３． 神经细胞的分离
运用膜片钳技术进行电生理学研究需要制备合适的单个细胞作标本，细胞制备的好坏直接影响实验的成功率。膜片钳实验要求细胞标本具有呼吸活性、耐钙、细胞膜完整、平滑、清洁度高的条件，以利于微电极与细胞膜进行高阻封接。活性好的细胞在形成全细胞模式后可以保持活性很长时间，足以保证实验的顺利进行。因此制备好的细胞标本是膜片钳实验的关键第一步。
七十年代以来，出现了许多分离各类细胞的分离技术，但是进行电生理学研究尤其是膜片钳实验多应用酶解分离细胞的方法。我们实验室曾分离过豚鼠心室肌细胞、大鼠肝脏细胞、大鼠脑皮层神经细胞、家兔肺动脉平滑肌细胞和人脑皮层神经细胞及人心房肌细胞。
这里重点介绍大鼠脑皮层神经细胞的分离技术。
(1) 用30mg/kg 戊巴比妥钠ip 麻醉后，断头开颅取出大脑半球放入冷的人工脑脊液中，轻轻剥离脑膜和血管等纤维组织，然后取脑皮层在人工脑脊液中剪成2mm×2mm 的组织块静止１小时，并通以氧气。
(2) 将脑组织块放入含有protease 16unit/ml ( type Ⅹ, sigma )和protease 2unit/ml (type ⅩⅣ, sigma)的人工脑脊液中，在36℃恒温震荡(60 次/min)水浴中孵育60 分钟左右。
(3) 将组织块取出，反复用人工脑脊液冲洗５次，以彻底清除消化酶，于室温下静止60 分钟并继续通氧，实验前将组织块轻柔吹打后即可分离出单一的神经细胞供实验使用。
膜片钳技术在神经药理方面的应用（5）
４． 千兆欧姆封接
取一滴细胞液，滴入浴槽中，用人工脑脊液进行灌流，将浮游的死细胞冲走，待细胞贴壁后即可进行封接吸引。通过PCLAMP 软件或电子刺激器，给予一个20mV，10~50ms的矩形波刺激，当电极进入浴槽溶液时，记录电流的直线变成与矩形波电压脉冲相对应的矩形波曲线，将电极尖轻轻压在细胞膜表面，此时电流曲线的高度变低，给电极以负压吸引，由于电极尖与细胞膜逐渐密接，细胞膜与电极间的电阻逐渐增加，电流曲线逐渐减小
直至变成一条直线，则形成了千兆欧姆封接。
膜片钳技术在神经药理方面的应用（6）
5．记录模式
根据研究目的选择记录模式，主要有下面叙述的前４种，后３种是依据前４种变更而来的。
(1) 细胞贴附式 (cell-attached 或on-cell mode)：千兆欧姆封接后的状态即为细胞贴附式模式，是在细胞内成分保持不变的情况下研究离子通道的活动，进行单通道电流记录。即使改变细胞外液对电极膜片也没有影响。
(2) 膜内面向外式 (inside-out mode)：在细胞贴附式状态下将电极向上提，电极尖端的膜片被撕下与细胞分离，形成细胞膜内面向外模式。此时膜片内面直接接触浴槽液，灌流液成分的改变则相当于细胞内液的改变。可进行单通道电流记录。此模式下细胞质容易渗漏(washout)，影响通道电流的变化，如Ca2+ 通道的run-down 现象。
(3) 全细胞式 (whole-cell mode) 记录： 在细胞贴附式状态下增加负压吸引或者给予电压脉冲刺激 (zapping)，使电极尖端膜片在管口内破裂，即形成全细胞记录模式。此时电极内液与细胞内液相通成为和细胞内电极记录同样的状态，不仅能记录一个整体细胞产生的电活动，并且通过电极进行膜电位固定，也可记录到全细胞膜离子电流。这种方式可研究直径小于20μm 以下的小细胞的电活动；也可在电流钳制 (current clamp)下测定细胞内电位。目前将这种方法形成的全细胞式记录称作常规全细胞模式 (conventional whole-cell
mode 或hole cell mode)。
(4) 膜外面向外式 (outside-out mode)：在全细胞模式状态下将电极向上提，使电极尖端的膜片与细胞分离后又粘合在一起，此时膜内面对电极内液，膜外接触的是灌流液。可在改变细胞外液的情况下记录单通道电流。
(5) 开放细胞贴附膜内面向外式 (open cell-attached inside-out mode)： 在细胞贴附式状态下，用机械方法将电极膜片以外的细胞膜破坏，从这个破坏孔调控细胞内液并在细胞贴附式状态下进行单通道电流记录。用这种方法时，细胞越大，破坏孔越小，距电极膜片越远，细胞因子的流出越慢。
