ClC-3氯通道和IK1钾通道在细胞容积调节和迁移中的作用

发布时间：2018-07-11 13:04

本文选题：ClC-3氯通道 + IK1钾通道　；参考：《暨南大学》2012年博士论文

【摘要】：目的：1）研究ClC-3氯通道和IK1钾通道在人低分化鼻咽癌（CNE-2Z）细胞和高转移人肝癌（MHCC97-H）细胞基础容积稳定和调节性容积减小（regulatory volume decrease，RVD）中的作用；2）探讨RVD过程中Cl~-和K~+跨膜转运动力学特征，以及H+跨膜转运及pH值改变对钾、氯通道活动和RVD的调节；3）研究IK1钾通道在细胞迁移过程中的作用，探讨IK1钾通道在细胞容积调节和迁移过程中的空间和极性分布及活动的改变。方法：全细胞膜片钳技术记录CNE-2Z细胞和MHCC97-H细胞氯电流和钾电流；分别将ClC-3siRNA和IK1siRNA转染细胞，Western blotting检测ClC-3氯通道和IK1钾通道蛋白表达；Scion image图像记录分析软件检测细胞容积；非损伤微测技术记录细胞K~+、Cl~-、H~+和Ca~2+跨膜流动；H~+选择性微电极检测细胞外表面pH值，pH敏感性荧光探针BCECF检测细胞内pH值；构建绿色荧光蛋白（enhanced green fluorescent protein，EGFP)与IK1钾通道的融合蛋白表达质粒，红色荧光探针DiI标记细胞膜，激光共聚焦显微镜观察IK1钾通道空间分布及动态变化过程；趋化因子诱导细胞迁移；细胞划痕实验检测细胞迁移能力。结果：1）等渗条件下，，用全细胞膜片钳技术可以在CNE-2Z细胞中分别记录到稳定的背景氯电流和背景钾电流，细胞外47%高渗液可使细胞缩小，抑制背景氯电流和背景钾电流；用siRNA技术下调ClC-3氯通道或IK1钾通道表达，分别抑制背景氯电流和背景钾电流，并使等渗液中的细胞容积增大；嘌呤受体阻断剂RB2在正常或细胞外无Ca~2+条件下均可使细胞容积增大；细胞外加入嘌呤受体阻断剂RB2阻断P2Y受体、用ATP酶分解细胞外ATP或用GdCl3抑制ATP经通道的释放均可显著抑制背景氯电流，细胞外微摩尔浓度的ATP可以激活氯电流并且该电流可被47%高渗诱导的细胞缩小抑制，ATP可逆转GdCl3对背景氯电流的抑制作用。 2）非损伤微测结果表明，在不接触细胞、不干扰细胞活动的条件下，低渗刺激下CNE-2Z细胞中K~+、Cl~-和H~+均为外流，但它们的跨膜转运动力学特征不同，K~+外流激活较早且持续时间较短，Cl~-和H~+外流激活较晚且持续时间较长；低渗刺激引起细胞内pH降低，激活H~+外流，使细胞外pH降低，质子泵抑制剂奥美拉唑可抑制低渗引起的H+外流和细胞外pH降低；改变细胞外pH对低渗激活性钾、氯电流的影响不同，适度降低细胞外pH值抑制低渗引起的K~+外流和RVD过程，而对低渗引起的Cl~-外流无抑制作用，抑制质子泵或者增强细胞外pH缓冲能力可以促进低渗引起的K~+外流和RVD过程；膜片钳技术观察到的低渗激活性钾、氯电流时程相似，低渗激活的K~+、Cl~-外流可能分别由钾通道和氯通道介导，低渗激活的K~+、Cl~-外流和电流可分别被钾通道和氯通道阻断剂抑制；IK1钾通道和ClC-3氯通道可能分别是低渗激活的钾通道和氯通道的主要成份，IK1siRNA和ClC-3siRNA分别抑制低渗引起的K~+外流和Cl~-外流。 3）MHCC97-H细胞中存在稳定的背景钾电流和较明显的低渗激活性钾电流，且均可被钾通道阻断剂克霉唑（CLT）抑制；用siRNA技术下调IK1钾通道蛋白表达，可以抑制背景钾电流和低渗激活性钾电流，并且使等渗条件下细胞容积增大；EGFP-IK1钾通道融合蛋白表达质粒转染后细胞可表达绿色荧光，背景钾电流增大，且该电流可被钾通道阻断剂CLT抑制；等渗条件下EGFP-IK1钾通道在细胞中可呈不均匀分布，且IK1钾通道数量多的区域细胞局部容积减小较明显；低渗刺激诱导胞浆中EGFP-IK1钾通道呈囊泡样聚集，并以胞吐方式转移到细胞膜上，使细胞膜上EGFP-IK1钾通道增多；钾通道阻断剂CLT和IK1siRNA均可抑制细胞迁移；K~+、H~+和Ca~2+跨膜转运在迁移细胞前、后极间不同，前极K~+内流，后极K~+外流，前极H+外流大于后极，前极无明显Ca~2+跨膜流动，后极有明显Ca~2+内流；迁移细胞后极的K~+外流主要由通道活动介导，前极的K~+内流可能与通道无关，钾通道阻断剂CLT抑制后极的K~+外流，不影响前极的K~+内流；IK1钾通道在迁移细胞后极多于前极，且在诱导活细胞迁移过程中连续动态观察到IK1钾通道在细胞后极分布逐渐增多，并在前极皱褶处聚集，IK1钾通道在胞浆与胞膜之间的转移现象在迁移活细胞前、后极也不同，在后极从胞浆向胞膜转移，在前极从胞膜向胞浆转移。结论：ClC-3氯通道和IK1钾通道在细胞基础容积稳定和RVD过程中发挥重要作用。等渗条件下细胞自分泌释放的ATP通过嘌呤受体信号途径激活ClC-3氯通道，参与细胞生理条件下的基础容积调节。低渗刺激下，细胞RVD过程中H~+外流致细胞外pH降低，引起K~+、Cl~-外流的非同步。IK1钾通道参与MHCC97-H细胞迁移过程，迁移细胞前、后极K~+跨膜转运不同，呈前极内流、后极外流的极化状态。IK1钾通道通过在胞浆与细胞膜间的转位以及在细胞膜上的运动，使整体或局部细胞膜上通道蛋白的数量发生改变，从而对细胞容积和迁移过程进行调节。迁移细胞前后两极H~+和Ca~2+跨膜流动呈极化分布，可能使IK1钾通道活动在细胞前后极分布不同，从而影响细胞迁移过程。
