膜片钳原理技术膜 片 钳 简 介膜 片 钳 记 录 方 式膜片钳几种基本工作方式Cell-attached recordingCellPatch-pipetteCell membraneExternal lipidInternal lipidWhole-cell recordingCellPatch-pipetteCell membraneInside-out recordingPatch-pipetteThe internal face of the lipid bi-layer faces the bath solutionOutside-out recordingPatch-pipetteThe external face of the lipid bi-layer faces the bath solution膜片钳实验步骤膜 片 钳 电 流 记 录膜 片 钳 的 应 用与细胞膜离子通道相关的药物作用药物 相关离子通道治疗糖尿病药物 钾离子通道 抗心律失常药 钠、钾钙通道 心功能调节及保护 钙、钾通道 平滑肌调节 钙、钾通道 影响神经冲动、传导 钠、钾、钙、氯通道 免疫细胞（功能）调节 钾、钙通道 降压药 钙通道-淀粉样肽对海马神经元钾通道的影响Fig . Effects of-AP25-35 3 mol.L-1 on the cultured rat hippocampal neurons . Cells viability was evaluated by the MTT reduction assay. Values are the mean SEM of three separate experiments performents in triplicate.Fig. Fig. Delayed Delayed rectiferingrectifering potassium potassium current current (I (IKK) ) in in cultured cultured rat rat hippocampalhippocampal neurons. neurons.（A A）I IKK recorded from normal neurons; recorded from normal neurons; （B B）I I KK recorded recorded from from neurons neurons after after exposed exposed to to 3 3 mol.Lmol.L-1-1 beta-beta-amyloidamyloid- - peptide peptide 25-3525-35 for 24h;for 24h; （C C）I IKK recorded recorded from from neurons neurons after after exposed exposed to to 10mol.L10mol.L-1-1 beta-beta-amyloidamyloid- - peptide peptide 25-3525-35 for 12h. for 12h. Kv2.1通道的稳定转染和高表达系统的建立 Fig. HEK293 cells were transiently transfected with pEGFP-N1 and pCR/CMV Kv2.1. Each of panels were captured by CCD under fluorescence microscopy to visualize GFP. (left 100X,right 400X) 2 nA100 msFig. Representative current of Kv2.1 channel in stable transfected HEK293 cell line. Wholecells currents evoked during 250ms depolarizing voltage steps 10mv to rest potentials between 50 and +40mV from a holding potential of 50mV.kv2.1Fig. Properties of steady state activation of Kv2.1 channel in stable transfected HEK293 cell. Wholecells currents evoked during 250ms depolarizing voltage steps to rest potentials between 50 and +40mV from a holding potential of 50mV.Fig. Effects of 10 M galantamine on I-V relationship (B) and the activation curve (C) of IK(DR) and IK(A). In (B), each point represents mean SEM of 7 cells for IK(DR) and 6 cells for IK(A). *P 0.05, *P 0.01 vs control. In (C), galantamine caused a 4 mV hyperpolarizing shift of the activation curve of IK(DR) (n = 9), and no effect on that of IK(A) (n = 6). Fig. Effect of 10 M rivastigmine on the voltage dependence of inactivation of IK(A) and IK(DR). (A) The original current traces before and after the addition of 10 M rivastigmine.(B) Rivastigmine caused both hyperpolarizing shifts of the steady-state inactivation of IK(A) and IK(DR) by 11 mV and 27 mV, respectively. n = 5.Ca2+3Na+Na+Na+ Cell model to record outward current of NaCell model to record outward current of Na+ +-Ca-Ca2+2+exchangerexchanger膜片钳全细胞记录膜片钳全细胞记录双向离子条件双向离子条件高高NaNa+ +电极内液电极内液Na/CaNa/Ca双向/生电性正 向 转 运SARCOLEMMAATPATPCa2+Na+Ca2+Ca2+ channelCa2+ATPATPCa2+Sarcoplasmic reticulumNCXNCXSARCOLEMMA2 K+3 Na3 Na+ +Na+H+3 Na+NCXCa2+ATPATPNCXNCXATPH+心肌缺血/再灌SARCOLEMMA2 K+3 Na3 Na+ +Na+H+3 Na+NCXCa2+Ca2+ overload心肌缺血诱发反向转运ATPNCXNCXNa+正常Na+-Ca2+交换电流在豚鼠心室肌细胞上的记录+60 mV-40 mV-100 mVA: current before Ni2+ 5 mM B: current after Ni2+ 5 mM A-B: Na+-Ca2+ exchange current (INa-Ca).5 mM Ni2+胞外Ca2+浓度为1.8 mM电极内液中Na+浓度为25 mM 5 mM Ni2+3 min左右达到最大持续5 min 未见“衰减”(run-down)现象细胞内Na+浓度对Na+-Ca2+交换电流的作用Current densities (pA/pF)（Journal of Cardiovascular Pharmacology 2002）Amiloride对豚鼠心室肌细胞Na+-Ca2+交换电流的作用Current densities (pA/pF)AmilorideCurrent densities (pA/pF)KB-R7943KB-R7943对豚鼠心室肌细胞Na+-Ca2+交换电流的作用（Journal of Cardiovascular PharmacologySZA对豚鼠心室肌细胞Na+-Ca2+交换电流的作用SZACurrent densities (pA/pF)膜片钳放大器的工作原理膜片钳基本电路原理膜片钳基本电路原理细胞等效电路基本电生理测量-伏安放大器n电压检测n电流检测电压钳电压钳单电极电压钳单电极膜片钳膜片钳实验装置膜片钳实验系统配置单细胞膜片钳实验配置1, 膜片钳放大器 (Patch clamp amplifier) 2, 显微操作仪（Micromanipulator） 3, 微电极拉制仪（Pipette Puller） 4, 打磨抛光仪（Microforge or Beveler） 5, 灌流系统（Perfusion System） 6, 细胞灌流槽（Chamber) 6, 显微镜（Microscope） 7, 摄像头和监视器（Camera , Monitor） 8, 防震台和静电屏蔽网（Antivibration and Cage） 9, 电脑和打印机 （Computer, Printer） 脑片/组织切片 膜片钳配置1, 显微镜移动台（X-Y Translation System） 2, 恒温槽 （Slicer Chamber） 3, 脑片/组织 切片机（Vibroslice） 4, IR 摄像头和显示器（IR Camera，Monitor） 5, 双道灌流泵（Pump)各种微电极放大器n膜片钳放大器(0.1pA- 20nA)n电压钳放大器(100pA- 100nA)n膜片钳/卵母细胞电压钳放大器防震台