穿孔膜片式 (perforated patch mode) 或缓慢全细胞式 (slow whole-cell mode)：在全细胞式记录时由于电极液与细胞内液相通，胞内可动小分子能从细胞内渗漏到电极液中。为克服此缺点，可在膜片电极内注入制霉菌素 (nystatin) 或二性霉素Ｂ (amphotericin 使电极膜片形成多数导电性小孔，进行全细胞膜电流记录，故被称为穿孔膜片式或制霉菌素膜片式 (nystatin-patch mode)。又因胞质渗漏极慢，局部串联阻抗较常规全细胞记录模式高，钳制速度慢，故也称为缓慢全细胞式。
(7) 穿孔囊泡膜外面向外式 (perforated vesicle outside-out mode)：在穿孔膜片式基础上，将电极向上提，使电极尖端的膜片与细胞分离后又粘合在一起形成一个膜囊泡。如果条件很好，在囊泡内可保留细胞质和线粒体等，能在比较接近正常的细胞内信号转导和代谢的条件下进行单通道记录。
6. 细胞内灌流方法：细胞内灌流是在全细胞式状态下利用电极内灌流法形成的。电极内灌流法的装置是由电极固定部、灌流液槽、注入管、流出管、电极记录用琼脂桥所组成。注入管是用直径2.5 mm 的塑料管经加热拉细制成，使其尖端能插到接近电极的尖顶部。灌流液槽注满实验用溶液，插入注入管，当千兆封接形成后，由于负压吸引的作用电极内液从流出管流入排液槽的同时，实验用溶液由流入管注入到电极内，电极充满实验用溶液
后关闭注入管，完成了液体的交换。
这种方法应用在“内面向外式”时，可同时改变细胞内液和细胞外液的组成；应用在“全细胞式”时就形成了细胞内灌流方法，直接改变了细胞内液。
膜片钳技术在神经药理方面的应用（7）
7. 全细胞记录模式离子通道电流记录
(1) 钠通道电流 (INa)： 灌流液即细胞外液同人工脑脊液液，也可加入CoCl2 3 mmol/L或nifidipine 10μmol/L 以阻断钙电流。电极液成分(mmol/L)：CsCl 150, EGTA 11, CaCl2 1,MgCl2 1, HEPES 10, 用CsOH 调pH 至7.4。
电压钳制方案，通常设保持电位 (holding potential) 为-80 mV，去极化电压为 -10~+40mV，步阶电压10 mV，去极化的保持时间（刺激脉冲宽度或钳制时间）10~40 ms。当全细胞记录方式形成后，利用上述电压钳制方案，即可记录出INa。根据实验数据制作电流-电压 (current-voltage, I-V) 关系曲线，从中找到Na+ 电流的激活电位、反转电位和最大电流的电压区域。
(2) 钙通道电流 (ICa)：灌流液有两种方案，一是人工脑脊液中加入TTX 10μmol/L，另一是将人工脑脊液中的NaCl 换成N-methyl-D-glucamine 130μmol/L, pH 用CsOH 调至7.4。电极液的组成(mmol/L)：Aspartic acid 60, CsOH 60, MgCl2 4, HEPES 10, EGTA 10, Na2ATP 3。用CsOH 调pH 至7.4。通常使用的电压钳制方案是设保持电位为-40 mV，去极化电压为10~110 mV，步阶电压10 mV, 钳制时间300 ms，此方案记录的是L 型Ca2+ 通道电流；但在此保持电位下记录到的Ca2+ 电流尚含有Na+ 通道电流或尚有T 型Ca2+ 通道电流。记录由于没有特异的T 型Ca2+ 通道阻滞剂，若想获得较纯净的L 型Ca2+ 通道电流，将保持电位抬高到-30 mV 即可。记录T 型Ca2+ 通道电流的电压钳制方案是设保持电位为-80 mV，仍以10 mV 的步阶电压去极，去极化电压为10~170 mV，应用L 型Ca2+ 通道阻滞剂，如：nitrendipine、nisodipine、nifedipine 等，即可得到较纯净的T 型Ca2+ 通道电流。两种方案得出的膜电流峰值，均可绘制I-V 曲线。
(3) 钾通道电流：神经细胞上亦存在多种K+ 通道，其研究也很复杂，通常最直观最容易观察和记录得到的K+ 通道电流不外乎几种。这里仅就延迟整流通道、内向整流通道及瞬间外向电流通道的电流记录加以介绍。
基本液体 灌流液的组成 (mmol/L)： N-methyl-D-glucamine 135, KCl 5.4, CaCl 1.8,
