暨南大学高级生理学研究生课程讲义离子通道20091030

离子通道(ion channel),第一节 离子通道概述 一、离子通道的分类 二、离子通道的结构 三、离子通道的特点 四、通道的激活 五、影响离子转运速率的因素 六、通道转运的特点 七、 K、Na+、Ca2+、 Cl-通道第二节 离子通道研究方法与技术 一、膜片钳技术 二、通道药理学 三、通道蛋白分离、通道重建 四、通道蛋白表达水平的检测 五、基因结构 六、射线晶体成像技术第三节 离子通道的生理功能第四节 离子通道与疾病第五节 离子通道的应用及展望,The Structure of the Cell Membrane,Transport of Substances Through the Cell Membrane,Simple diffusionMembrane protein mediated transportEndocytosis and exocytosis,Simple diffusion,Membrane Protein Mediated transport,Facilitated diffusion via CarrierFacilitated diffusion via ChannelPrimary active transportSecondary active transport,Secondary active transport,Endocytosis and exocytosis(need energy from ATP),Endocytosis Phagocytosis; PinocytosisExocytosis,第一节 离子通道概述,概念： 离子通道是细胞膜上的一类特殊的蛋白质，可以选择性让离子进行跨膜转运。,离子通道的发现,1890年，德国物理化学家 WilhelmOstwald首先提出，生物体内的电信号是离子进出细胞膜而产生的。 1909年诺贝尔化学奖 20世纪20年代，科学家证实存在一些供离子出入的细胞膜通道。1950年代初，两位英国科学家AlanHodgkin和 AndrewHuxley用枪乌鰂大神经轴突的电压钳等技术发现，钾离子和钠离子进出神经细胞的细胞膜，从而让神经信号传递下去。 他们因此获得1963年诺贝尔生理学或医学奖。但那时科学家未知离子通道结构和工作原理。,电压钳技术记录膜电流,内向电流,外向电流,0mV,20多年前，德国科学家萨克曼（Bert Sakman）和内尔（Erwin Neher）用膜片钳技术发现了细胞膜上单离子通道，发现当离子通过细胞膜上的离子通道的时候，产生十分微弱的电流。他们因此重大成就而获1991年诺贝尔生理学奖。,获2003年诺贝尔化学奖的两位科学家是美国人阿格雷（PeterAgre）和麦金农（RoderickMacKinnon），他们的发现都涉及到了“细胞膜上的通道”。1998年罗德里克麦金农利用射线晶体成像技术获得了世界第一张离子通道的高清晰度照片，绘制出了世界上第一张离子通道（钾离子通道蛋白质）的三维结构图。第一次从原子层次揭示了离子通道的工作原理。这张照片上的离子通道取自青链霉菌。麦金农的方法是革命性的，它可以让科学家观测离子在进入离子通道前在通道中，以及穿过通道后的状态。 1980年代中期，阿格雷发现了镶嵌在细胞膜上的一种专门供水分子进出的蛋白质。这种蛋白质就是30多年来科学家一直寻找的“水通道”水分子进出细胞的通道。,钾离子通道蛋白质的三维结构图,一、分类,门控: 控制通道功能状态的通道蛋白分子内部“闸门”样结构的运动过程,根据门控机制的不同而分: 电压门控通道(voltage-gated) 化学门控通道(ligand-gated) 机械性通道 (stress-activated)根据离子选择性的不同而分为：钠通道钾通道钙通道氯通道,1 电压门控性通道(voltage gated),-由膜两侧电位差而控制开启和关闭的通道。膜内负电荷减少 Na+通道开放 胞外Na +进入胞内以最容易通过的离子命名： K+、Na+、Ca 2+、Cl-通道4种主要类型。,2 化学门控通道 (chemical gated),由化学信号控制开启和关闭的通道。又称配体门控性通道(ligand-gated)由化学物质与通道蛋白质受体分子上的结合位点结合而开启。 递质受体以递质受体命名，如 乙酰胆碱受体通道 谷氨酸受体通道 门冬氨酸受体通道. 非选择性阳离子通道 (non-selective cation channels)是由配体作用于相应受体而开放，同时允许Na+、Ca2+ 或K+ 通过。,3 机械门控性通道(mechanically-gated),是一类感受细胞膜表面应力变化，实现胞外机械信号向胞内转导的通道。,二、通道的结构（一）电压门控通道,电压门控钠、钾、钙通道有相似结构:a b 亚单位, a 亚单位是形成孔道的单位；a亚单位含- 共4个结构域;每个结构域含6个跨膜 a螺旋. S1-S6.,结构域和之间有使通道失活的结构,跨膜a螺旋: S1-S6S5 和 S6共同形成孔道，决定通道的离子选择性和通透性,S4是电压传感器，与通道的激活机制有关（带正电的aa) 结构域和之间有使通道失活的结构,三、离子通道的特点,1、离子选择性 选择性让某种或某些离子通过通道 对离子的选择性取决于：通道、离子的直径离子的种类孔道上带电的氨基酸Ach 阳离子通道口径0.65nm,水合K+直径0.396nm, 水合 Na+直径0.512nm, K+和Na+都可通过通道,孔道外口和内口有带负电的氨基酸,故阴离子不能通过.,2、门控性 通道开放或关闭取决于：膜电位（电压门控） 化学信号（化学门控） 机械刺激 （机械门控）,离子通道不同的功能状态：,静息 关闭，可激活激活 开放失活 关闭，不可激活,Characteristics of the voltage-gated sodium (top) and potassium (bottom) channels, showing successive activation and inactivation of the sodium channels and delayed activation of the potassium channels when the membrane potential is changed from the normal resting negative value to a positive value.,四 通道的激活1 跨膜电位差的改变,AP传来, 神经未梢膜去极化，电压门控Ca2 +通道开放，Ca2 +内流， Ach释放,2 化学信号分子(递质或第二信使)与特异结合位点（受体）结合,受体本身即是通道 例如在终板膜上：Ach 与终板膜Ach受体(化学门控通道)结合；Ach受体阳离子通道开放（受体构型改变）；钠内流为主，少量钾外流， 产生终板电位；后者扩布使邻近肌膜去极化达阈电位，引发AP。