细胞膜去极化调节细胞膜PIP_2水平及KCNQ2/Q3通道电流的研究
|
摘要
越来越多的实验结果证明了细胞膜磷脂酰肌醇4,5二磷酸(PtdIns (4,5) P2,PIP2)在细胞内信号转导过程中发挥着重要的作用。细胞膜上的PIP2在诸如众多的离子通道及离子转运体功能调节、细胞骨架、囊泡输送、融合,及出胞和入胞等细胞过程中起重要作用。细胞膜PIP2不仅是某些重要信号分子,如二酰甘油(DAG)和三磷酸肌醇(IP3)的前体。PIP2本身作为信号分子发挥着自己的功能,它的水平被动态调节。细胞膜PIP2水平的一个重要的调节机制是磷脂酶C (PLC)介导的PIP2水解,这种事件依赖性调节机制是众多受体调节离子通道功能的重要机制。此外,在细胞中存在对稳态PIP2水平调节的精细机制,主要通过多种肌醇磷脂酰肌酶和磷酸酶来实现。在这项研究中,我们描述了一个细胞膜PIP2代谢调节的新机制。细胞膜电位去极化引起细胞膜PIP2水平增加,进而增加表达于非洲爪蟾卵母细胞中功能依赖PIP2的KCNQ2/Q3电流。进一步的实验证据显示细胞膜去极化引起的PIP2水平提高是通过激活蛋白激酶C (PKC)进而增加PI 4-激酶(PI4 kinase)的活性引起的。
一、细胞膜去极化对表达于非洲爪蟾卵母细胞的KCNQ2/Q3通道电流的影响
目的:在对KCNQ/M电流的研究过程中,我们发现表达在爪蟾卵母细胞的KCNQ2/Q3的功能对膜电位的变化敏感,而这种反应又有别于通道本身的电压依赖性,本部分实验对此现象进行详细描述,并对其机制进行探讨。
方法:将KCNQ和Kir通道的质粒线性化,体外转录成相应的cRNA,注入非洲爪蟾卵母细胞,表达为相应通道,用双电极电压钳记录的方法,观察细胞膜去极化及细胞外液钾离子浓度的改变对KCNQ电流的调节。
结果:(1)长时间膜电位去极化(-40 mV ~ +40 mV),可观察到表达于非洲爪蟾卵母细胞中的KCNQ2/Q3电流呈时间依赖性及电压依赖性增加。当膜电压再极化回到静息电位水平时,KCNQ2/Q3电流恢复到原水平,这说明去极化引起的KCNQ2/Q3电流增加具有可逆性特征。用M/KCNQ通道特异阻断剂linopirdine (30μM)可完全阻断初始电流和增加的电流,这说明增加的电流是KCNQ2/Q3电流,而不是其它的电流成分。(2)通过测定电压钳制(0 mV,15 min)前后, KCNQ2/Q3电流在0 mV激活及-60 mV去活的时程,观察到膜电位去极化不改变KCNQ2/Q3电流动力学时程。通过测定膜电位去极化(0 mV, 15 min)前后KCNQ2/Q3电流的电导-电压关系曲线,可观察到膜电位去极化不改变KCNQ2/Q3电流激活特点。(3)在卵母细胞上分别表达不同亚型的KCNQ通道,观察膜电位去极化(0 mV,15 min)对其电流幅度及电流特征的影响,发现去极化对KCNQ电流的调节具有亚型特异性。去极化可增大KCNQ2和KCNQ2/Q3电流,但不影响KCNQ1和KCNQ1/KCNE1电流。去极化对KCNQ2作用的电压依赖性特点与对KCNQ2/Q3影响的特点相同,二者半最大增大电压无明显差异。同样,去极化不改变KCNQ2通道电流的激活特征。(4)在卵母细胞上分别表达Kir2.1和Kir2.3通道,观察膜电位去极化(+40 mV)对二者电流的影响,发现去极化可不同程度地增大Kir电流,去极化增大Kir2.3电流的程度大于Kir2.1。我们以前的研究显示Kir2.1对PIP2亲和力较Kir2.3大,提示去极化可能通过影响PIP2水平调节通道功能。(5)观察了细胞外钾离子浓度对KCNQ2/Q3电流的影响,目的是为了检测去极化引起的KCNQ2/Q3电流增大是否是因为钾离子大量外流引起的细胞外钾浓度升高所致。发现提高细胞外液钾离子浓度(10 mM)不影响去极化对KCNQ2/Q3电流的作用。(6)使用高钾外液(ND96K:含96 mM K+)使膜电位去极化,来模拟电压钳制的效果,观察其是否也可以引起KCNQ2/Q3电流增大。发现ND96K孵育(15 min)引起的电流增加幅度与ND96下电压钳制在0 mV(15 min)无明显差异。高钾同样不影响KCNQ2通道的电流动力学和激活特征。这些结果证明去极化本身可以使KCNQ2/Q2电流增加。
结论:(1)膜电位去极化可增大表达于非洲爪蟾卵母细胞中的KCNQ2/Q3通道电流。这种增大作用具有时间依赖性及电压依赖性的特征。(2)膜电位去极化没有影响KCNQ2/Q3通道电流动力学时程及电导-电压关系曲线。(3)膜电位去极化对KCNQ电流的调节具有亚型特异性。(4)膜电位去极化可不同程度增大Kir亚型通道电流。提示去极化可能通过影响PIP2水平调节通道功能。(5)提高细胞外液钾离子浓度(10 mM )不能取消或降低去极化钳制引起的KCNQ2/3电流增大的现象,表明去极化引起的电流增大与细胞外钾浓度的增加无关,即:KCNQ2/3电流增大由膜电位去极化本身引起。(6)细胞外高钾使膜电位去极化并可模拟电压钳制引起的KCNQ2/Q3电流增大,同样不影响通道的激活特性。这进一步证明了膜电位去极化本身可增大KCNQ2/Q3电流。
二、细胞膜去极化提高细胞膜PIP2水平进而增大KCNQ2/3通道电流的分子学机制
目的:上部分实验提示细胞膜去极化可能通过提高细胞膜PIP2水平,进而增大了表达于非洲爪蟾卵母细胞的KCNQ2/Q3。这部分我们主要研究其分子学机制。
方法:(1)将KCNQ2/Q3通道和Ci-VSP共表达于非洲爪蟾卵母细胞,用双电极电压钳记录的方法,观察Ci-VSP和去极化对KCNQ2/Q3电流的调节,探讨去极化过程对PIP2水平的影响。通过使用高渗溶液预提高膜PIP2含量,观察膜电位去极化对PIP2水平的影响。(2)通过使用PI 4-激酶的拮抗剂阻断PIP2的合成,观察PI 4-激酶是否介导了膜电位去极化引起PIP2水平提高的过程。(3)通过使用PKC的激动剂和拮抗剂,观察PKC是否介导了膜电位去极化引起PIP2水平提高的过程。(4)通过薄层色谱的方法直接测定PIP2含量的变化,观察膜电位去极化和激活PKC对细胞膜PIP2水平的影响。(5)通过使用PKA的拮抗剂,观察PKA是否介导了膜电位去极化引起PIP2水平提高的过程。(6)用膜片钳记录的方法,观察去极化对DRG神经元中内源性表达的M电流和CHO细胞中异源性表达的KCNQ2/Q3电流的作用。
结果:(1)通过共表达Ci-VSP,推测去极化可激活PIP2合成。通过薄层色谱的方法证明了膜电位去极化可引起PIP2和PIP水平升高。(2)高渗溶液可通过激活β型PIP 5-激酶而提高PIP2水平。所以如果去极化引起的电流增加依赖PIP2的合成增加,那么用高渗溶液预孵育应可减弱去极化增大KCNQ2/Q3电流的作用。实验结果显示高渗溶液可减弱去极化引起的电流增加。(3)采用wortmannin阻断PI 4-激酶,可基本取消去极化引起的KCNQ2/Q3电流增大作用。(4)PKC的激动剂PMA同样可增大KCNQ2/Q3电流而对KCNQ1无作用。PMA引起的电流增大的时程与去极化引起电流增大的时程相似,且其作用可被去极化的作用所阻断。PKC的阻断剂Bis可取消去极化引起的电流增大作用。但是PMA增大电流的同时,引起KCNQ2/Q3通道的电导-电压关系曲线右移,而对KCNQ1无作用。薄层色谱的方法证明了用PMA激活PKC同样可以提高卵母细胞PIP2和PIP的水平。(5)结果显示:PKA的抑制剂H-89不能阻断去极化引起的KCNQ2/Q3电流增大。提示PKA不是去极化引起的KCNQ2/Q3电流增大的分子学机制(6)去极化(-20 mV)不能增大DRG神经元中内源性表达的M电流及CHO细胞中异源性表达的KCNQ2/KCNQ3电流。PMA(100 nM)可抑制CHO细胞中异源性表达的KCNQ2/KCNQ3电流,而不是激活作用。更大程度的去极化(+20 mV)可逆性抑制DRG神经元中内源性表达的M电流。
结论:(1)膜电位去极化引起KCNQ2/Q3增大是通过提高PIP2水平实现。(2)高渗溶液预孵育减弱了去极化增大KCNQ2/Q3电流的作用。(3)去极化引起的PIP2水平升高是由于激活了PI 4-激酶。(4)PKC的激活介导了去极化引起的KCNQ2/Q3电流增大。(5)PKA不是去极化引起的KCNQ2/Q3电流增大的分子学机制。(6)去极化(-20 mV)不能增大DRG神经元中内源性表达的M电流及CHO细胞中异源性表达的KCNQ2/KCNQ3电流。激活PKC可抑制了CHO细胞中异源性表达的KCNQ2/KCNQ3电流。
Growing body of evidences show membrane phosphatidylinostol 4,5-bisphosphates (PtdIns(4,5)P2, PIP2) plays important role in cell signaling. Presence of PIP2 is fundamentally important for maintaining function of a large number of ion channels and transporters, and for other cell processes such as vesicle trafficking, mobility and endo- and exocytosis. Membrane PIP2 is not only the precursor of some signaling molecules such as diacylglycerol (DAG) and inositol trisphosphate (IP3), PIP2 is a signaling molecule in its own right and its level in the membrane is dynamically modulated. A major metabolic modulation of membrane PIP2 level is phospholipase C (PLC)-mediated PIP2 hydrolysis. This event related mechanism has been contributed to be the underlying mechanism for receptor-induced modulation of function of ion channels. The membrane PIP2 level is maintained by many phosphoinositides kinases and phosphotases. In this study, we describe a novel mechanism of membrane PIP2 modulation. Membrane depolarization induces elevation of membrane PIP2 and subsequently increases KCNQ2/Q3 currents expressed in Xenopus oocytes. Further evidence suggests the depolarization-induced elevation of membrane PIP2 is through activation of PKC and increased activity of PI4 kinase.
