膜片钳讲课.ppt

单击此处编辑母版标题样式,单击此处编辑母版文本样式,第二级,第三级,第四级,第五级,*,膜片钳技术简介,丁仲如,C,E,K,M,G,K,Out,In,K,channel,Bilayer,Ion gradient,Channel,=capacitor,=battery,=conductance,Molecules vs.circuits,Structure of Na+Channel,Methods for studying ion channels,Functional,Current clamp,(record membrane potential),Voltage clamp,(record ionic currents),Macroscopic,2-electrode,Whole-cell patch clamp,Microscopic(single channels),Cell-attached,Inside-out,Outside-out,Gating current,Spectroscopy,Structural,Biochemistry,Molecular biology,Structural(x-ray,etc.,离子通道研究简史,1903,年,Einthoven,第一份体表心电图,1952,年,Hodgken,、,Huxley,电压钳技术 枪乌贼巨突轴动作电位,1963,年诺贝尔医学或生理学奖,1980,1981,年,Neher,、,sakeman,膜片钳技术,1991,年诺贝尔医学或生理学奖,电压钳技术（,voltage clamp),钳制细胞膜电位于指定水平（反馈电路）,改变电压敏感性离子通道的开放或关闭,欧姆定律：,I,m,g,V,m,膜电导（膜对离子通透性，,g,）,膜电位（,V,m,）膜电流（,I,m,),电压钳技术,2 ms,Stimulus,40,0,G,Na,G,K,cm,2,mS,Conductance,E,Na,E,K,40,0,-40,-80,V,rest,Voltage,mV,Action potential squid giant axon,电压钳技术,首次记录了动作电位及离子通道电流,Hodgken,Huxley,电压门控动力学模型,缺点：,双微电极，要求细胞大，细胞小不能进行,背景噪声大（,1000pA,）,无法记录单个离子通道（,1,2pA,）,Closed,Open,Blocked,Open channel block,C,O,OB,B,Closed,Open,Inactivated,Ball-and-chain model for inactivation,C,O,I,膜片钳技术,要求电极与细胞良好封接,减少电极尖端与细胞膜之间产生漏流，以减少背景噪音,措施：小膜片；电极双步拉制；尖端热抛光；,50M,欧姆,负压吸引：偶然发现，使阻抗达,10,9,欧姆（,giga,seal,）,Cytoplasm,Ion channels,Cell membrane,Giga-seal,Glass,microelectrode,Suction,1 m,Patch clamp recording,膜片钳技术,利用一个微电极同时完成膜片或全细胞电位的监测、钳制和膜电流的记录,通过膜电流的变化分析个体或群体离子通道分子的电活动,精度：电流,1pA,（,10,9,A,）,时间：,10mS,空间：,1,m,膜片钳系统组成,膜片钳负反馈放大器,Data/digital adapter,玻璃微电极操纵系统,Pclamp,软件系统：发放脉冲、采集数据、分析数据,accueil,techniques,culture,tranches,electrodes,equipement,scellement,Les,Appareils,Leur,fonctionnement,Pour en savoir plus,sur,les,amplis,oprationnels,accueil,techniques,culture,tranches,electrodes,equipement,scellement,Comment,obtenir,des,tranches,de,cerveau,Stimulated Cell Types5,13,14,Human Atrial Cardiomyocyte,Human Liver Fibroblasts,Human Ventricular Cardiomyocyte,Human Aortic Endothelial Cells,细胞准备：来源于动物,双酶酶解分离法（胶原酶蛋白酶）,心脏冰盐水主动脉逆行插管,langendoff,灌流装置台氏液,无钙台氏液,低钙胶原酶台氏液灌流,剪成,1mm,大小心肌碎片,蛋白酶孵化,过滤,冰箱保存备用（大约可成活,12,小时）,细胞准备：其它来源,来源于培养细胞：直接蛋白酶消化,来源于人心肌手术标本剪刀剪碎，蛋白酶胶原酶孵化,电极要求,尖端,1,2,m,（加热拉制）,电阻,2,4,欧姆（双步拉制）,尖端光滑（热抛光）,电极内充填（电极内液）细胞内液,尖端无气泡、灰尘,电极内有银丝与膜片钳系统相连接,电极由液压三维操纵器操纵（精确致,m,）,液压三维操纵器,accueil,techniques,culture,tranches,electrodes,equipement,scellement,Electrodes,accueil,techniques,culture,tranches,electrodes,equipement,scellement,全细胞记录示意图,enregistr,.,膜片钳记录四种模式,全细胞记录：,whole cell recording,单通道模式,cell attached,外面向外：,outside out,内面向外：,inside out,CELL-ATTACHED,Patch clamp configurations,WHOLE-CELL,Suction,Pull,OUTSIDE-OUT,Pull,INSIDE-OUT,Cell-attached patch-clamp recording,Whole-cell patch-clamp recording,单通道记录,记录单个离子通道开放或关闭,通道门控动力学分析,开放时间 概率,单通道电导测定,Corriente,elemental:2.69,pA,V=90 mV,单通道记录,KSJ2-F12.9,2.69,pA,V=130 mV,3.9,pA,Single Channel Patch Clamp,全细胞记录,动作电位,宏膜电流分析：实质为单通道电流的叠加,根据电压依赖性或特异性通道阻断药物区分不同的离子通道,电压敏感性离子通道,化学门控性通道,Action potential diversity,C,O,O,C,C,O,C,O,1,100,100,1,Voltage,Energy levels,Rate constants,Current through,single channels,10,ms,Current through,many channels,K,+,channel,Voltage,Current through,many channels,Current through,single channels,1 ms,I,O,C,C,I,O,C,O,I,20000,10,2,1000,C,O,I,10,1000,20,2000,Energy levels,Rate constants,Na,+,channel,O,C,Depolarization,K,+,channel,10 ms,Voltage,Conductance,I,O,C,Depolarization,Na,+,channel,1 ms,钾通道电流电压曲线,Current-voltage curves,-100,-50,50,100,-15,-10,-5,5,10,15,pA,-100,-50,0.5,100,0 mV,1.0,G/,G,max,50,p,i,K,Na,-100,-50,50,100,-15,-10,-5,5,10,15,nA,I=N p i,K,Na,化学门控通道,膜片钳技术进展,与分子生物学结合研究离子通道不同的亚基,与,PCR,技术结合研究单个细胞电生理及某种表达产物的,PCR,研究,与绿色荧光蛋白标记显像技术结合,活体组织直接行膜片钳研究,Ion,Name,Genes,Physiologically defined currents,K,+,Kv,27,Neuronal delayed rectifier;A-current,Eag,8,Cardiac fast delayed rectifier,KCNQ,5,M-current;cardiac slow delayed rectifier,SK,4,Low conductance Ca,2+,-dependent,Slo,3,High conductance Ca,2+,-and voltage-dependent,Kir,16,Inward rectifiers,G protein-activated,2P,15,Leakage(resting potential),Na,+,TTX-s,6,Neurons,adult skeletal muscle,glia,TTX-r,4,Cardiac muscle,some sensory neurons,Ca,2+,S,C,D,F,4,High voltage-activated(L-type),A,B,E,3,High voltage-activated(N,P/Q,R-types),G,H,I,3,Low voltage-activated(T-type),Cation,Pacemaker,4,Cardiac muscle,some neurons,CNG,6,Cyclic nucleotide-gated,Trp,17,Capacitative,Ca,2+,entry;capsaicin receptor,Molecular diversity of voltage-dependent ion channels,Ca,2+,channel diversity,Na,+,channel diversity,K,+,channel,diversity,谢谢！,