氯通道的电生理研究.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,*,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,第一部分,电生理研究基础理论,不同种类心肌细胞的生物电特性,不同层次心肌的动作电位,心外膜下心肌,心室壁中层,心内膜下心肌,跨室壁复极化离散,基 因,疾病,长,QT,综合征,SCN5A,KCNQ1,KCNJ2,通道类别,Na,+,通道,Na,+,通道,组织,/,器官,心 脏,骨 骼 肌,高血钾性周期性麻痹,低血钾性周期性麻痹,与人类遗传性疾病相关的离子通道,Cl,-,通道,先天性肌强直,神经系统,SCN4A,CLC1,Na,通道,SCN1A,癫痫,K,+,通道,K,+,通道,KCNQ2,Voltage Clamp,Na,+,电流记录,V,m,：,transmembrane potential,，,跨膜电位,Holding potential,，,保持电位,Clamping potential,，,钳制电位,Command potential,V,c,，,指令电位,基本名词,玻璃微电极技术,电子学技术,Patch Clamp,Inward current&outward current,内向电流：阳离子由细胞外向细胞膜内流动,外向电流：,K,+,外流，,Cl,-,内流,Slow delayed rectified potassium current,I,K1,I,-,V,curve,I-V curve(current-voltage relationship),Inward rectified current,（内向整流）,内向电导大于外向电导,Outward rectified current,（外向整流）,外向电导大于内向电导,反转电位（,reversal potential,E,rev,）,电流为,0,的电位,电流在此改变方向,I,s,=,g,s,(,E,-,E,s,),内向整流（,I,K1,）的生理意义,动作电位,3,期快速复极过程,维持心肌细胞的静息电位,心肌钠电流测定,心肌钠通道失活的研究,心肌阴离子（氯）通 道,Anion/chloride channel in heart,第二部分,电解质,细胞外液,细胞内液,阳离子,Na,+,140,10,K,+,5,150,Ca,2+,1.25,极低,阴离子,Cl,-,112,10 20,HCO,3,-,28,10,体液中主要电解质的含量（单位：,mEq/L,）,Anion Conductance of Cardiac Muscle,By Carmeliet,Hutter&Noble in 1961,Nernst,方程,R,气体常数,Z,离子价,T,绝对温度,F,Farady,常数,ZF,E,Cl,=,RT,In,Cl,-,i,Cl,o,=61,lg,15,112,=,-,65,-,40 mV,Cl,-,o,=61,lg,Cl,-,i,（,37,C,）,Current,Activation,pS,I-V(,Cl,o,=Cl,i,),Gene,I,Cl,PKA,AC-cAMP-PKA,7-13,Linear,CFTR,I,Cl,PKC,PKC,7-13,Linear,CFTR,I,Cl,ATP,ATP(P2 R),12,Linear,CFTR?,I,Cl,Ca,Ca,2+,i,2,Linear,ClCA1?,I,Cl,Vol,Cell swelling,30-60,Out Rec,ClC-3?,I,Cl,b,Basally active,30-60,Out Rec,ClC-3?,Out Rec,Outwardly rectifying,Properties of,functionally,identified,sarcolemmal Cl,-,channels in heart,Anion channel,Physiological roles of cardiac Cl-channels,1.Electrical activities of cardiac myocytes,2.Regulation of cell volume,3.Regulation of cardiac cation channels4.Pathophysiological significance of cardiac Cl,-,channels,Difficulties in studying chloride channel,1.The molecular basis of some Cl,-,channels,2.Gating of Cl,-,channels by anions,3.Nonselective effect of Cl,-,blockers,CFTR Cl,-,channel,结构与功能关系研究,第三部分,CFTR Cl,-,通道的发现,1980s Cl,-,conductance activated by cAMP,Cystic Fibrosis Transmembrane conductance Regulator,Outwardly Rectifying Chloride Channel,Epithelial Sodium Channel,Renal Outer Medullary Potassium Channel,ATP-Binding Cassette Protein,1989 Identification of gene encoding CFTR,CFTR function