1.The effect of membrane depolarization on KCNQ/Q3 current expressed in Xenopus oocytes.
Objective: In previous studies, we noticed that KCNQ2/Q3 current expressed in Xenopus oocytes was sensitive to membrane potential, in a manner different from classical voltage-dependent gating of channels. In this part of the study, we studied this phenomenon in details, and investigated possible underlying molecular mechanisms.
Methods: Plasmids encoding KCNQ and Kir were linearized and cRNA were synthesized by in vitro transcription, and expressed in Xenopus oocytes, Two electrodes voltage clamp (TEVC) recording was used to measure the modulation of depolarization and extracellular K+ on KCNQ currents.
Results: (1) KCNQ2/Q3 currents expressed in Xenopus oocytes could be increased by a continuous depolarization (-40 mV~ +40 mV) with the characteristics of time- and voltage-dependency. When the membrane was repolarized back to -80 mV, the depolarization-induced increase was reversed, indicating that voltage-dependent current increase is reversible. The increased current could be inhibited by linopirdine (30μM), a specific M/KCNQ blocker, indicating that the increased current is KCNQ2/Q3 current. (2) Depolarization did not affect the activation and deactivation kinetics of KCNQ2/Q3 currents when the activation was measured at 0 mV and deactivation was measured at -60 mV either before or after a 0 mV depolarization for 15 min. Similarly, the conductance-voltage relationship of KCNQ2/Q3 activation was not affected by the depolarization. (3) Subunit specificity for effects of depolarization (0 mV, 15 min) was studied. Depolarization could augment KCNQ2 and KCNQ2/Q3 currents, but not KCNQ1 or KCNQ1/KCNE1 currents. The pattern of effects of depolarization on KCNQ2 current is similar to that on KCNQ2/Q3. The half-maximum increase voltages (V1/2) for KCNQ2 and KCNQ2/Q3 were similar. Similar to KCNQ2/Q3, depolarization did not affect the kinetics of KCNQ2 currents. (4) Kir2.1 and Kir2.3 channels were expressed in Xenopus oocytes to study the effects of depolarization (+40 mV) on these channels. Both Kir currents were increased but Kir 2.3 currents were increased by depolarization do greater degree than Kir2.1 currents. This result is consistent with our earlier study that indicates Kir2.1 channel has higher affinity with PIP2 than Kir2.3. This result is also in accordance with the notion that modulation of currents by depolarization may be a result of increased PIP2 level. (5) We tested the effect of external K+ on KCNQ2/Q3 currents to exclude the possibility that the depolarization-induced enhancement of KCNQ2/Q3 currents is due to an increased out flux of K+ and increased concentration of external K+. Increasing external K+ to 10 mM did not affect depolarization-induced potentiation of KCNQ2/Q3 currents. (6) High external K+ solution (ND96K: 96 mM K+) was used to depolarize the membrane to mimic the effect of voltage-clamped depolarization. ND96K incubation (membrane depolarization) also led to enhancement of KCNQ2/Q3 currents. The average folds of increase by ND96K incubation were not significantly different from the voltage-clamped experiments (0 mV, 15 min). The results suggest the depolarization per se contributes to the observed enhancement of KCNQ2/Q3 currents.
Conclusions: (1) Membrane depolarization augments amplitude of KCNQ2/Q3 currents expressed in Xenopus oocytes with the characteristic of time- and voltage-dependency. (2) Depolarization did not affect he activation and deactivation kinetics and the conductance-voltage relationship of KCNQ2/Q3 current. (3) Depolarization-induced increase of KCNQ currents is subunits sensitive. Depolarization had no effects on KCNQ1 and KCNQ1/KCNE1. (4) Kir2.1 and Kir2.3 currents were also sensitive to depolarization. However depolarization increases Kir2.1 and Kir2.3 current in a manner relating to their affinity to PIP2. (5) An increase of external K+ by depolarization could not be causal factor for potentiation of KCNQ2/Q3 currents. (6) External high K+ could mimic the effect of membrane depolarization in increasing KCNQ2/Q3 currents and in a similar manner in regarding the effect of kinetics and voltage-dependent activation of KCNQ2/Q3 currents. The results strongly suggest the depolarization per se contributes to the observed enhancement of KCNQ2/Q3 currents.
2. The mechanism of depolarization-induced elevation of PIP2 level and augment of KCNQ2/Q3 current.
Objective: The first part of the study demonstrated that depolarization induced KCNQ2/Q3 current and an elevation of PIP2 level may be involved. We focused our study of this part on the underlying molecular mechanism.
Methods: (1) Ci-VSP was co-expressed with KCNQ2/Q3 in Xenopus oocytes, and TEVC recordings were used to study the modulation of KCNQ2/Q3 current and PIP2 level by activation of Ci-VSP and depolarization. (2) Blocker of PI4 kinase was used to test whether PI4 kinase was involved in the depolarization-induced PIP2 elevation. (3) Activator and blocker of PKC were used to test whether PKC was involved in the depolarization-induced PIP2 elevation. (4) TLC method was used to measure phosphoinositides level directly to study the effect of depolarization and activation of PKC on PIP2 level. (5) Blocker of PKA was used to test whether PKA was involved in the depolarization-induced PIP2 elevation. (6) Perforated patch clamp was used to study the effect of depolarization on M current in DRG neurons and KCNQ2/3 currents expressed in CHO cells.
Results: (1) Depolarization-induced potentiation of KCNQ2/Q3 currents can be prevented and reversed by activation Ci-VSP, indicating an involvement of PIP2. Measurement of PIP2 using TLC method directly demonstrated that depolarization increased PIP and PIP2 level. (2) Hypertonic stress increases PIP2 level by activating PIP5KIβ. If our depolarization-induced increase of KCNQ2/Q3 currents depends on extra PIP2 synthesis, pre-incubating the oocytes with hypertonic solution should blunt the increase. The result shows that depolarization-induced increase was significantly reduced by hypertonic stress. (3) Wortmannin, which inhibits PI4 kinase, greatly reduced the depolarization-induced enhancement of KCNQ2/Q3 currents. (4) Activation of PKC by PMA increased KCNQ2/Q3 current in a similar manner as depolarization, and similarly had no effect on KCNQ1 and KCNQ1/KCNE1. PMA also increased PIP and PIP2 level measured by TLC. (5) H-89, a inhibitor of PKA, could not inhibit depolarization-induced KCNQ2/Q3 current increase, indicating that PKA is not involved in depolarization-induced potentiation of KCNQ2/Q3 currents. (6) Depolarization did not augument M current in DRG neurons and KCNQ2/3 currents expressed in CHO cells; Activation of PKC inhibited KCNQ2/3 currents expressed in CHO cells. Large depolarization (+20 mV) inhibited M current in DRG neurons reversibly.
Conclusions: (1) The membrane depolarization increases KCNQ2/Q3 currents through increasing membrane PIP2 level. (2) Pre-incubating the oocytes with hypertonic solution blunt the depolarization-induced KCNQ2/Q3 current increase. (3) The depolarization increases PIP2 level through activation of PI4 kinase. (4) Activation of PKC mediates the depolarization- induced KCNQ2/Q3 current increase. (5) PKA is not involved in the depolarization-induced KCNQ2/Q3 current increase. (6) Depolarization do not affect M current in DRG neurons and KCNQ2/3 currents expressed in CHO cells. Activation of PKC inhibits KCNQ2/3 currents expressed in CHO cells.