MgCl2 0.5, HEPES 10, Glucose 5.5。用HCl 调pH 至7.4。也可在灌流液中加入TTX
(10-6mol/L)和Cd+ (0.2~0.5mmol/L)，以阻断Na+ 和Ca2+ 通道。电极溶液为通常的细胞内液。
① 延迟外向整流电流 (delayed rectifier outward current, Ikr)：设保持电位为 –80 mV，去极化电压为 -20~+170 mV，步阶电压10 mV, 钳制时间可这在100~400 ms，时间间隔为2~3ms 以上。有时可将保持电位设在 –30 mV 或 –40 mV，这样不仅可以使T 型Ca2+ 通道失活，记录出Ikr，而且也可以同时记录到尾电流 (Itail)，尾电流也是延迟外向整流电流的一种表现形式，当钳制方波从 +70 mV 或 +90 mV 复极到保持电位时，这个电流并不紧随，而是延迟于复极的钳制方波，以指数衰减方式，逐渐回至电流基线。现认为Itail 和Ikr 使用
同一通道。Ikr 值的表示，通常是测定钳制方波就要结束时的外向电流幅值；Itail 的测定是在钳制初期上升的幅值。
② 内向整流电流 (inward rectifier current, Ikir)： 在膜超极化时内向整流通道开放，K＋流入细胞内，当膜电位近于静息电位或更正时，该通道趋于关闭。一般情况下保持电位的设置与细胞的静息电位相当，设在 –80 mV，在这个电位下，膜电位为“零”，保持电位是“零”电流电位。然后令钳制电位从正于保持电位的方向，向超极化方向复极，超极化可达-140 mV 到 –160 mV，过度超极化可能会损伤细胞，步阶电压仍为10 mV
在神经细胞，Ikir 于钳制初期可表现出一个瞬间内向电流，很快衰减，之后趋于平衡，形成时间不依赖性或称为持续性电流。测量电流幅度是测瞬间电流峰值和持续性电流峰值。分别绘制其I-V 曲线，再做分析。
③ 瞬间外向电流 (transient outward current, IA 或Ito)： 用于记录Ito 的电压钳制方案与延迟整流电流的方案基本一样，通常将保持电位设在 –80 mV，但这种电压钳制方案在记录到的Ito 中，一定混有Ikir。目前可用两种方法将其分开。第一，设置两个电压钳制方案，
即：第一个方案中的保持电位为 –80 mV，钳制电位为 +50 mV 或更高，时间为80~100ms，目的在于最大程度地记录到Ito。第二，如用改变保持电位的方法仍不能分开Ito 和Ikir，则可用某些阻断剂 (如E-4031、TEA 等) 阻断Ikir，然后利用方案一记录Ito。然而，实际上要得到较纯净的Ito 是相当不容易的，这其中包括：在某些细胞Ito 和Ikir 对膜电位的依赖性太接近，以及到目前为止尚未有十分特异的Ikir 阻断剂。由于TEA 这类阻断剂的特异性差，
所有应用时要格外小心，应使用特异性较好的阻断剂。在K+ 通道的研究中，也可应用斜坡 (ramp) 钳制方案。其基本要点是膜电位斜坡除极
的速度不要太快。通常将膜电位从 –110 mV 斜坡除极到 +70 mV 或更高，旨在使这一钳制方案覆盖整个生理电位活动范围。在神经细胞，斜坡钳制所得到的K+ 电流包含几种类型K+ 通道电流，其中有Ikr、Ito 、Ikir 等，也就是记录到的应是一条多类型的K+ 电流组成的电流轨迹。不同类型的K+ 通道阻滞剂可以分别阻断这一电流轨迹的不同部分。实验者可在上述原则基础上设计适用于不同K+ 通道研究的斜坡钳制方案。
膜片钳技术在神经药理方面的应用（8）
8. 单通道电流记录
(1) 钠通道电流 (INa)： 用“细胞贴附式”膜片时，细胞外液为人工脑脊液，电极液为无钙的人工脑脊液 (Na+ 140mmol/L) 保持电位与去极化电压的设置同全细胞记录方式，施加50ms 的去极化脉冲，可记录到单通道INa。为便于观察，使通道开闭的速度变慢，实验需在较低的温度(22~24℃)下进行。INa 表现为内向（向下）的矩形波状的变化。将保持电位从静息电位钳制到 –130 mV，处于超极化状态下，给予10 mV 步阶电压的去极化脉冲，钠通道开放数逐渐增多，可得到一个近似于用全细胞模式记录出现的INa 波形。