,五 影响离子转运速率的因素,膜两侧离子浓度差和电位差 正变膜通道的密度 正变膜通道开放的数量 正变离子的性质和通道的选择性和通透性,六 通道转运的特点,1. 顺电-化学差扩散 （RP和AP的产生）离子转运的方向:顺化学梯度 (从高浓度向低浓度侧) 细胞外(mmol/L) 细胞内 (mmol/L) Na+ 145 12 K+ 4 155顺电梯度2. 不直接耗能需要Na+-K+泵建立和维持细胞膜内外的离子的电化学梯度,细胞的静息电位 (Resting Potential),静息电位：细胞未受刺激时膜两侧的电位差。RP -10-100mV,安静时膜对钾的通透性较大; 钾顺浓度差外流, 促使钾外流的钾浓度势能差 = 阻碍钾外流的电势能差 （钾外流导致的外正内负）RP相当于Ek。,Nernst公式,内,外,动作电位(action potential),膜对Na+通透性增大，Na+内流 （膜外高Na+ ，膜内负电）引起AP上升支,钠内流动力: 电驱动力 膜内负膜外正 化学驱动力 膜外高钠钠内流阻力 钠内流形成的膜内正外负的电场力,Na+-K+泵建立和维持细胞膜内外Na+和K+的化学梯度 泵出3 Na+ ，泵入2 K+,七、K、Na+、Ca2+、 Cl-通道,（一） K通道,分二大类：内向优势K通道 (inward rectifiers K (ir) ） 电压门控K通道 （voltage-gated K (v)）,内向优势K通道 ： a voltage driving a current into the cell produces a larger current than an identical voltage driving current out of the cell 时间依赖性失活 电压依从性 电导 29pS in 145 mM K+, 3.6 pS in normal K+ 高度 K+ 选择性 存在于骨骼肌细胞、上皮细胞,钾离子通道作用:,细胞的生物电的产生调节神经递质释放调节心率胰岛素分泌、神经细胞分泌、骨骼肌收缩细胞容积调节K通道阻断剂：内向优势K通道 Cs、Ba2+ 电压门控性K通道 TEA,电压依赖性钾电流（Ikr）,临床应用： 抗心律失常药-钾通道阻滞药（延长动作电位时程药物）： 主要抑制Ikr钾电流，延长心肌细胞动作电位时程，降低自律性，延长有效不应期。,（二）Na+通道,Na+通道是细胞产生动作电位的关键性通道。Na+通道阻断剂：河豚毒 （tetrodotoxin，TTX) 普鲁卡因,电压依赖性钠电流,Na+通道阻断剂临床应用：抗心律失常药 (作用于心肌细胞）局部麻醉药和抗癫痫药(作用于神经细胞,阻抑AP传导和产生）,Composed of , -1 and -2 subunits, but, as with the calcium channel, the large -subunits carries most of the functional properties. E.g. when this subunit is expressed in Xenopus oocytes, a voltage-gated, toxin sensitive Na+ current is produced. The -subunit is structurally similar to Ca2+ channels (see above) with longer intracellular linking loops but a shorter C-terminal. Within the -subunit a motif of 300-400 amino acids is repeated 4 times, each repeated motif contains 6 predicted transmembrane domains, each of the 4 repeated motifs has an S4 domain containing positively charged residues that acts as the voltage sensor.The alpha subunit has the voltage sensing S4 region in each of the 4 repeats. View structure. The intracellular loops have phosphorylation sites (P), and the extracellular loops have glycosylation sites (Y) and saxitoxin (ScTx) and tetrodotoxin (TTX) binding sites. The P-segments (a.k.a. H5 loops) form the lining of the pore. Voltage-gated sodium channels are responsible for the current underlying the rapid upstroke of the nerve and muscle action potential. They are very voltage-sensitive - at the resting potential the open probability of Na+ channels is very low. Depolarization can increase open probability dramatically - Na+ enters causing further depolarization, opening more Na+ channels,细胞外 Ca 2+浓度 (1-2mM)显著高于细胞内 Ca 2+浓度 (10-7M) 。Ca 2+ 作为第二信使激活许多生理过程，与递质释放,细胞分泌，收缩，运动等功能密切相关。