一、细胞膜去极化对表达于非洲爪蟾卵母细胞的KCNQ2/Q3通道电流的影响
目的:在对KCNQ/M电流的研究过程中,我们发现表达在爪蟾卵母细胞的KCNQ2/Q3的功能对膜电位的变化敏感,而这种反应又有别于通道本身的电压依赖性,本部分实验对此现象进行详细描述,并对其机制进行探讨。
方法:将KCNQ和Kir通道的质粒线性化,体外转录成相应的cRNA,注入非洲爪蟾卵母细胞,表达为相应通道,用双电极电压钳记录的方法,观察细胞膜去极化及细胞外液钾离子浓度的改变对KCNQ电流的调节。
结果:(1)长时间膜电位去极化(-40 mV ~ +40 mV),可观察到表达于非洲爪蟾卵母细胞中的KCNQ2/Q3电流呈时间依赖性及电压依赖性增加。当膜电压再极化回到静息电位水平时,KCNQ2/Q3电流恢复到原水平,这说明去极化引起的KCNQ2/Q3电流增加具有可逆性特征。用M/KCNQ通道特异阻断剂linopirdine (30μM)可完全阻断初始电流和增加的电流,这说明增加的电流是KCNQ2/Q3电流,而不是其它的电流成分。(2)通过测定电压钳制(0 mV,15 min)前后, KCNQ2/Q3电流在0 mV激活及-60 mV去活的时程,观察到膜电位去极化不改变KCNQ2/Q3电流动力学时程。通过测定膜电位去极化(0 mV, 15 min)前后KCNQ2/Q3电流的电导-电压关系曲线,可观察到膜电位去极化不改变KCNQ2/Q3电流激活特点。(3)在卵母细胞上分别表达不同亚型的KCNQ通道,观察膜电位去极化(0 mV,15 min)对其电流幅度及电流特征的影响,发现去极化对KCNQ电流的调节具有亚型特异性。去极化可增大KCNQ2和KCNQ2/Q3电流,但不影响KCNQ1和KCNQ1/KCNE1电流。去极化对KCNQ2作用的电压依赖性特点与对KCNQ2/Q3影响的特点相同,二者半最大增大电压无明显差异。同样,去极化不改变KCNQ2通道电流的激活特征。(4)在卵母细胞上分别表达Kir2.1和Kir2.3通道,观察膜电位去极化(+40 mV)对二者电流的影响,发现去极化可不同程度地增大Kir电流,去极化增大Kir2.3电流的程度大于Kir2.1。我们以前的研究显示Kir2.1对PIP2亲和力较Kir2.3大,提示去极化可能通过影响PIP2水平调节通道功能。(5)观察了细胞外钾离子浓度对KCNQ2/Q3电流的影响,目的是为了检测去极化引起的KCNQ2/Q3电流增大是否是因为钾离子大量外流引起的细胞外钾浓度升高所致。发现提高细胞外液钾离子浓度(10 mM)不影响去极化对KCNQ2/Q3电流的作用。(6)使用高钾外液(ND96K:含96 mM K+)使膜电位去极化,来模拟电压钳制的效果,观察其是否也可以引起KCNQ2/Q3电流增大。发现ND96K孵育(15 min)引起的电流增加幅度与ND96下电压钳制在0 mV(15 min)无明显差异。高钾同样不影响KCNQ2通道的电流动力学和激活特征。这些结果证明去极化本身可以使KCNQ2/Q2电流增加。
结论:(1)膜电位去极化可增大表达于非洲爪蟾卵母细胞中的KCNQ2/Q3通道电流。这种增大作用具有时间依赖性及电压依赖性的特征。(2)膜电位去极化没有影响KCNQ2/Q3通道电流动力学时程及电导-电压关系曲线。(3)膜电位去极化对KCNQ电流的调节具有亚型特异性。(4)膜电位去极化可不同程度增大Kir亚型通道电流。提示去极化可能通过影响PIP2水平调节通道功能。(5)提高细胞外液钾离子浓度(10 mM )不能取消或降低去极化钳制引起的KCNQ2/3电流增大的现象,表明去极化引起的电流增大与细胞外钾浓度的增加无关,即:KCNQ2/3电流增大由膜电位去极化本身引起。(6)细胞外高钾使膜电位去极化并可模拟电压钳制引起的KCNQ2/Q3电流增大,同样不影响通道的激活特性。这进一步证明了膜电位去极化本身可增大KCNQ2/Q3电流。
二、细胞膜去极化提高细胞膜PIP2水平进而增大KCNQ2/3通道电流的分子学机制
目的:上部分实验提示细胞膜去极化可能通过提高细胞膜PIP2水平,进而增大了表达于非洲爪蟾卵母细胞的KCNQ2/Q3。这部分我们主要研究其分子学机制。
方法:(1)将KCNQ2/Q3通道和Ci-VSP共表达于非洲爪蟾卵母细胞,用双电极电压钳记录的方法,观察Ci-VSP和去极化对KCNQ2/Q3电流的调节,探讨去极化过程对PIP2水平的影响。通过使用高渗溶液预提高膜PIP2含量,观察膜电位去极化对PIP2水平的影响。(2)通过使用PI 4-激酶的拮抗剂阻断PIP2的合成,观察PI 4-激酶是否介导了膜电位去极化引起PIP2水平提高的过程。(3)通过使用PKC的激动剂和拮抗剂,观察PKC是否介导了膜电位去极化引起PIP2水平提高的过程。(4)通过薄层色谱的方法直接测定PIP2含量的变化,观察膜电位去极化和激活PKC对细胞膜PIP2水平的影响。(5)通过使用PKA的拮抗剂,观察PKA是否介导了膜电位去极化引起PIP2水平提高的过程。(6)用膜片钳记录的方法,观察去极化对DRG神经元中内源性表达的M电流和CHO细胞中异源性表达的KCNQ2/Q3电流的作用。
结果:(1)通过共表达Ci-VSP,推测去极化可激活PIP2合成。通过薄层色谱的方法证明了膜电位去极化可引起PIP2和PIP水平升高。(2)高渗溶液可通过激活β型PIP 5-激酶而提高PIP2水平。所以如果去极化引起的电流增加依赖PIP2的合成增加,那么用高渗溶液预孵育应可减弱去极化增大KCNQ2/Q3电流的作用。实验结果显示高渗溶液可减弱去极化引起的电流增加。(3)采用wortmannin阻断PI 4-激酶,可基本取消去极化引起的KCNQ2/Q3电流增大作用。(4)PKC的激动剂PMA同样可增大KCNQ2/Q3电流而对KCNQ1无作用。PMA引起的电流增大的时程与去极化引起电流增大的时程相似,且其作用可被去极化的作用所阻断。PKC的阻断剂Bis可取消去极化引起的电流增大作用。但是PMA增大电流的同时,引起KCNQ2/Q3通道的电导-电压关系曲线右移,而对KCNQ1无作用。薄层色谱的方法证明了用PMA激活PKC同样可以提高卵母细胞PIP2和PIP的水平。(5)结果显示:PKA的抑制剂H-89不能阻断去极化引起的KCNQ2/Q3电流增大。提示PKA不是去极化引起的KCNQ2/Q3电流增大的分子学机制(6)去极化(-20 mV)不能增大DRG神经元中内源性表达的M电流及CHO细胞中异源性表达的KCNQ2/KCNQ3电流。PMA(100 nM)可抑制CHO细胞中异源性表达的KCNQ2/KCNQ3电流,而不是激活作用。更大程度的去极化(+20 mV)可逆性抑制DRG神经元中内源性表达的M电流。
结论:(1)膜电位去极化引起KCNQ2/Q3增大是通过提高PIP2水平实现。(2)高渗溶液预孵育减弱了去极化增大KCNQ2/Q3电流的作用。(3)去极化引起的PIP2水平升高是由于激活了PI 4-激酶。(4)PKC的激活介导了去极化引起的KCNQ2/Q3电流增大。(5)PKA不是去极化引起的KCNQ2/Q3电流增大的分子学机制。(6)去极化(-20 mV)不能增大DRG神经元中内源性表达的M电流及CHO细胞中异源性表达的KCNQ2/KCNQ3电流。激活PKC可抑制了CHO细胞中异源性表达的KCNQ2/KCNQ3电流。
Growing body of evidences show membrane phosphatidylinostol 4,5-bisphosphates (PtdIns(4,5)P2, PIP2) plays important role in cell signaling. Presence of PIP2 is fundamentally important for maintaining function of a large number of ion channels and transporters, and for other cell processes such as vesicle trafficking, mobility and endo- and exocytosis. Membrane PIP2 is not only the precursor of some signaling molecules such as diacylglycerol (DAG) and inositol trisphosphate (IP3), PIP2 is a signaling molecule in its own right and its level in the membrane is dynamically modulated. A major metabolic modulation of membrane PIP2 level is phospholipase C (PLC)-mediated PIP2 hydrolysis. This event related mechanism has been contributed to be the underlying mechanism for receptor-induced modulation of function of ion channels. The membrane PIP2 level is maintained by many phosphoinositides kinases and phosphotases. In this study, we describe a novel mechanism of membrane PIP2 modulation. Membrane depolarization induces elevation of membrane PIP2 and subsequently increases KCNQ2/Q3 currents expressed in Xenopus oocytes. Further evidence suggests the depolarization-induced elevation of membrane PIP2 is through activation of PKC and increased activity of PI4 kinase.
1.The effect of membrane depolarization on KCNQ/Q3 current expressed in Xenopus oocytes.
Objective: In previous studies, we noticed that KCNQ2/Q3 current expressed in Xenopus oocytes was sensitive to membrane potential, in a manner different from classical voltage-dependent gating of channels. In this part of the study, we studied this phenomenon in details, and investigated possible underlying molecular mechanisms.