as both a regulator and a Cl,-,channel,CFTR,的拓扑结构,Transmembrane Regions(TMs),1,12,N,C,NBD1,NBD2,R Domain,ATP,ATP,PKA,(PKC),Out,In,NBD,结构域：,ATP,结合（,gating,）,R,结构域：磷酸化（,PKA,和,PKC,）,孔道区,CFTR,的功能结构域,Single channel&Macroscopic Current Recording,How does this make a channel?,How does Cl get through?,?,研究手段,-DNA,重组技术与膜片钳技术的结合,pIRES/CFTR,WT DNA,（,extracted from E.Coli,）,Mutagenesis(PCR),Transfect(BHK cells),Patch clamp(compare WT and mutant),WT,R303Q/A/,E,/,K,WT,K95Q,etc,Patch Clamp Recording of CFTR Channel Currents,Inward rectification,Outward rectification,K95Q,Wild,Macroscopic Current Recording(Cl,-,o,=Cl,-,i,=154 mM),R334E,非极性中性氨基酸：,丙氨酸,（,A,）,等,极性中性氨基酸：,谷氨酰胺,（,Q,）、,半胱氨酸,（,C,）,等,碱性氨基酸：,赖氨酸,（,K,）,、精氨酸,（,R,）,、组氨酸,（,H,）,酸性氨基酸：,谷氨酸,（,E,）,、天门冬氨酸,（,D,）,氨基酸分类,CFTR,一级结构模式图,不同位点突变后对氯电流的影响,突变为中性,或 酸性氨基酸残基后,I,Cl,CFTR,整流特性的变化,改变,Cl,-,浓度对,I,Cl,CFTR,整流特性的变化,突变为 酸性氨基酸残基后对,I,Cl,CFTR,单通道电流的影响,突变为中性,或 酸性氨基酸残基后单通道,I-V,curve,的变化,不同位点突变后对,I,Cl,CFTR,整流特性的影响,采用MTSET,或,MTSES,孵育细胞,改变单通道,I-V,curve,电极内液加入,MTSET,或,MTSES,改变单通道,I-V,curve,CFTR,通道的孔道结构模式图,CFTR Cl,-,channel,药物作用机制研究,第四部分,Molecular Structure,Inhibition of CFTR Cl,-,currents by intracellular suramin,Cl,-,o,=4 mM,Cl,-,i,=154 mM,-0 mv,-100 mV,Suramin inhibition is independent of extracellular Cl,-,Cl,o=154 mM,Cl,-,o=4 mM,Out,In,+,K95,+,R303,NPPB,Cl,-,Cl,-,Extracellular Cl,-,Electrical repulsion?,Effects of intracellular suramin on CFTR single-channel currents,Flickering,Suramin,6,M,Voltage,-30 mV,Suramin inhibition of CFTR channel mutants,Wild,K95Q,R303Q,Effect of channel mutnts on the affinity of suramin inhibition,Mean,K,d,value was estaminated at-100 mV,Relative roles of pore-forming positively charged amino acids on channel inhibition by intracellular glibenclamide,mutant,wild,Relative effect of removal of positive charges in the pore on the inhibitory effects of different channel blockers,Blocker,K95Q,R303Q,Suramin,0,+,Glibenclamide,+,+,DNDS,+,+,Lonidamine,+,0,NPPB,+,0,TLCS,+,0,Proposed arrangement of blocker binding sites,in the CFTR pore,Small drug,Big drug,Summary,2.,分子生物学与膜片钳技术的结合是研究离子通道生物物理学特性的重要手段,3.,膜片钳技术仍然是当前离子通道药物筛选的重要工具,4.,现阶段对氯通道的分子结构已有一定认识，但其生理功能仍不十分清楚,5.,氯通道的工具药物虽然很多，但仍没有选择性强的抑制剂,1.,离子通道功能异常是多种疾病发生、发展的重要原因,参考资料,1.,心血管生理学与临床,，,P,515-519,，,P,527-543,2.Duan D(2009).,Phenomics of cardiac chloride channels:the systematic study of chloride channel function in the heart.,J Physiol 587:2163-2177.,