(2) 钙通道电流 (ICa)： 为准确控制膜电位，在记录Ca2+ 电流时，应将灌流液换成高钾 (K+ 浓度130mmol/L) 溶液，此时细胞静息电位约为0 mV，通过保持电位的设置可以较准确的控制细胞膜电位；也可用人工脑脊液灌流，但此时细胞的静息电位约为 –80 mV，设置保持电位时应考虑到这一点。电极液与全细胞记录时应用的细胞外液类似。电压条件与全细胞记录相同。
用细胞贴附式或膜内面向外式记录单通道ICa 时，常用的高钾灌流液的组成（mmol/L）：
K-aspartate 90，KCI 30， KH2PO4 10，EGTA 1，MgCI2 0.5，CaCI2 0.5。用KOH 调pH 至7.4。电极液组成 (mmol/L)：BaCl2 50, Choline Cl 或TEA Cl 70, HEPES 10, EGTA 0.5。用CsOH 调pH 至7.4。
应注意的是，并不是每次去极化都能使钙通道开放，常常见到钙通道全部不开的情况(blank trace)，肾上腺素能神经激动剂、钙通道激动剂如Bay K 8644 能延长通道开放时间。
(3) 钾通道电流： 细胞K+ 在正常生理浓度时，钾通道的电导很小，K+ 电流也非常小，测定很困难，因此在记录钾通道电流时，应将电极内（膜片外）液的K+ 浓度增加到140~150mmol/L。电压条件与全细胞模式记录时相同。
单通道电流记录的主要观察指标包括：单通道电导 (conductance)，开放概率 (open probability)，平均开放时间 (mean open time)，平均关闭时间 (mean close time)。一般说来，单通道记录和分析均较全细胞电流的记录和分析的难度大且更复杂．
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膜片钳 膜片钳简介
膜片钳发展与原理
膜片钳原理图
膜片钳三种形式
膜片钳三种形式关系图
通道的概述 离子通道的分类
离子通道的本质
配体基门控通道简单示意
膜片钳对乙酰胆碱通道的研究 通道亚型1
通道亚型2
通道亚型3
通道的结构与功能
电流记录
膜片钳对电压门控通道的研究 钠离子通道的研究
一般通道特性
离子通道闸控机制1
通道闸控机制2
膜片钳对细胞信号传导的研究 膜片钳的用途1
膜片钳的用途2
研究细胞内钙的变化1
研究细胞内钙的变化2
膜片钳实验所需的仪器 实验室全貌
拉控器
工作台
计算机
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膜片钳操作
The Practice of Patch Clamping
In this Chapter the actual procedure of making a gigaseal on a cell
membrane and establishing the desired configuration is discussed. The
purpose of this is to indicate good practice. Practitioners might find that
this Chapter helps to increase the success rate, facilitates trouble-shooting
and reduces having to ‘reinvent the wheel’. Novices hopefully will be saved
much time. The method used in this Chapter is to provide a step-by-step
account of how to establish a patch clamp configuration, using the
background information outlined in the previous Chapters. Electrophysiology
is one of the disciplines within biology that allow many instant
quality controls; these will be indicated where appropriate.