Methods: Plasmids encoding KCNQ and Kir were linearized and cRNA were synthesized by in vitro transcription, and expressed in Xenopus oocytes, Two electrodes voltage clamp (TEVC) recording was used to measure the modulation of depolarization and extracellular K+ on KCNQ currents.
Results: (1) KCNQ2/Q3 currents expressed in Xenopus oocytes could be increased by a continuous depolarization (-40 mV~ +40 mV) with the characteristics of time- and voltage-dependency. When the membrane was repolarized back to -80 mV, the depolarization-induced increase was reversed, indicating that voltage-dependent current increase is reversible. The increased current could be inhibited by linopirdine (30μM), a specific M/KCNQ blocker, indicating that the increased current is KCNQ2/Q3 current. (2) Depolarization did not affect the activation and deactivation kinetics of KCNQ2/Q3 currents when the activation was measured at 0 mV and deactivation was measured at -60 mV either before or after a 0 mV depolarization for 15 min. Similarly, the conductance-voltage relationship of KCNQ2/Q3 activation was not affected by the depolarization. (3) Subunit specificity for effects of depolarization (0 mV, 15 min) was studied. Depolarization could augment KCNQ2 and KCNQ2/Q3 currents, but not KCNQ1 or KCNQ1/KCNE1 currents. The pattern of effects of depolarization on KCNQ2 current is similar to that on KCNQ2/Q3. The half-maximum increase voltages (V1/2) for KCNQ2 and KCNQ2/Q3 were similar. Similar to KCNQ2/Q3, depolarization did not affect the kinetics of KCNQ2 currents. (4) Kir2.1 and Kir2.3 channels were expressed in Xenopus oocytes to study the effects of depolarization (+40 mV) on these channels. Both Kir currents were increased but Kir 2.3 currents were increased by depolarization do greater degree than Kir2.1 currents. This result is consistent with our earlier study that indicates Kir2.1 channel has higher affinity with PIP2 than Kir2.3. This result is also in accordance with the notion that modulation of currents by depolarization may be a result of increased PIP2 level. (5) We tested the effect of external K+ on KCNQ2/Q3 currents to exclude the possibility that the depolarization-induced enhancement of KCNQ2/Q3 currents is due to an increased out flux of K+ and increased concentration of external K+. Increasing external K+ to 10 mM did not affect depolarization-induced potentiation of KCNQ2/Q3 currents. (6) High external K+ solution (ND96K: 96 mM K+) was used to depolarize the membrane to mimic the effect of voltage-clamped depolarization. ND96K incubation (membrane depolarization) also led to enhancement of KCNQ2/Q3 currents. The average folds of increase by ND96K incubation were not significantly different from the voltage-clamped experiments (0 mV, 15 min). The results suggest the depolarization per se contributes to the observed enhancement of KCNQ2/Q3 currents.
Conclusions: (1) Membrane depolarization augments amplitude of KCNQ2/Q3 currents expressed in Xenopus oocytes with the characteristic of time- and voltage-dependency. (2) Depolarization did not affect he activation and deactivation kinetics and the conductance-voltage relationship of KCNQ2/Q3 current. (3) Depolarization-induced increase of KCNQ currents is subunits sensitive. Depolarization had no effects on KCNQ1 and KCNQ1/KCNE1. (4) Kir2.1 and Kir2.3 currents were also sensitive to depolarization. However depolarization increases Kir2.1 and Kir2.3 current in a manner relating to their affinity to PIP2. (5) An increase of external K+ by depolarization could not be causal factor for potentiation of KCNQ2/Q3 currents. (6) External high K+ could mimic the effect of membrane depolarization in increasing KCNQ2/Q3 currents and in a similar manner in regarding the effect of kinetics and voltage-dependent activation of KCNQ2/Q3 currents. The results strongly suggest the depolarization per se contributes to the observed enhancement of KCNQ2/Q3 currents.
2. The mechanism of depolarization-induced elevation of PIP2 level and augment of KCNQ2/Q3 current.
Objective: The first part of the study demonstrated that depolarization induced KCNQ2/Q3 current and an elevation of PIP2 level may be involved. We focused our study of this part on the underlying molecular mechanism.
Methods: (1) Ci-VSP was co-expressed with KCNQ2/Q3 in Xenopus oocytes, and TEVC recordings were used to study the modulation of KCNQ2/Q3 current and PIP2 level by activation of Ci-VSP and depolarization. (2) Blocker of PI4 kinase was used to test whether PI4 kinase was involved in the depolarization-induced PIP2 elevation. (3) Activator and blocker of PKC were used to test whether PKC was involved in the depolarization-induced PIP2 elevation. (4) TLC method was used to measure phosphoinositides level directly to study the effect of depolarization and activation of PKC on PIP2 level. (5) Blocker of PKA was used to test whether PKA was involved in the depolarization-induced PIP2 elevation. (6) Perforated patch clamp was used to study the effect of depolarization on M current in DRG neurons and KCNQ2/3 currents expressed in CHO cells.
Results: (1) Depolarization-induced potentiation of KCNQ2/Q3 currents can be prevented and reversed by activation Ci-VSP, indicating an involvement of PIP2. Measurement of PIP2 using TLC method directly demonstrated that depolarization increased PIP and PIP2 level. (2) Hypertonic stress increases PIP2 level by activating PIP5KIβ. If our depolarization-induced increase of KCNQ2/Q3 currents depends on extra PIP2 synthesis, pre-incubating the oocytes with hypertonic solution should blunt the increase. The result shows that depolarization-induced increase was significantly reduced by hypertonic stress. (3) Wortmannin, which inhibits PI4 kinase, greatly reduced the depolarization-induced enhancement of KCNQ2/Q3 currents. (4) Activation of PKC by PMA increased KCNQ2/Q3 current in a similar manner as depolarization, and similarly had no effect on KCNQ1 and KCNQ1/KCNE1. PMA also increased PIP and PIP2 level measured by TLC. (5) H-89, a inhibitor of PKA, could not inhibit depolarization-induced KCNQ2/Q3 current increase, indicating that PKA is not involved in depolarization-induced potentiation of KCNQ2/Q3 currents. (6) Depolarization did not augument M current in DRG neurons and KCNQ2/3 currents expressed in CHO cells; Activation of PKC inhibited KCNQ2/3 currents expressed in CHO cells. Large depolarization (+20 mV) inhibited M current in DRG neurons reversibly.
Conclusions: (1) The membrane depolarization increases KCNQ2/Q3 currents through increasing membrane PIP2 level. (2) Pre-incubating the oocytes with hypertonic solution blunt the depolarization-induced KCNQ2/Q3 current increase. (3) The depolarization increases PIP2 level through activation of PI4 kinase. (4) Activation of PKC mediates the depolarization- induced KCNQ2/Q3 current increase. (5) PKA is not involved in the depolarization-induced KCNQ2/Q3 current increase. (6) Depolarization do not affect M current in DRG neurons and KCNQ2/3 currents expressed in CHO cells. Activation of PKC inhibits KCNQ2/3 currents expressed in CHO cells.