[Abstract]:Objective: 1) to study the role of ClC-3 chloride channel and IK1 potassium channel in the volume stability and regulatory volume reduction (regulatory volume decrease, RVD) of human low differentiated nasopharyngeal carcinoma (CNE-2Z) cells and high metastatic human hepatoma (MHCC97-H) cells (regulatory volume decrease, RVD). (2) to explore the mechanical characteristics of Cl~- and K~+ trans membrane transmembrane movement in RVD process, and the transfer of H+ transmembrane and alteration value Change to potassium, chloride channel activity and regulation of RVD; 3) study the role of IK1 potassium channel during cell migration, and explore the spatial and polar distribution and activity changes of IK1 potassium channel during cell volume regulation and migration.
Methods: whole cell patch clamp technique was used to record the chlorine current and potassium current of CNE-2Z and MHCC97-H cells, ClC-3siRNA and IK1siRNA were transfected into cells, Western blotting was used to detect the expression of ClC-3 chloride channel and IK1 potassium channel protein, Scion image image recording analysis software was used to detect cell volume, and cell K~+ and Cl~-, H~+ and Ca~2+ were transmembrane flow; H~+ selective microelectrode was used to detect the pH value on the outer surface of the cell, and the pH sensitivity fluorescence probe BCECF was used to detect the intracellular pH value; the fusion protein expression plasmid of the green fluorescent protein (enhanced green fluorescent protein, EGFP) and the potassium channel was constructed, the red fluorescent probe labeled cell membrane, and the laser confocal microscope view The spatial distribution and dynamic process of IK1 potassium channel were observed; chemokines induced cell migration; cell scratch test was used to detect cell migration ability.
Results: 1) under the isosmotic condition, the stable background chlorine current and background potassium current can be recorded in CNE-2Z cells by whole cell patch clamp technique, and the 47% hypertonic liquid can reduce the cell, inhibit the background chlorine current and background potassium current, and reduce the expression of ClC-3 chloride channel or IK1 potassium channel with the technique of siRNA, and inhibit the background chlorine electricity respectively. The flow and background potassium current can increase the cell volume in the isosotic fluid, and the purinergic receptor blocker RB2 can increase the cell volume in normal or extracellular Ca~2+, and the addition of the purinergic receptor blocker RB2 to the P2Y receptor, the ATP enzyme decomposition of the extracellular ATP or the release of the ATP via the channel can be significantly inhibited by the ATP enzyme. ATP can activate the chlorine current and the current can be reduced by 47% hyperosmotic cells, and ATP can reverse the inhibitory effect of GdCl3 on the background chlorine current.