4 The Practice of Patch Clamping 95
4.1 Preparing the Experiment and Making a Seal 95
4.1.1 Setting up 95
4.1.2 Bringing the pipette near the preparation 98
4.1.3 Making the seal 101
4.2 Whole-cell Modes 104
4.2.1 Conventional whole-cell recording 104
4.2.2 Perforated patch recording 108
4.3 Single-channel Modes 110
4.3.1 General notes 110
4.3.2 Cell-attached patch 112
4.3.3 Excised patches 113
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Although patch clamp set-ups range from a simple rig to the most
elaborate patch clamp arrangements in which a large number of variables
are carefully controlled, there is a basic set of conditions that must be met
in all cases for patch clamping to work. These conditions will be discussed
in this Chapter. In brief, because patch clamping involves the placement of
a glass micropipette onto a cell to form a tight seal, the basic elements of a
set-up are
• a platform with minimal mechanical interference;
• a microscope for visualisation of the preparation;
• manipulators to position the micropipette;
• electronics to perform stimulation, recording and analysis in an
electrically clean environment.
These elements will be discussed in turn below.
3 Requirements 43
3.1 The Platform 43
3.1.1 Stability: vibrations and drift 43
3.1.2 Where in the building should the set-up be placed? 44
3.1.3 Anti-vibration tables 45
3.2 Mechanics and Optics 47
3.2.1 The microscope 48
3.2.2 Micromanipulators 52
3.2.3 Pipette pressure 56
3.2.4 Baths and superfusion systems 57
3.3 Electrodes and Micropipettes 64
3.3.1 Solid–liquid junction potentials and polarisation 65
3.3.2 The bath electrode 67
3.3.3 Micropipettes 67
3.3.4 Liquid junction potentials 74
3.4 Electronics 75
3.4.1 External noise and Faraday cages 76
3.4.2 Patch clamp amplifiers 81
3.4.3 Noise prevention and signal conditioning 84
3.4.4 Data acquisition and digitisation 90
3.4.5 Computers and software 93
先贴一页大家可以看看。
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于近日将分页上传刘振伟编的实用膜片钳技术一书中的某些重要章节
膜片钳记录技术
膜片钳记录技术.pdf (30.2k)
海马脑片盲法膜片钳全细胞记录技术
海马脑片盲法膜片钳全细胞记录技术.pdf (216.19k)
实验原因 要学习讨论膜片钳技术 还望大侠多多指教。
也希望自己早日进入高层。
提供几个网站吧 不知是否已有了 如重复请删帖
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图片2.png (28.23k)
The patch clamp consists of an electrode inside a glass pipette. The pipette, which contains a salt solution resembling the fluid normally found within the cell, is lowered to the cell membrane where a tight seal is formed. When a little suction is applied to the pipette, the "patch" of membrane within the pipette ruptures, permitting access to the whole cell. The electrode, which is connected to specialized circuitry, can then be used to measure the currents passing through the ion channels of the cell. Furthermore, we can use our electrical circuitry to "clamp" the membrane potential to any voltage that we desire: very handy when measuring the activity of voltage-dependent channels.