引文
1 McLaughlin S, Wang J, Gambhir A, et al. PIP(2) and proteins: interactions, organization, and information flow. Annu Rev Biophys Biomol Struct, 2002, 31: 151-175
2 Xu C, Watras J, Loew LM. Kinetic analysis of receptor-activated phosphoinositide turnover. J Cell Biol, 2003, 161: 779-791
3 Di Paolo G, De Camilli P. Phosphoinositides in cell regulation and membrane dynamics. Nature, 2006, 443: 651-657
4 Krauss M, Haucke V. Phosphoinositide-metabolizing enzymes at the interface between membrane traffic and cell signalling. EMBO Rep, 2007, 8: 241-246
5 Hilgemann DW, Feng S,Nasuhoglu C. The complex and intriguing lives of PIP2 with ion channels and transporters. Sci STKE, 2001, 2001: RE19
6 Suh BC, Hille B. Regulation of ion channels by phosphatidylinositol 4,5-bisphosphate. Curr OpinNeurobiol, 2005, 15: 370-378
7 Gamper N, Shapiro MS. Regulation of ion transport proteins by membrane phosphoinositides. Nat Rev Neurosci, 2007, 8: 921-934
8 Suh BC, Hille B. Recovery from muscarinic modulation of M current channels requires phosphatidylinositol 4,5-bisphosphate synthesis. Neuron, 2002, 35: 507-520
9 Zhang H, Craciun LC, Mirshahi T, et al. PIP(2) activates KCNQ channels, and its hydrolysis underlies receptor-mediated inhibition of M currents. Neuron, 2003, 37: 963-975
10 Ford CP, Stemkowski PL, Light PE, et al. Experiments to test the role of phosphatidylinositol 4,5-bisphosphate in neurotransmitter-induced M-channel closure in bullfrog sympathetic neurons. J Neurosci, 2003, 23: 4931-4941
11 Delmas P, Brown DA. Pathways modulating neural KCNQ/M (Kv7) potassium channels. Nat Rev Neurosci, 2005, 6: 850-862
12 Jia Q, Jia Z, Zhao Z, et al. Activation of epidermal growth factor receptor inhibits KCNQ2/3 current through two distinct pathways: membrane PtdIns(4,5)P2 hydrolysis and channel phosphorylation. J Neurosci, 2007, 27: 2503-2512
13 Jia Z, Bei J, Rodat-Despoix L, et al. NGF inhibits M/KCNQ currents and selectively alters neuronal excitability in subsets of sympathetic neurons depending on their M/KCNQ current background. J Gen Physiol, 2008,131: 575-587
14 Murata Y, Iwasaki H, Sasaki M, et al. Phosphoinositide phosphatase activity coupled to an intrinsic voltage sensor. Nature, 2005, 435: 1239-1243
15 Murata Y, Okamura Y. Depolarization activates the phosphoinositide phosphatase Ci-VSP, as detected in Xenopus oocytes coexpressing sensors of PIP2. J Physiol, 2007, 583: 875-889
16 Wang HS, Pan Z, Shi W, et al. KCNQ2 and KCNQ3 potassium channel subunits: molecular correlates of the M-channel. Science, 1998, 282: 1890-1893
17 Barhanin J, Lesage F, Guillemare E, et al. K(V)LQT1 and lsK (minK) proteins associate to form the I(Ks) cardiac potassium current. Nature, 1996, 384: 78-80
18 Sanguinetti MC, Curran ME, Zou A, et al. Coassembly of K(V)LQT1 and minK (IsK) proteins to form cardiac I(Ks) potassium channel. Nature, 1996, 384: 80-83
19 Loussouarn G, Park KH, Bellocq C, et al. Phosphatidylinositol-4,5-bisphosphate, PIP2, controls KCNQ1/KCNE1 voltage-gated potassium channels: a functional homology between voltage-gated and inward rectifier K+ channels. Embo J, 2003, 22: 5412-5421
20 Ding WG, Toyoda F, Matsuura H. Regulation of cardiac IKs potassium current by membrane phosphatidylinositol 4,5-bisphosphate. J Biol Chem, 2004, 279: 50726-50734
21 Du X, Zhang H, Lopes C, et al. Characteristic interactionswith phosphatidylinositol 4,5-bisphosphate determine regulation of kir channels by diverse modulators. J Biol Chem, 2004, 279: 37271-37281
22 Wood MJ, Korn SJ. Two mechanisms of K(+)-dependent potentiation in Kv2.1 potassium channels. Biophys J, 2000, 79: 2535-2546
23 Billups D, Billups B, Challiss RA, et al. Modulation of Gq-protein-coupled inositol trisphosphate and Ca2+ signaling by the membrane potential. J Neurosci, 2006, 26: 9983-9995
24 Suh BC, Inoue T, Meyer T, et al. Rapid chemically induced changes of PtdIns(4,5)P2 gate KCNQ ion channels. Science, 2006, 314: 1454-1457
25 Selyanko AA, Hadley JK, Brown DA. Properties of single M-type KCNQ2/KCNQ3 potassium channels expressed in mammalian cells. J Physiol, 2001, 534: 15-24
26 Li Y, Gamper N, Shapiro MS. Single-channel analysis of KCNQ K+ channels reveals the mechanism of augmentation by a cysteine-modifying reagent. J Neurosci, 2004, 24: 5079-5090
27 Li Y, Gamper N, Hilgemann DW, et al. Regulation of Kv7 (KCNQ) K+ channel open probability by phosphatidylinositol 4,5-bisphosphate. J Neurosci, 2005, 25: 9825-9835
28 Park KH, Piron J, Dahimene S, et al. Impaired KCNQ1-KCNE1 andphosphatidylinositol-4,5-bisphosphate interaction underlies the long QT syndrome. Circ Res, 2005, 96: 730-739
1 Rameh LE, Tolias KF, Duckworth BC, et al. A new pathway for synthesis of phosphatidylinositol-4,5- bisphos- phate. Nature, 1997, 390: 192-196
2 Di Paolo G,De Camilli P. Phosphoinositides in cell regulation and membrane dynamics. Nature, 2006, 443: 651-657
3 Krauss M,Haucke V. Phosphoinositide-metabolizing enzymes at the interface between membrane traffic and cell signalling. EMBO Rep, 2007, 8: 241-246
4 Raucher D, Stauffer T, Chen W, et al. Phosphatidylinositol 4,5-bisphosphate functions as a second messenger that regulates cytoskeleton-plasma membrane adhesion. Cell, 2000, 100: 221-228
5 Wenk MR, Pellegrini L, Klenchin VA, et al. PIP kinase Igamma is the major PI (4,5) P (2) synthesizing enzyme at the synapse. Neuron, 2001, 32: 79-88
6 Suh BC,Hille B. Recovery from muscarinic modulation of M current channels requires phosphatidylinositol 4,5-bisphosphate synthesis. Neuron, 2002, 35: 507-520
7 Zhang H, Craciun LC, Mirshahi T, et al. PIP (2) activates KCNQ channels, and its hydrolysis underlies receptor-mediated inhibition of M currents. Neuron, 2003,37: 963-975
8 Li Y, Gamper N, Hilgemann DW, et al. Regulation of Kv7 (KCNQ) K+ channel open probability by phosphatidylinositol 4,5-bisphosphate. J Neurosci, 2005, 25: 9825-9835
9 Suh BC, Inoue T, Meyer T, et al. Rapid chemically induced changes of PtdIns (4,5) P2 gate KCNQ ion channels. Science, 2006, 314: 1454-1457
10 Winks JS, Hughes S, Filippov AK, et al. Relationship between membrane phosphatidylinositol-4,5-bisphosphate and receptor-mediated inhibition of native neuronal M channels. J Neurosci, 2005, 25: 3400-3413
11 Murata Y, Iwasaki H, Sasaki M, et al. Phosphoinositide phosphatase activity coupled to an intrinsic voltage sensor. Nature, 2005, 435: 1239-1243
12 Murata Y,Okamura Y. Depolarization activates the phosphoinositide phosphatase Ci-VSP, as detected in Xenopus oocytes coexpressing sensors of PIP2. J Physiol, 2007, 583: 875-889
13 Folch J, Lees M,Sloane Stanley GH. A simple method for the isolation and purification of total lipides from animal tissues. J Biol Chem, 1957, 226: 497-509
14 Yamamoto M, Chen MZ, Wang YJ, et al. Hypertonic stress increases phosphatidylinositol 4,5-bisphosphate levels by activating PIP5KIbeta. J Biol Chem, 2006, 281: 32630-32638
15 Nakanishi S, Catt KJ, Balla T. A wortmannin-sensitive phosphatidylinositol 4-kinase that regulates hormone-sensitive pools of inositolphospholipids. Proc Natl Acad Sci U S A, 1995, 92: 5317-5321
16 Hoshi N, Zhang JS, Omaki M, et al. AKAP150 signaling complex promotes suppression of the M-current by muscarinic agonists. Nat Neurosci, 2003, 6: 564-571
17 Nakajo K,Kubo Y. Protein kinase C shifts the voltage dependence of KCNQ/M channels expressed in Xenopus oocytes. J Physiol, 2005, 569: 59-74
18 Hilgemann DW, Feng S,Nasuhoglu C. The complex and intriguing lives of PIP2 with ion channels and transporters. Sci STKE, 2001, 2001: RE19
19 Suh BC,Hille B. Regulation of ion channels by phosphatidylinositol 4,5-bisphosphate. Curr Opin Neurobiol, 2005, 15: 370-378
20 Gamper N,Shapiro MS. Regulation of ion transport proteins by membrane phosphoinositides. Nat Rev Neurosci, 2007, 8: 921-934
21 Zeng WZ, Li XJ, Hilgemann DW, et al. Protein kinase C inhibits ROMK1 channel activity via a phosphatidylinositol 4,5-bisphosphate-dependent mechanism. J Biol Chem, 2003, 278: 16852-16856
22 Zhang L, Lee JK, John SA, et al. Mechanosensitivity of GIRK channels is mediated by protein kinase C-dependent channel-phosphatidylinositol 4,5-bisphosphate interaction.J Biol Chem, 2004, 279: 7037-7047