,（三）Ca2通道,Calcium channels are composed of several subunits. Ten 1 subunits have been cloned. Voltage-dependent calcium channels exist as a complex of subunits including 1, 2, , , and . These subunits control the structure and activity of the 1 subunit. 1 has most of the functional properties. The 1 subunit is structurally similar to Na+ channels (see below) - with shorter intracellular linking loops but a longer C-terminal. Within the 1 subunit a motif of 300-400 amino acids is repeated 4 times, each repeated motif contains 6 predicted transmembrane domains, each of the 4 repeated motifs has an S4 domain containing positively charged residues that acts as the voltage sensor. P-segments from each repeat line the pore.,At least 6 types of Ca2+ current have been identified: N, L, T, P，Q and R N-type: Activation : strong depolarization, inactivation: slow location : presynaptic nerve terminals, Major contribution in cortical neurons function: involved in “classical” transmitter release (e.g. ACh, NAdr). Blocker: CgTx (omega conus toxin)P-type: Activation: strong depolarization, inactivation : very slow. Blocker: Ftx (funnel web spider toxin). Insensitive to dihydropyridines. Now usually referred to as P/Q-type T-type: Activation: small (20mV) depolarizations, Inactivation: rapid (T = transient). location :Widely distributed in excitable and non-excitable cells. Function: Support pacemaker activity and, in conjunction with IK,Ca, allow oscillations of membrane potential. Regulate intracellular Ca2+ concentration. Blocker: Ni2+.,L-type: Location: all excitable and many non-excitable cells.Blocker: dihydropyridines and verapamil. Insensitive to CgTx. function: transmitter release (peptide transmitters) in endocrine cells; contraction in heart and smooth muscle (E-C coupling). Q-type: Activation: strong depolarizationinactivating: FastLocation: cerebellar granule cells, hippocampal pyramidal neurones, heart, pancreas, pituitaryBlocker: -AgaIVA spider toxin Function: transmitter release, excitation-secretion coupling in pancreatic. R-type: Location: neurones (brain), heart, testes, pituitary.Function: transmitter release.Blocker: CgTx MVllC.,高电压激活的钙通道（high voltage activated，HVA) 包括L- , P/Q-, N-, and R-型 通道激活需要约50mV以上的去极化。低电压激活的钙通道（Low voltage activated，LVA) 指T-型通道 通道激活仅需要约20mV的去极化.,Ca 2+ 作为第二信使激活许多生理过程，与递质释放,细胞分泌，收缩等功能密切相关。,钙通道抑制剂的临床应用： 治疗冠心病，心律不齐，高血压,Action potentials of Cardiac Muscles and Smooth muscles,1. Ca2 channels (sodium-calcium channels) responsible for the depolarization of action potentials in sinus node cells of the heart and in smooth muscle cells.responsible for the formation of AP plateau in ventricular and atrial cells. responsible for the autorhythmicity. 