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这个网站是从刚才那个ppt里弄出来得
看起来很不错啊 很多演示movie
patch clamp technique (flash movie, 825 kb)
3D-movie combined with navigation elements
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METHODS BASED ON ELECTRONICS AND PHARMACOLOGY
Some Background: The galvanometer was invented around 1820 by Schweigger, and was a necessary instrument for detecting currents in Galvanii's famous twitching frog leg experiment. Without a galvanometer, no fundamental understanding into the mechanisms of electrical phenomena in animal tissue could ever have been uncovered. The invention also coincided with the discovery by Faraday of electric and magnetic forces being tied together. "...although it could not detect bioelectrical currents, it led the way to a succession of contrivances for the detection and recording of them, invented over the next century and a half, and ranging from the astatic galvanometer, to the reflecting-mirror galvanometer, the capillary electrometer, the string galvanometer, the cathode-ray oscilloscope, and eventually to the sophisticated apparatus used today." from the book entitled Nineteenth-Century Origins of Neuroscience Concepts by Clarke. Nobili in 1825 devised an ingenious instrument in which he made use of the astatic needle that had been invented in 1820 by Ampere. It neutralized, or greatly reduced the background effects of terrestrial magnetism, and with the addition of his own modifications, he constructed an improved Schweigger astatic galvanometer that provided a sensitivity denied earlier workers. (note: "although early multipliers, or galvanoscopes, could detect the presence of a current, they still could not measure it" as they were too insensitive for quantitation) Nobili's instrument was still too insensitive to register the presence of the brief and minute electrical signal produced in nerve conduction. But later he would detect the presence in the frog of an electric current in 1827. Matteucci used the "rheoscopic frog" to detect currents from the membranes of injured muscle cells. This current detector consisted of a frog's leg in a jar. The nerve in the leg acted as a simple readout device. In the period of approximately 150 years, from 1800s to 1970s, electrophysiologists would progress from these methods to devices so sensitive, that it is now possible to routinely detect the tiny current emanating from a single ion channel of a cell.
Voltage-Clamp: Invented in the late 1940s by Kenneth Cole, voltage clamping involves placing a second glass electrode inside the cell in order to "voltage clamp" the interior of the cell. Voltage clamping made it possible for the first time to keep constant the membrane potential on the interior of the cell even during the sodium influx during the action potential. Researchers from that point on could distinguish the voltage effects caused by influx of sodium or efflux of potassium from changes those made deliberately by the experimenter. Voltage clamp essentially means to control the potential across a cell membrane. Typically, a current is applied and changes in the cell membrane voltage potential are recorded. Applied current flows locally across the cm both as ionic and capacity current. A voltage is applied and the current is measured. The measure of ionic current is the ionic movements thru ion channels. Most methods use two intracellular electrodes. One to record voltage and the other to send the current. It is important to measure cell membrane potential changes at the cell membrane itself and not just at the electrodes. It wasn't until voltage clamping was invented that a quantitative measurement of ionic currents was possible.
Voltage clamping is often needed due to the capacitance-caused current (the cell membrane can be thought of as a capacitor). This can happen whenever a change in voltage occurs across a membrane. I(total) = I(ion channels) + Cm(dV/dt) When doing voltage clamping, (which was invented by Marmont and Cole and more fully developed by Huxley and Hodgkin in 1952) a current is injected which is equal in amplitude but opposite in sign to that which flows across cell membrane. Therefore, there is no NET current across a cell membrane and the membrane potential is therefore kept constant. By measuring the current that has to be injected to clamp potential, one also measures current flowing across cell membrane.
Patch Clamp: A very versatile and powerful technique which can be used to study individual ion channel activity in an isolated patch of cell membrane. The invention of the patch clamp in the late 1970s allowed for the first time, the underlying kinetics of ion channels to be measured. Patch clamping is undoubtedly the most important technique we have to study ion channels, and is often used in conjunction with other techniques such as mutagenesis in order to determine what effects these changes made in an ion channel have on its activity. From these types of studies, structural and functional detail can be obtained about ion channels.