2) the results of noninvasive micromeasurement showed that K~+, Cl~- and H~+ were Exodus in CNE-2Z cells under hypotonic stimulation without contact cell and cell activity, but their transmembrane movement mechanics characteristics were different, K~+ Exodus activated early and duration was shorter, Cl~- and H~+ Exodus activated late and lasted longer; low permeability stimulation was caused by low permeability. PH decreased in cells, activated H~+ Exodus and reduced extracellular pH. Omeprazole, a proton pump inhibitor, inhibited H+ Exodus induced by hypotonic and extracellular pH; the effects of pH on hypotonic activation potassium, chlorine current were different, and the extracellular pH values were moderated to inhibit the K~+ Exodus and RVD process induced by low permeability, and Cl~- for hypotonic Cl~-. The external flow has no inhibitory effect. The inhibition of the proton pump or the enhancement of the pH buffer capacity can promote the K~+ Exodus and RVD processes caused by low permeability; the patch clamp technique has observed the hypotonic activation potassium, the chlorine current time history similar, the low permeability activated K~+, the Cl~- Exodus may be mediated by the potassium channel and the chloride channel, the low permeability activated K~+, the Cl~- Exodus and the electricity. The flow can be inhibited by potassium channel and chloride channel blocker respectively; IK1 potassium channel and ClC-3 chloride channel may be the main components of low permeability activated potassium channel and chlorine channel respectively. IK1siRNA and ClC-3siRNA inhibit the K~+ Exodus and Cl~- Exodus caused by hypotonic respectively.
3) there are stable background potassium current and obvious hypotonic activation potassium current in MHCC97-H cells, and can be inhibited by potassium channel blocker clotrimazole (CLT). Down regulation of IK1 potassium channel protein expression by siRNA technology can inhibit the background potassium current and hypotonic activated potassium current, and increase the cell volume under isosmotic conditions; EGFP-IK1 potassium. After transfection of the channel fusion protein expression plasmid, the cells could express green fluorescence, the background potassium current increased, and the current could be inhibited by potassium channel blocker CLT. Under isosmonic conditions, the EGFP-IK1 potassium channel could be distributed inhomogeneous in the cells, and the local volume of the cells in the region with more IK1 potassium channels decreased obviously, and the hypotonic stimulation induced the cytoplasm. The EGFP-IK1 potassium channel is vesicular aggregation and is transferred to the cell membrane by exocytosis, which increases the EGFP-IK1 potassium channel on the cell membrane, the potassium channel blocker CLT and IK1siRNA can inhibit cell migration, and K~+, H~+ and Ca~2+ transtransport before the migration of cells, the posterior polar K ~ + influx, the backward K~+ Exodus, and the exodus of the front pole H+ are larger than the posterior pole. There is no obvious Ca~2+ transmembrane flow in the front, and there is an obvious Ca~2+ inflow in the posterior pole; the K~+ exodus of the migratory cells is mainly mediated by channel activity, and the K~+ influx of the front pole may not be related to the channel. The potassium channel blocker CLT inhibits the K~+ exodus of the pole, and does not affect the K~+ inflow in the front pole; IK1 potassium channel is more than the anterior pole after the migration of the cells and is induced to live. In the process of cell migration, it was observed continuously that the IK1 potassium channel increased gradually in the post pole distribution, and gathered at the front polar fold. The transfer of IK1 potassium channel between the cytoplasm and the membrane was very different before the migration of the living cells, and transferred from the cytoplasm to the cytoplasm at the back pole and transferred from the membrane to the cytoplasm at the front pole.
Conclusion: ClC-3 chloride channel and IK1 potassium channel play an important role in cell base volume stability and RVD process. Under isosmotic conditions, the autocrine and released ATP activates the ClC-3 chloride channel through the purinergic receptor signaling pathway and participates in the basal volume regulation under the cell physiological conditions. Under the hypotonic stimulation, the H~+ Exodus induces the extracellular pH in the cell RVD process. The non synchronous.IK1 potassium channel of K~+, Cl~- exodus is involved in the migration process of MHCC97-H cells. Before the migration of the cells, the trans membrane transport of the K~+ transmembrane is different, and the transmembrane of the posterior polar exodus is in the forward flow. The polarization state of the posterior polar Exodus,.IK1 potassium channel, through the transposition between the cytoplasm and the cell membrane and the movement on the cell membrane, makes the whole or local cell membrane protein on the membrane. The number of the cell volume and the migration process are regulated. The polarization distribution of the H~+ and Ca~2+ transmembrane flow in the two poles of the migrated cells may lead to the different distribution of the IK1 potassium channel activity before and after the cell, thus affecting the cell migration process.
【学位授予单位】：暨南大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R739.63

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