2. Na+ channels: responsible for the depolarization of action potentials in atrial and ventrical cells of the heart 3. K+ channels: responsible for the depolarization of action potentials,The action potential and ionic flows in a ventriclar myocardial cell,The action potential and ionic flows in a sinus node cell,钙参与形成(临床应用基础):,心肌细胞收缩心室肌细胞动作电位平台期钙内流窦房结细胞0期和4期去极化血管平滑肌0期去极化(平滑肌细胞紧张性),（四）Cl-通道,分类： CLC CFTR (cystic fibrosis transmembrane conductance regulator) calcium-activated Cl channel (CLCA-n) ligand-gated anion channels (GABAA and glycine receptors) Cl-通道调节物： MDR1, pICln, mat-8, phospholemman,Cl-通道参与膜电位的形成,细胞内PH的调节,容积调节,上皮细胞分泌等.,1、ClC family,ClC-0; torpedo electroplax; 10pS ClC-1; mammalian muscle; 1pS ClC-2; ubiquitous; 3-5pS and also ClC-K1 and ClC-K2 which are rat kindey-specific transcripts of ClC-2 ClC-3; volume-activated chloride channel (present in all cells?) - however recent ClC-3 -/- knock-out mouse still has volume regulation ClC-4; mammalian muscle, brain, liver, kidney, spleen; 140pS ClC-5; kidney ClC-6 and -7; present in most tissues but not functionally expressed as Cl- channels (intracellular organelle channels),Recently the structure of the CLC family of channels has been determined by X-ray crystallography of a bacterial version of the protein. There are 18 -helices of which 17 are in the membrane. The -helices lie at different angles to each other and to the membrane surface so they have a variety of different lengths.,容积激活性氯通道抑制剂：,NPPBTamoxifenATPDIDS,2 囊性纤维化跨膜转导因子(cystic fibrosis transmembrane conductance regulator，CFTR),分布：哺乳动物上皮细胞电导：6-10pS 功能：上皮细胞液体跨膜转运 其基因突变时导致囊性纤维化因 cAMP或细胞内的钙升高而激活 12-transmembrane domains, 2 NBF (nucleotide binding folds) and 1 R-domain (regulatory domain),3 钙激活的氯通道 （ICl,Ca and CaCC）,分布：腺细胞、上皮细胞作用： 与内分泌腺和外分泌腺分泌有关 与上皮细胞跨膜液体转运有关 影响神经元和肌细胞的兴奋性 卵母细胞受精,4 g -氨基丁酸促离子型受体 （g -aminobutyric acid, GABAA）,属配体门控通道，氨基丁酸激活GABAA受体，受体本身是氯通道分布于几乎所有神经元, 突触前和突触后,作用: 介导 CNS 中的快速突触抑制临床应用：抗惊厥剂，催眠药，肌松药，镇静药苯(并)二氮卓类（BENZODIAZEPINE）促进通道开放巴比妥酸盐 （Barbiturate）延长通道开放时间,5 甘氨酸受体（ Glycine receptors ）,甘氨酸激活甘氨酸受体 (受体本身是氯通道）主要分布于脊髓和脑干作用：引起抑制 -subunits contain the agonist and antagonist binding sites.拮抗剂：士的宁（Strychnine）,MDR1,MDR与耐药性有关（药物泵）。 Cl- 通道调节物。,第二节 离子通道研究方法与技术,综合应用各种技术：膜片钳技术通道蛋白分离、纯化等生化技术人工膜离子通道重建技术通道药物学基因重组技术射线晶体成像技术,一、膜片钳技术,膜片钳技术是用来测量离子通道跨膜电流和研究分子的非中性跨膜转运的技术。获1991年诺贝尔生理科学奖。膜片钳技术是将尖端直径仅1mm 的玻璃微电极接触细胞膜，施加负压使微电极与细胞膜之间形成10G以上的高阻抗封接，使电极尖端开口处相接的细胞膜的膜片与周围环境在电学上隔离，并通过外加命令电压钳制膜电位，对此膜片上的离子通道电流进行监测。,两种工作模式：,电压钳模式（voltage mode）： 对细胞进行电压钳制， 即利用电子学技术施加一跨膜电压并把膜电位固定于某一数值，从而实验性调节或控制膜片或全细胞电压，可以测定该膜电位条件下离子电流随时间变化的动态过程。研究电压依赖性离子通道。电流钳模式（current mode）： 通过电流钳制，改变跨膜离子流来监测细胞膜电位的变化，从而可以观察各种离子通道电流及其调控,膜片钳技术的应用：,利用药物或改变细胞内外的溶液成分，使其他离子通道失效，即可测定被研究的某种离子通道的功能性参量，分析离子电流的稳态和动力学与膜电位、离子浓度等之间的关系，可推断该种通道的电导、活化和失活速率、离子选择性等，并能测量和分析通道的门控电流的特性。膜片钳技术广泛应用于生命科学各个学科，尤其在递质、激素、药物作用机制研究、寻找药物最佳靶点、新药开发等方面更具有其独特优越性。