23 Du X, Zhang H, Lopes C, et al. Characteristic interactions with phosphatidylinositol 4,5-bisphosphate determine regulation of kir channels by diverse modulators. J Biol Chem, 2004, 279: 37271-37281
24 Etkovitz N, Rubinstein S, Daniel L, et al. Role of PI3-kinase and PI4-kinase in actin polymerization during bovine sperm capacitation. Biol Reprod, 2007, 77: 263-273
25 Eltit JM, Hidalgo J, Liberona JL, et al. Slow calcium signals after tetanic electrical stimulation in skeletal myotubes. Biophys J, 2004, 86: 3042-3051
26 Cardenas C, Muller M, Jaimovich E, et al. Depolarization of skeletal muscle cells induces phosphorylation of cAMP response element binding protein via calcium and protein kinase Calpha. J Biol Chem, 2004, 279: 39122-39131
27 Jaimovich E, Carrasco MA. IP3 dependent Ca2+ signals in muscle cells are involved in regulation of gene expression. Biol Res, 2002, 35: 195-202
28 Araya R, Liberona JL, Cardenas JC, et al. Dihydropyridine receptors as voltage sensors for a depolarization-evoked, IP3R-mediated, slow calcium signal in skeletal muscle cells. J Gen Physiol, 2003, 121: 3-16
29 Maasch C, Wagner S, Lindschau C, et al. Protein kinase calpha targeting is regulated by temporal and spatial changes in intracellular free calcium concentration [Ca (2+) ] (i) . Faseb J, 2000, 14: 1653-1663
30 Audigier SM, Wang JK,Greengard P. Membrane depolarization and carbamoylcholine stimulate phosphatidylinositol turnover in intact nerve terminals. Proc Natl Acad Sci U S A, 1988, 85: 2859-2863
31 Billups D, Billups B, Challiss RA, et al. Modulation of Gq-protein-coupled inositol trisphosphate and Ca2+ signaling by the membrane potential. J Neurosci, 2006, 26: 9983-9995
32 Ben-Chaim Y, Tour O, Dascal N, et al. The M2 muscarinic G-protein-coupled receptor is voltage-sensitive. J Biol Chem, 2003, 278: 22482-22491
33 Martinez-Pinna J, Gurung IS, Vial C, et al. Direct voltage control of signaling via P2Y1 and other Galphaq-coupled receptors. J Biol Chem, 2005, 280: 1490-1498
34 Ben-Chaim Y, Chanda B, Dascal N, et al. Movement of 'gating charge' is coupled to ligand binding in a G-protein-coupled receptor. Nature, 2006, 444: 106-109
35 Nasuhoglu C, Feng S, Mao Y, et al. Modulation of cardiac PIP2 by cardioactive hormones and other physiologically relevant interventions. Am J Physiol Cell Physiol, 2002, 283: C223-234
36 Micheva KD, Holz RW, Smith SJ. Regulation of presynaptic phosphatidylinositol 4,5-biphosphate by neuronal activity. J Cell Biol, 2001, 154: 355-368
1 Doupnik CA, Davidson N, Lester HA. The inward rectifier potassium channel family. Curr Opin Neuro biol, 1995, 5:268-277
2 Krapivinsky G, Medina I, Eng L, Krapivinsky L, et al. A novel inward rectifier K+ channel with unique pore properties. Neuron, 1998, 20:995-l005
3 Qu Z, Zhu G, Yang Z, et al. Identification of a critical motif responsible for gating of Kir2.3 channel by intracellular protons. J Biol Chem, 1999, 274(20):13783-13789
4 Logothetis DE, Jin T, Lupyan D,et al. Phosphoinositide-mediated gating of inwardly rectifying K(+) channels. Pflugers Arch, 2007, 455(1):83-95
5 Du XN, Zhang HL, Lopes C, et al. Characteristic interactions with PIP2 determine regulation of Kir channels by diverse modulators. J Biol Chem, 2004, 279(36):37271-37281
6 Wang C, Mirshahi UL, Liu BY, et al. Arachdonic acid activates Kir2.3 channels by enhancing channel-PIP2interactions. Mol Pharmacol, 2008, 73:1185-1194
7 Leaney JL, Dekker LV and Tinker A. Regulation of a G protein-gated inwardly rectifying K+ channel by a Ca2+-independent protein kinase C. J Physiol, 2001, 534:367-379
8 Perillan PR, Chen M, Potts EA, et al. Transforming growth factor-beta 1 regulates Kir2.3 inward rectifier K+ channels via phospholipase C and protein kinase C-delta in reactive astrocytes from adult rat brain. J Biol Chem, 2002, 277(3):1974-1980
9 Doyle DA, Cabral JM, Pfuetzner RA, et al. The structure of the potassium channel: molecular basis of K+ conduction and selectivity. Science, 1998, 280:69-76
10 Hebert SC. Roles of Na-K-2Cl and NaCl cotransporters and ROMK potassium channels in urinary concentration mechanism. Am J Physiol, 1998, 275:F325-F327
11 Fakler B, Braèndle U, Glowatzki E, et al. Strong voltage-dependent inwardrectification of inward rectifier K+ channels is caused by intracellular spermine. Cell, 1995, 80:149-154
12 Lopatin AN, Makhina EN, Nichols CG. Potassium channel block by cytoplasmic polyamines as the mechanism of intrinsic rectification. Nature, 1994, 372:366-369
13 Hibino H, Horio Y, Inanobe A, et al. An ATP-dependent inwardly rectifying potassium channel, KAB-2 (Kir4.1), in cochlear stria vascularis of inner ear: its specific subcellularlocalization and correlation with the formation of endocochlear potential. J Neurosci, 1997, 17:4711-4721
14 Aguilar-Bryan L, Clement JP, Gonzalez G, et al. Toward understanding the assembly and structure of KATP channels. Physiol Rev, 1998, 78:227-245
15 NishizukaY. Protein kinase C and lipid signaling for sustained cellular responses. FASEB J, 1995, 9:484-496
16 Hoffman J. The potential for isoemzyme-selective modulation of protein kinase C. FASEB J, 1997, 11:649-669
17 Zhu GY, Qui ZQ, Cui NR, et al. Suppression of Kir2.3 Activity by Protein Kinase C Phosphorylation of the Channel Protein at Threonine 53. J Biol Chem, 1999, 274(17): 11643-11646
18 Shi Y, Cui N, Shi WW, et al. A short motif in Kir6.1 consisting of 4 phosphorylation repeats underlies the vascular KATP channel inhibition by protein kinase C. J Biol Chem, 2008, 283:2488-2494
19 Mao JZ, Wang XR, Chen FC, et al. Molecular basis for the inhibition of G protein-coupled inward rectifier K+ channels by protein kinase C. PNAS, 2004, 101(4):1087-1092
20 McLaughlin S, Wang J, Gambhir A, et al. PIP2 and proteins: interactions, organization, and information flow. Annu Rev BioPhys Biomol Struct, 2002, 31:151-175
21 Xu C, Watras J, Loew LM. Kinetic analysis of receptor-activated phosphoinositide turnover. The Journel ofCell Biology, 2003, 161(4):779-791
22 Di Paolo G, De Camilli P. Phosphoinositides in cell regulation and membrane dynamics. Nature, 2006, 443: 651–657
23 Krau? M & Haucke V. Phosphoinositide-metabolizing enzymes at the interface between membrane traffic and cell signaling EMBO reports, 2007, 8(3):241–246
24 Zhang HL, He C, Yan XX, et al. Activation of inwardly rectifying K+ chanels by distinct PtdIns(4,5)P2 interactions. Nature Cell Biology, 1999, 1:183-188
25 Lopes C MB, Zhang H, Rohacs T, et al. Alterations in Conserved Kir Channel-PIP2 Interactions Underlie Channelopathies. Neuron, 2002, 34, 933–944
26 Quinn KV, Cui Y, Giblin JP, et al. Do Anionic phospholipids Serve as Cofactors or Second Messengers for the Regulation of Activity of Cloned ATP-Sensitive K+ Channels?Circ Res, 2003, 93:646-655
27 Thorneloe KS, Maruyama Y, Malcolm AT, et al. Protein kinase C modulation of recombinant ATP-sensitive K+ channels composed of Kir6.1 and/or Kir6.2 expressed with SUR2B. J. Physiol, 2002, 541;65-80
28 Zhang L, Lee JK, John SA, et al. Mechanosensitivity of GIRK Channels Is Mediated by Protein Kinase C-dependent Channel-Phosphatidylinositol 4,5-Bisphosphate Interaction. JBC, 2004, 279:7037-7047
29 John R Apgar. Activation of Protein Kinase C in RatBasophilic Leukemia Cells Stimulates Increased Production of Phosphatidylinositol 4-Phosphate and Phosphatidylinositol 4,5-Bisphosphate: Correlation with Actin Polymerization. Molecular Biology of the Cell, 1995, 6:97-108
30 Light PE, Bladen C, Winkfein RJ, et al. Molecular basis of protein kinase C-induced activation of ATP-sensitive potassium channels. PNAS, 2000, 97(16):9058-9063
31 Freesia L. Huang, Kuo-Ping Huang. Interaction of Protein Kinase C Isozymes with Phosphatidylinositol 4,5-Bisphosphate. J Biol Chem, 1991, 266(14):8727-8733
32 Sonia SB, Consuelo MV, Juan C GF, et al. The C2 Domain of PKCαIs a Ca2+-dependent PtdIns(4,5)P2 Sensing Domain: A New Insight into an Old Pathway. J Mol Biol, 2006, 362:901-914
33 Marta GV, Consuelo MV, Juan C GF, et al. The C2 Domains of Classical PKCs are Specific PtdIns(4,5)P2-sensing Domains with Different Affinities for Membrane Binding. J Mol Biol, 2007, 371:608-621