Procedure: The cell membrane is first clamped so the voltage potential across it isn't changed. The amount of electric current needed to maintain the cell membrane at a constant voltage is used as a measure to quantify the movement of ions thru the ion channel, or patch. Patch clamping requires filtering of random high-frequency noise. This noise arises primarily from charging currents inherent to the voltage clamping of a membrane. Patch clamp has 3 main configurations: 1) inside out (pipette simply touches outside of membrane), 2) whole cell (where membrane is broken. Allows "summed" activity of all channels to be measured), 3) outside out, where pipette is pulled out (as in 2) and outside of cm side is now outside patch clamp. 4) like 1 but membrane is permeabilized by antibiotics). An important difference between patch clamp and the two-electrode voltage clamp method is that patch clamp uses a single electrode both to control membrane potential and to measure current. Another difference is that the patch clamp amplifier is highly sensitive and is able to resolve the tiny currents (pA) flowing thru single channels.
With a patch clamp, one can also measure 1) synaptic transmission between neurons in brain, 2) monitor change in cell membrane area during secretion. Note: there are two critical noise sources: seal and current-to-voltage amplifier. Patch clamp is now sensitive enough to measure capacitance change due to a single vesicle fusion event. Note also that single-channel recording (i.e. patch clamping) gives only 2 basic pieces of information: 1) current amplitude, and 2) dwell times. One of the drawbacks of using patch clamp when looking for new channels is that it will bias the discovery process towards higher conducting channels, while lower ones (less than 1 pS) will be underrepresented. The frequency response of most electronics is ~10 microseconds, however the actual transition (opening and closing) of ion channel gating is less than that, and will therefore remain invisible to these techniques.
Voltage and Patch clamp facts, etc:
Differing results have been obtained by various groups studying CFTR depending on the exact type of equipment or the cells used for protein expression. Standardization is very important when using these techniques. Whether or not an open channel is able to pass ions may depend on such factors as the absolute voltage, the concentrations of the ions, and the type of ions present on each side. This means that agreement on basis for comparison is important.
When using artificial bilayers and purified ion channels, it is important to remember that these bilayers are usually more noisy. However, with artificial bilayers, one can vary more details during the experiment, for example: lipid type, toxins, etc.
In theory, the two most direct ways to investigate ion channels is to record ion current that flows directly thru them (patch-clamping) or to measure a change in membrane potential that ion flow makes (voltage-clamping).
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Channel
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膜通道有离子通道和水通道，前者又包括钙通道（电压依赖性、对钙离子选择性通过的细胞膜糖蛋白）、钠通道、钾通道、氯通道。其中钙通道又分为L、T、N、P、Q、和R型。前两者存在于心血管系统和中枢神经系统，后四者存在于神经元组织中。美国的两位科学家因为在离子通道和水通道结构方面的杰出成就获得了2003年诺贝尔化学奖，关于膜通道的研究和发现对于我们在分子水平理解离子和水的转运机制至关重要，进而对于理解基本生物过程和相关疾病的分子基础、以及将来可能的治疗方法的结构基础都有重要意义。为此CMBI特准备了Channel的特别报道，供研究参考，更多文献请用相关关键词通过CMBI文献查询获取。
Syntaxin 1A Regulates ENaC Channel Activity
Physicochemical Features of the hERG Channel Drug Binding Site
Molecular Determinants Responsible for Differential Cellular Distribution of G Protein-gated Inwardly Rectifying K+ Channels
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膜片钳记录技术
膜片钳记录技术.pdf (30.2k)
噪声来源及解决在膜片钳实验中的作用
膜片钳实验将电生理实验推向了分子水平,其记录的信号是皮安级的通道电流,因此,排除噪声在膜片钳实验中极其重要。结合工作实践,分析膜片钳实验中噪声的原因及种类,介绍常用的解决措施。
噪声来源及解决在膜片钳实验中的作用.pdf (68.35k)
膜片钳技术简介
简要介绍１９９１年诺贝尔生理学和医学奖获得者ＥｒｗｉｎＮｅｈｅｒ和ＢｅｒｔｔＳａｋｗａｎｎ所发明的膜片钳技术及其在研究胰腺腺泡细胞外分泌机制中的应用。
膜片钳技术简介.pdf (173.91k)