,膜片钳方法,全细胞电流（容积激活性氯电流）,Whole-cell currents under isotonic and 47% hypotonic conditionsTypical current traces, representative of 30 cells, recorded under isotonic bath conditions and at the peak of the hypotonic response are showed in A and B. The protocol of voltage clamp was given in C. The voltage was held at 0 mV and then stepped to 0, 40, 80 mV with an interval of 4 sec between steps.,250 ms,2 nA,B. Hypo,A. Iso,C.,0,Time course of volume-activated whole-cell current The value of the whole-cell current taken 10 ms after the start of each voltage step (0, 40, 80 mV) is plotted as a function of time. Similar results were observed in 30 cells. Currents in response to all these voltage steps were small and stable when the cells were bathed in isotonic solution (indicated by the first Iso bar). The currents began to increase in 1-2 min after the application of a 47% hypotonic solution (indicated by the Hypo bar). The activated currents reached the peak and levelled off 3-5 min after the exposure to hypotonic solution and then recovered partially after returning to the isotonic solution (indicated by the second Iso bar). The residual currents were abolished by a 47% hypertonic solution (indicated by the Hyper bar).,单通道记录,用特制的玻璃微吸管吸附于细胞表面,使之形成10100G的密封(giga-seal)，被孤立的小膜片面积为m2量级,内中仅有少数离子通道。然后对该膜片实行电压钳位，可测量单个离子通道开放产生的pA（10-12安培）量级的电流，这种通道开放是一种随机过程。通过观测单个通道开放和关闭的电流变化，可直接得到各种离子通道开放的电流幅值分布、开放几率、开放寿命分布等功能参量，并分析它们与膜电位、离子浓度等之间的关系。还可把吸管吸附的膜片从细胞膜上分离出来，以膜的外侧向外或膜的内侧向外等方式进行实验研究。这种技术对小细胞的电压钳位、改变膜内外溶液成分以及施加药物都很方便。,二、通道药理学研究,通道拮抗剂、激动剂等 区分离子流 通道功能 钙通道阻断剂 钙电流变化 心肌细胞反应,药物影响通道的方式:,改变激活延缓或加快通道的失活阻抑离子流动,影响电压门控性钠通道的药物及毒素,1. 通道阻断剂 : 河豚毒 (tetrodotoxin，)与神经细胞，肌细胞有高亲合力结合位点在2. 持续的通道激活：蛙毒（batrachotoxin）3. 延缓通道的失活蝎毒素 a- scorpion toxins4. 改变通道失活的电压依赖性: 腰鞭毛虫毒素brevetoxin,Time course of NPPB inhibition of volume-activated chloride currents in CNE-2Z cellsThe whole-cell current, measured 10 ms after the start of each 200 ms voltage step (0, 40, 80 mV; 4s interval), was plotted against time. A 47% hypotonic solution was introduced into bath, as indicated by the Hypo bar. Once currents began to plateau, cells were perfused with 47% hypotonic solution containing 100 mM NPPB, as indicated by the NPPB bar.,三、通道蛋白分离、通道重建,利用与通道特异结合的毒剂标记，可把通道蛋白质从膜上分离下来，经过纯化，然后做通道蛋白质的序列测定。把人工合成或分离的通道蛋白质装在人工膜，观察通道功能。,20世纪80年代中期，美国科学家PeterAgre发现一种被称为水通道蛋白的细胞膜蛋白就是人们寻找已久的水通道。为了验证自己的发现， PeterAgre把含有水通道蛋白的细胞和去除了这种蛋白的细胞进行了对比试验，结果前者能够吸水，后者不能。为进一步验证， PeterAgre制造了两种人造细胞膜，一种含有水通道蛋白，一种则不含这种蛋白。他将这两种人造细胞膜分别做成泡状物，然后放在水中，结果第一种泡状物吸收了很多水而膨胀，第二种则没有变化。这些充分说明水通道蛋白具有吸收水分子的功能，就是水通道。,四、通道蛋白表达水平的检测,Western blot免疫荧光检测,五、研究编码离子通道的基因结构,基因定位克隆、聚合酶链反应、Northern 、 Western 、核酸杂交等分子生物学方法。,六、射线晶体成像技术,1998年，罗德里克麦金农利用射线晶体成像技术获得了世界第一张离子通道的高清晰度照片，并第一次从原子层次揭示了离子通道的工作原理。这张照片上的离子通道取自青链霉菌。麦金农的方法是革命性的，它可以让科学家观测离子在进入离子通道前的状态，在通道中的状态，以及穿过通道后的状态。