.2mM chloroform could obviously induce outward openings in cultured astrocytes. The open probabilities of10μM arachidonic acid-activated and0.2mM chloroform-activated outward channels were0.173±0.01and0.255±0.02. The calculated single channel conductance of the recorded channels was107±16pS, which was close to previous literature. These results indicated that arachidonic acid and chloroform could activate outward channel openings with a similar conductance to TREK-1in astrocytes.
2Outward single channel currents activated by stretch (negative pressure) in astrocytes
Stretch was elicited via suction of a syringe connected to a manometer. The results showed in inside-out recordings little outward current could be recorded when no stretch was applied to the excised patches. When a-30cmH2O negative pressure was applied, obvious outward openings with an open probability of0.142±0.02could be detected. After the negative pressure was removed, the openings were almost reversed. It meant stretch could activate an outward single channel in cardiomyocytes.
3.2Outward single channel currents induced by intracellular acidification in astrocytes
Observe the effects of intracellular acidification on outward single channel currents in cultured astrocytes. The results showed in inside-out recordings when the pH of the external solution was7.3, no obvious openings could be recorded in cardiomyocytes. When the pH was6.8and6.3, visible opening could be seen. Outward channel openings could be increased pH-dependently. PH6.8and6.3solutions elicited an outward single channel in astrocytes with open probabilities of0.064±0.01and0.176±0.01, respectively. These results indicated that intracellular acidification could activate an outward current in cardiomyocytes.
According to the above results, the outward channels we recorded in astrocytes could be activated by stretch, intracellular acidification, AA and chloroform and had a similar conductance to TREK-1, so it could be proved that the current we recorded in astrocytes was native TREK-1.
引文
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