2 Xu C, Watras J, Loew LM. Kinetic analysis of receptor-activated phosphoinositide turnover. J Cell Biol, 2003, 161: 779-791
3 Di Paolo G, De Camilli P. Phosphoinositides in cell regulation and membrane dynamics. Nature, 2006, 443: 651-657
4 Krauss M, Haucke V. Phosphoinositide-metabolizing enzymes at the interface between membrane traffic and cell signalling. EMBO Rep, 2007, 8: 241-246
5 Hilgemann DW, Feng S,Nasuhoglu C. The complex and intriguing lives of PIP2 with ion channels and transporters. Sci STKE, 2001, 2001: RE19
6 Suh BC, Hille B. Regulation of ion channels by phosphatidylinositol 4,5-bisphosphate. Curr OpinNeurobiol, 2005, 15: 370-378
7 Gamper N, Shapiro MS. Regulation of ion transport proteins by membrane phosphoinositides. Nat Rev Neurosci, 2007, 8: 921-934
8 Suh BC, Hille B. Recovery from muscarinic modulation of M current channels requires phosphatidylinositol 4,5-bisphosphate synthesis. Neuron, 2002, 35: 507-520
9 Zhang H, Craciun LC, Mirshahi T, et al. PIP(2) activates KCNQ channels, and its hydrolysis underlies receptor-mediated inhibition of M currents. Neuron, 2003, 37: 963-975
10 Ford CP, Stemkowski PL, Light PE, et al. Experiments to test the role of phosphatidylinositol 4,5-bisphosphate in neurotransmitter-induced M-channel closure in bullfrog sympathetic neurons. J Neurosci, 2003, 23: 4931-4941
11 Delmas P, Brown DA. Pathways modulating neural KCNQ/M (Kv7) potassium channels. Nat Rev Neurosci, 2005, 6: 850-862
12 Jia Q, Jia Z, Zhao Z, et al. Activation of epidermal growth factor receptor inhibits KCNQ2/3 current through two distinct pathways: membrane PtdIns(4,5)P2 hydrolysis and channel phosphorylation. J Neurosci, 2007, 27: 2503-2512
13 Jia Z, Bei J, Rodat-Despoix L, et al. NGF inhibits M/KCNQ currents and selectively alters neuronal excitability in subsets of sympathetic neurons depending on their M/KCNQ current background. J Gen Physiol, 2008,131: 575-587
14 Murata Y, Iwasaki H, Sasaki M, et al. Phosphoinositide phosphatase activity coupled to an intrinsic voltage sensor. Nature, 2005, 435: 1239-1243
15 Murata Y, Okamura Y. Depolarization activates the phosphoinositide phosphatase Ci-VSP, as detected in Xenopus oocytes coexpressing sensors of PIP2. J Physiol, 2007, 583: 875-889
16 Wang HS, Pan Z, Shi W, et al. KCNQ2 and KCNQ3 potassium channel subunits: molecular correlates of the M-channel. Science, 1998, 282: 1890-1893
17 Barhanin J, Lesage F, Guillemare E, et al. K(V)LQT1 and lsK (minK) proteins associate to form the I(Ks) cardiac potassium current. Nature, 1996, 384: 78-80
18 Sanguinetti MC, Curran ME, Zou A, et al. Coassembly of K(V)LQT1 and minK (IsK) proteins to form cardiac I(Ks) potassium channel. Nature, 1996, 384: 80-83
19 Loussouarn G, Park KH, Bellocq C, et al. Phosphatidylinositol-4,5-bisphosphate, PIP2, controls KCNQ1/KCNE1 voltage-gated potassium channels: a functional homology between voltage-gated and inward rectifier K+ channels. Embo J, 2003, 22: 5412-5421
20 Ding WG, Toyoda F, Matsuura H. Regulation of cardiac IKs potassium current by membrane phosphatidylinositol 4,5-bisphosphate. J Biol Chem, 2004, 279: 50726-50734
21 Du X, Zhang H, Lopes C, et al. Characteristic interactionswith phosphatidylinositol 4,5-bisphosphate determine regulation of kir channels by diverse modulators. J Biol Chem, 2004, 279: 37271-37281
22 Wood MJ, Korn SJ. Two mechanisms of K(+)-dependent potentiation in Kv2.1 potassium channels. Biophys J, 2000, 79: 2535-2546
23 Billups D, Billups B, Challiss RA, et al. Modulation of Gq-protein-coupled inositol trisphosphate and Ca2+ signaling by the membrane potential. J Neurosci, 2006, 26: 9983-9995
24 Suh BC, Inoue T, Meyer T, et al. Rapid chemically induced changes of PtdIns(4,5)P2 gate KCNQ ion channels. Science, 2006, 314: 1454-1457
25 Selyanko AA, Hadley JK, Brown DA. Properties of single M-type KCNQ2/KCNQ3 potassium channels expressed in mammalian cells. J Physiol, 2001, 534: 15-24
26 Li Y, Gamper N, Shapiro MS. Single-channel analysis of KCNQ K+ channels reveals the mechanism of augmentation by a cysteine-modifying reagent. J Neurosci, 2004, 24: 5079-5090
27 Li Y, Gamper N, Hilgemann DW, et al. Regulation of Kv7 (KCNQ) K+ channel open probability by phosphatidylinositol 4,5-bisphosphate. J Neurosci, 2005, 25: 9825-9835
28 Park KH, Piron J, Dahimene S, et al. Impaired KCNQ1-KCNE1 andphosphatidylinositol-4,5-bisphosphate interaction underlies the long QT syndrome. Circ Res, 2005, 96: 730-739
1 Rameh LE, Tolias KF, Duckworth BC, et al. A new pathway for synthesis of phosphatidylinositol-4,5- bisphos- phate. Nature, 1997, 390: 192-196
2 Di Paolo G,De Camilli P. Phosphoinositides in cell regulation and membrane dynamics. Nature, 2006, 443: 651-657
3 Krauss M,Haucke V. Phosphoinositide-metabolizing enzymes at the interface between membrane traffic and cell signalling. EMBO Rep, 2007, 8: 241-246
4 Raucher D, Stauffer T, Chen W, et al. Phosphatidylinositol 4,5-bisphosphate functions as a second messenger that regulates cytoskeleton-plasma membrane adhesion. Cell, 2000, 100: 221-228
5 Wenk MR, Pellegrini L, Klenchin VA, et al. PIP kinase Igamma is the major PI (4,5) P (2) synthesizing enzyme at the synapse. Neuron, 2001, 32: 79-88
6 Suh BC,Hille B. Recovery from muscarinic modulation of M current channels requires phosphatidylinositol 4,5-bisphosphate synthesis. Neuron, 2002, 35: 507-520
7 Zhang H, Craciun LC, Mirshahi T, et al. PIP (2) activates KCNQ channels, and its hydrolysis underlies receptor-mediated inhibition of M currents. Neuron, 2003,37: 963-975
8 Li Y, Gamper N, Hilgemann DW, et al. Regulation of Kv7 (KCNQ) K+ channel open probability by phosphatidylinositol 4,5-bisphosphate. J Neurosci, 2005, 25: 9825-9835
9 Suh BC, Inoue T, Meyer T, et al. Rapid chemically induced changes of PtdIns (4,5) P2 gate KCNQ ion channels. Science, 2006, 314: 1454-1457
10 Winks JS, Hughes S, Filippov AK, et al. Relationship between membrane phosphatidylinositol-4,5-bisphosphate and receptor-mediated inhibition of native neuronal M channels. J Neurosci, 2005, 25: 3400-3413
11 Murata Y, Iwasaki H, Sasaki M, et al. Phosphoinositide phosphatase activity coupled to an intrinsic voltage sensor. Nature, 2005, 435: 1239-1243
12 Murata Y,Okamura Y. Depolarization activates the phosphoinositide phosphatase Ci-VSP, as detected in Xenopus oocytes coexpressing sensors of PIP2. J Physiol, 2007, 583: 875-889
13 Folch J, Lees M,Sloane Stanley GH. A simple method for the isolation and purification of total lipides from animal tissues. J Biol Chem, 1957, 226: 497-509
14 Yamamoto M, Chen MZ, Wang YJ, et al. Hypertonic stress increases phosphatidylinositol 4,5-bisphosphate levels by activating PIP5KIbeta. J Biol Chem, 2006, 281: 32630-32638
15 Nakanishi S, Catt KJ, Balla T. A wortmannin-sensitive phosphatidylinositol 4-kinase that regulates hormone-sensitive pools of inositolphospholipids. Proc Natl Acad Sci U S A, 1995, 92: 5317-5321
16 Hoshi N, Zhang JS, Omaki M, et al. AKAP150 signaling complex promotes suppression of the M-current by muscarinic agonists. Nat Neurosci, 2003, 6: 564-571
17 Nakajo K,Kubo Y. Protein kinase C shifts the voltage dependence of KCNQ/M channels expressed in Xenopus oocytes. J Physiol, 2005, 569: 59-74
18 Hilgemann DW, Feng S,Nasuhoglu C. The complex and intriguing lives of PIP2 with ion channels and transporters. Sci STKE, 2001, 2001: RE19
19 Suh BC,Hille B. Regulation of ion channels by phosphatidylinositol 4,5-bisphosphate. Curr Opin Neurobiol, 2005, 15: 370-378
20 Gamper N,Shapiro MS. Regulation of ion transport proteins by membrane phosphoinositides. Nat Rev Neurosci, 2007, 8: 921-934
21 Zeng WZ, Li XJ, Hilgemann DW, et al. Protein kinase C inhibits ROMK1 channel activity via a phosphatidylinositol 4,5-bisphosphate-dependent mechanism. J Biol Chem, 2003, 278: 16852-16856
22 Zhang L, Lee JK, John SA, et al. Mechanosensitivity of GIRK channels is mediated by protein kinase C-dependent channel-phosphatidylinositol 4,5-bisphosphate interaction.J Biol Chem, 2004, 279: 7037-7047
23 Du X, Zhang H, Lopes C, et al. Characteristic interactions with phosphatidylinositol 4,5-bisphosphate determine regulation of kir channels by diverse modulators. J Biol Chem, 2004, 279: 37271-37281