,2003年诺贝尔化学奖获得者,荧光探针钙图像分析技术,荧光探针钙图像分析技术为检测细胞内游离钙离子浓度提供了有效手段，常用的荧光探针有Fura-2/AM、Indo-1/AM、Fluo-3/AM、Calcium Green等，常用的检测仪器有双波长显微荧光光度计、激光扫描共聚焦显微镜等.,第三节 离子通道的生理功能,容积调节细胞生物电细胞运动细胞收缩细胞分泌细胞周期细胞增殖细胞死亡,一、容积调节,（一）调节性容积回缩 (RVD)概念：当细胞处于低渗环境发生肿胀时，诱发细胞自身容积调节过程使细胞容积回缩， 趋向于肿胀之前的容积。机理： 主要通过激活K+和/或Cl-通道 ， K+和/或Cl-外流 KCl的同向转运体、K+/H+与Cl-/HCO3-交换和Na+/Ca 2+交换等。,Swelling-activated K+ and Cl- conductanceThe figure illustrates opening of independent K+ and Cl- channels induced by osmotic swelling and accompanying outflow of water. Efflux of K+, Cl- and water results in decrease of cell volume towards the normal level.,A. Iso,B. Hypo 3min,C. Hypo 20 min,20mM,Light micrographs of CNE-2Z cells following exposure to a hypotonic solution,Effects of Cl- channel blocker on RVD in CNE-2Z cellsA 47% hypotonic solution activated RVD (control). The RVD was inhibited by extracellular applications of Cl- channel blocker NPPB (100 mM). The blocker was added to the bath solutions 5 min before and during the hypotonic shocks (indicated by the bars). Data are mean standard error of at least 15 cells from 5 or more experiments.,（二）调节性容积增大 (RVI),概念：当细胞处于高渗环境发生皱缩时，诱发细胞自身容积调节过程使细胞容积增大， 趋向于皱缩之前的容积机理： 主要通过Na+-K+-2Cl-协同转运、Na+/H+交换和Cl-/HCO3-交换、Na+/K+ATP酶的激活以及Cl- 和K+通道的抑制,Shrinkage-activated Na+-K+-2Cl- cotransport The figure illustrates the activation of Na+-K+-2Cl- cotransport induced by cell shrinkage and the accompanying influx of water. The cell accumulates Na+, Cl- and K+. The uptake of Na+, Cl- and K+ and the influx of water result in cell volume increase towards the normal level. Na+-K+ pump extrudes the Na+ that enters during RVI and intakes K+, to maintain the ion gradient need for the cotransport and other cell functions, resulting in a gain of Cl- and K+ by the cell.,二、细胞生物电,Na+内流,安静时K+ 外流产生静息电位Na+通道开放， Na+内流，产生动作电位,三、细胞运动,细胞迁移受钙通道，钾通道，氯通道等影响鼻咽癌细胞迁移与氯通道开放有关。,氯离子通道阻断剂对CNE-2Z细胞迁移的抑制作用,四、细胞收缩,钙是兴奋收缩耦联的耦联因子。,五 细胞分泌,兴奋分泌耦联因子 (Ca 2+) 递质释放等Cl-，K+与液体分泌等有关,六 细胞周期,阻抑氯通道、钾通道使细胞周期停滞于细胞周期G1期.,Arrest of proliferating CNE-2Z cells in G0/G1 phase by NPPBFlow cytometric analysis was used to detect the distribution of cells in different cell cycle phases. Cells were plated at a density of 2.5 105 cells in 25 cm2 culture flask and incubated for 24 h. The cells were cultured in the medium without (control) or with 100 M NPPB for 2 days and then harvested for flow cytometric analysis. Data represent the means standard error of 4 experiments. Note the increase of cells at G0/G1 phase and the decrease of cells in S and M phases (P 0.01).,七 细胞增殖,氯通道阻抑剂抑制鼻咽癌细胞增殖,Effects of NPPB on cell proliferation in CNE-2Z cellsCells were plated at a density of 2500 cells /well in 96 well culture plates and cultured for 24 h. The cells were then cultured in the medium without (control) or with various co