24 Etkovitz N, Rubinstein S, Daniel L, et al. Role of PI3-kinase and PI4-kinase in actin polymerization during bovine sperm capacitation. Biol Reprod, 2007, 77: 263-273
25 Eltit JM, Hidalgo J, Liberona JL, et al. Slow calcium signals after tetanic electrical stimulation in skeletal myotubes. Biophys J, 2004, 86: 3042-3051
26 Cardenas C, Muller M, Jaimovich E, et al. Depolarization of skeletal muscle cells induces phosphorylation of cAMP response element binding protein via calcium and protein kinase Calpha. J Biol Chem, 2004, 279: 39122-39131
27 Jaimovich E, Carrasco MA. IP3 dependent Ca2+ signals in muscle cells are involved in regulation of gene expression. Biol Res, 2002, 35: 195-202
28 Araya R, Liberona JL, Cardenas JC, et al. Dihydropyridine receptors as voltage sensors for a depolarization-evoked, IP3R-mediated, slow calcium signal in skeletal muscle cells. J Gen Physiol, 2003, 121: 3-16
29 Maasch C, Wagner S, Lindschau C, et al. Protein kinase calpha targeting is regulated by temporal and spatial changes in intracellular free calcium concentration [Ca (2+) ] (i) . Faseb J, 2000, 14: 1653-1663
30 Audigier SM, Wang JK,Greengard P. Membrane depolarization and carbamoylcholine stimulate phosphatidylinositol turnover in intact nerve terminals. Proc Natl Acad Sci U S A, 1988, 85: 2859-2863
31 Billups D, Billups B, Challiss RA, et al. Modulation of Gq-protein-coupled inositol trisphosphate and Ca2+ signaling by the membrane potential. J Neurosci, 2006, 26: 9983-9995
32 Ben-Chaim Y, Tour O, Dascal N, et al. The M2 muscarinic G-protein-coupled receptor is voltage-sensitive. J Biol Chem, 2003, 278: 22482-22491
33 Martinez-Pinna J, Gurung IS, Vial C, et al. Direct voltage control of signaling via P2Y1 and other Galphaq-coupled receptors. J Biol Chem, 2005, 280: 1490-1498
34 Ben-Chaim Y, Chanda B, Dascal N, et al. Movement of 'gating charge' is coupled to ligand binding in a G-protein-coupled receptor. Nature, 2006, 444: 106-109
35 Nasuhoglu C, Feng S, Mao Y, et al. Modulation of cardiac PIP2 by cardioactive hormones and other physiologically relevant interventions. Am J Physiol Cell Physiol, 2002, 283: C223-234
36 Micheva KD, Holz RW, Smith SJ. Regulation of presynaptic phosphatidylinositol 4,5-biphosphate by neuronal activity. J Cell Biol, 2001, 154: 355-368
1 Doupnik CA, Davidson N, Lester HA. The inward rectifier potassium channel family. Curr Opin Neuro biol, 1995, 5:268-277
2 Krapivinsky G, Medina I, Eng L, Krapivinsky L, et al. A novel inward rectifier K+ channel with unique pore properties. Neuron, 1998, 20:995-l005
3 Qu Z, Zhu G, Yang Z, et al. Identification of a critical motif responsible for gating of Kir2.3 channel by intracellular protons. J Biol Chem, 1999, 274(20):13783-13789
4 Logothetis DE, Jin T, Lupyan D,et al. Phosphoinositide-mediated gating of inwardly rectifying K(+) channels. Pflugers Arch, 2007, 455(1):83-95
5 Du XN, Zhang HL, Lopes C, et al. Characteristic interactions with PIP2 determine regulation of Kir channels by diverse modulators. J Biol Chem, 2004, 279(36):37271-37281
6 Wang C, Mirshahi UL, Liu BY, et al. Arachdonic acid activates Kir2.3 channels by enhancing channel-PIP2interactions. Mol Pharmacol, 2008, 73:1185-1194
7 Leaney JL, Dekker LV and Tinker A. Regulation of a G protein-gated inwardly rectifying K+ channel by a Ca2+-independent protein kinase C. J Physiol, 2001, 534:367-379
8 Perillan PR, Chen M, Potts EA, et al. Transforming growth factor-beta 1 regulates Kir2.3 inward rectifier K+ channels via phospholipase C and protein kinase C-delta in reactive astrocytes from adult rat brain. J Biol Chem, 2002, 277(3):1974-1980
9 Doyle DA, Cabral JM, Pfuetzner RA, et al. The structure of the potassium channel: molecular basis of K+ conduction and selectivity. Science, 1998, 280:69-76
10 Hebert SC. Roles of Na-K-2Cl and NaCl cotransporters and ROMK potassium channels in urinary concentration mechanism. Am J Physiol, 1998, 275:F325-F327
11 Fakler B, Braèndle U, Glowatzki E, et al. Strong voltage-dependent inwardrectification of inward rectifier K+ channels is caused by intracellular spermine. Cell, 1995, 80:149-154
12 Lopatin AN, Makhina EN, Nichols CG. Potassium channel block by cytoplasmic polyamines as the mechanism of intrinsic rectification. Nature, 1994, 372:366-369
13 Hibino H, Horio Y, Inanobe A, et al. An ATP-dependent inwardly rectifying potassium channel, KAB-2 (Kir4.1), in cochlear stria vascularis of inner ear: its specific subcellularlocalization and correlation with the formation of endocochlear potential. J Neurosci, 1997, 17:4711-4721
14 Aguilar-Bryan L, Clement JP, Gonzalez G, et al. Toward understanding the assembly and structure of KATP channels. Physiol Rev, 1998, 78:227-245
15 NishizukaY. Protein kinase C and lipid signaling for sustained cellular responses. FASEB J, 1995, 9:484-496
16 Hoffman J. The potential for isoemzyme-selective modulation of protein kinase C. FASEB J, 1997, 11:649-669
17 Zhu GY, Qui ZQ, Cui NR, et al. Suppression of Kir2.3 Activity by Protein Kinase C Phosphorylation of the Channel Protein at Threonine 53. J Biol Chem, 1999, 274(17): 11643-11646
18 Shi Y, Cui N, Shi WW, et al. A short motif in Kir6.1 consisting of 4 phosphorylation repeats underlies the vascular KATP channel inhibition by protein kinase C. J Biol Chem, 2008, 283:2488-2494
19 Mao JZ, Wang XR, Chen FC, et al. Molecular basis for the inhibition of G protein-coupled inward rectifier K+ channels by protein kinase C. PNAS, 2004, 101(4):1087-1092
20 McLaughlin S, Wang J, Gambhir A, et al. PIP2 and proteins: interactions, organization, and information flow. Annu Rev BioPhys Biomol Struct, 2002, 31:151-175
21 Xu C, Watras J, Loew LM. Kinetic analysis of receptor-activated phosphoinositide turnover. The Journel ofCell Biology, 2003, 161(4):779-791
22 Di Paolo G, De Camilli P. Phosphoinositides in cell regulation and membrane dynamics. Nature, 2006, 443: 651–657
23 Krau? M & Haucke V. Phosphoinositide-metabolizing enzymes at the interface between membrane traffic and cell signaling EMBO reports, 2007, 8(3):241–246
24 Zhang HL, He C, Yan XX, et al. Activation of inwardly rectifying K+ chanels by distinct PtdIns(4,5)P2 interactions. Nature Cell Biology, 1999, 1:183-188
25 Lopes C MB, Zhang H, Rohacs T, et al. Alterations in Conserved Kir Channel-PIP2 Interactions Underlie Channelopathies. Neuron, 2002, 34, 933–944
26 Quinn KV, Cui Y, Giblin JP, et al. Do Anionic phospholipids Serve as Cofactors or Second Messengers for the Regulation of Activity of Cloned ATP-Sensitive K+ Channels?Circ Res, 2003, 93:646-655
27 Thorneloe KS, Maruyama Y, Malcolm AT, et al. Protein kinase C modulation of recombinant ATP-sensitive K+ channels composed of Kir6.1 and/or Kir6.2 expressed with SUR2B. J. Physiol, 2002, 541;65-80
28 Zhang L, Lee JK, John SA, et al. Mechanosensitivity of GIRK Channels Is Mediated by Protein Kinase C-dependent Channel-Phosphatidylinositol 4,5-Bisphosphate Interaction. JBC, 2004, 279:7037-7047
29 John R Apgar. Activation of Protein Kinase C in RatBasophilic Leukemia Cells Stimulates Increased Production of Phosphatidylinositol 4-Phosphate and Phosphatidylinositol 4,5-Bisphosphate: Correlation with Actin Polymerization. Molecular Biology of the Cell, 1995, 6:97-108
30 Light PE, Bladen C, Winkfein RJ, et al. Molecular basis of protein kinase C-induced activation of ATP-sensitive potassium channels. PNAS, 2000, 97(16):9058-9063
31 Freesia L. Huang, Kuo-Ping Huang. Interaction of Protein Kinase C Isozymes with Phosphatidylinositol 4,5-Bisphosphate. J Biol Chem, 1991, 266(14):8727-8733
32 Sonia SB, Consuelo MV, Juan C GF, et al. The C2 Domain of PKCαIs a Ca2+-dependent PtdIns(4,5)P2 Sensing Domain: A New Insight into an Old Pathway. J Mol Biol, 2006, 362:901-914
33 Marta GV, Consuelo MV, Juan C GF, et al. The C2 Domains of Classical PKCs are Specific PtdIns(4,5)P2-sensing Domains with Different Affinities for Membrane Binding. J Mol Biol, 2007, 371:608-621
© 2004-2018 中国地质图书馆版权所有 京ICP备05064691号 京公网安备11010802017129号 地址：北京市海淀区学院路29号 邮编：100083 电话：办公室：(+86 10)66554848；文献借阅、咨询服务、科技查新：66554700 |