膜片箝技術(shù)基本原理

1、The Principal of Patch Clamp Technique膜片箝技術(shù)基本原理 神經(jīng)生物學(xué)系 陸 巍 實(shí)驗(yàn)室主頁：1ContentIntroductionThe equivalent circuits of membrane & patch recordingsDifferent configurationsConfigurations to be usedCase study23 What is patch-clamp?Patch-clamping is an electrophysiological technique in which we are able to CLA

2、MP the VOLTAGE of an isolated piece of cell membrane (or whole-cell). By clamping the voltage we are able to observe CURRENTS that flow through ION CHANNELS. It is the current that the patch-clamp amplifier supplies to hold the voltage steady (clamped) that we measure. Patch-clamp recording allows u

3、s to measure very small currents - in the pA range (10-12 A).4細(xì)胞膜5細(xì)胞膜液態(tài)的脂質(zhì)雙層中鑲嵌著球形蛋白骨架是磷脂雙分子層。蛋白質(zhì)分子以附著、鑲嵌、貫穿的形式存在于磷脂雙分子層上67The patch-clamp circuitPatch of cell membrane with ion channelFBR_+AmplifierTechnicalThe high gain operational amplifier isconnected in the circuit so that the currentflowing th

4、rough the ion channel is measuredas a voltage drop across the feedback resistor(FBR). The FBR has a resistance of 50 Gallowing very small currents (10-12 A)to be measured.8 膜片箝原理示意圖9 Patch clampers win Nobel Prize Sakmann, Neher et al revolutionized the field of electrophysiology in 1981 with their

5、paper “Improved patch-clamp techniques for high resolution current recording from cells and cell-free membrane patches” (Pflgers Arch. 391, 85-100). With patch-clamp recording we can observe the movement of single molecules (strictly macromolecular complexes) in real time. In 1991, Neher and Sakmann

6、 were rewarded for their pioneering efforts in patch-clamp recording when they jointly won the Nobel Prize in Physiology or Medicine.10Patch clamping Hamill, O. P., Marty, A., Neher, E., Sakmann, B. and Sigworth, F. J. (1981) Improvedpatch-clamp techniques for high resolution current recording from

7、cells and cell freemembrane patches. Pflugers Arch. 391: 85100. 1991-Nobel Prize Winners in Physiology & Medicine: Drs. Neher & Sakmann Development of Patch Clamp (1976)11121314151617Its all to down to suction!By increasing the seal resistance we reduce the noise Level and increase temporal resoluti

8、on.From Hamill et al 19811819Patch-clamp configurationsFrom Hamill et al 198120Cell-attached recordingCellPatch-pipetteCell membraneExternal lipidInternal lipid21 Cell-attached recordingThe simplest patch clamp configuration (in terms of physical manipulation) is cell-attached patch mode. Every patc

9、h clamp experiment starts with this situation. The micropipette, unlike intracellular recording, is positioned against the cell membrane where the glass makes a very strong connection, resulting in a tight (high resistance) seal. Ion channel activity in the tiny patch of membrane surrounded by the t

10、ip of the pipette can now be studied, as we shall see in the next section. The cell-attached patch mode is therefore a single-channel configuration.22 Cell-attached recording23 Cell-attached recordingThis configuration leaves the cell intact, and is therefore the most physiological configuration to

11、study single channels and the simplest to obtain. However, it does not allow easy manipulation of the media on either side of the membrane, and control over the potential over the patch (voltage clamp) has uncertainties because the membrane potential, which cannot be measured directly in this config

12、uration, is still intact.24The equivalent circuit for the cell-attached patch configuration25The equivalent circuit for the cell-attached patch configurationThe resistors present in this circuit differ from the intracellular recording situation only in that the pipette resistance is relatively low,

13、but the resistance of the patch of membrane is very high. We have seen that the highest resistance in a series circuit determines the current flow, therefore if the patch resistance Rpatch is high compared with the resistance of the rest of the cell (Rm) and the pipette resistance (Rpipette), then t

14、he circuit effectively monitors current flow through the patch and any ion channels in it.26The equivalent circuit for the cell-attached patch configuration There is one parallel resistor in the circuit, with the potential of shortcircuiting, i.e. draining away current. Leak resistance Rleak represe

15、nts the quality of the seal between the glass of the micropipette and the membrane. If the seal is good, then Rleak is very high and no significant current will leak away.27Voltage clamp in the cell-attached patch configuration.To obtain the actual potential over the patch, the command voltage (hold

16、ing potential, HP) must be subtracted from the cell membrane potential28Whole-cell recordingCellPatch-pipetteCell membrane29 Whole-cell recording If the patch of membrane under the pipette tip in cell-attached patch mode is ruptured, then the pipette solution and the electrode make direct electrical

17、 contact with the cytoplasm. The situation is very similar (but not quite identical) to intracellular recording: the patch electrode, electrically, is on one side of the plasma membrane and the ground electrode is on the other, therefore the membrane potential can be recorded directly.30 Whole-cell

18、recording A patch pipette tip is sufficiently wide to allow washout of the cytoplasm by the pipette-filling solution. Because the volume of the cell is negligible compared with that of the patch pipette, the composition of the intracellular fluid can be considered equal to that of the pipette-fillin

19、g solution. If a physiological situation is desired, then the pipette is filled with a solution of ionic composition that resembles the cytoplasm as closely as possible.31 Whole-cell recording Washout can be a blessing or a curse: the experimenter can manipulate the intracellular milieu, but unknown

20、 cytosolic factors relevant to the subject of study can be unwittingly washed out. To avoid the latter, intracellular recording was used, but in many cases it is now possible to apply perforated patch clamp, where electrical contact with the cytosol is established by adding a membrane-perforating ag

21、ent to the pipette solution. The agent (nystatin or amphoteracin B) perforates the membrane so that only small molecules such as ions can pass through, leaving the cytoplasms organic composition largely intact.32The equivalent circuit for the whole-cell configuration33 The equivalent circuit for the

22、 whole-cell configuration The series circuit consists of the pipette resistance Rpipette, the access resistance Raccess and the membrane resistance Rm. The latter is the largest (current-limiting) resistor, so this configuration allows the observation of currents through Rm. Because these currents a

23、re the sum of currents through all activated single channels of the cell, they are named whole-cell currents or macro-currents. Parallel to the circuit is the leak resistance Rleak , which should be as high as possible to minimize short-circuiting of the membrane current.34The equivalent circuit for

24、 the whole-cell configuration The membrane capacitance plays an important role in whole-cell recording, mainly because it affects the voltage clamp time characteristics. Any change in holding potential will be delayed because Raccess and Rpipette in series with Cm form a significant RC circuit. The

25、sum of Raccess and Rpipette is sometimes referred to as series resistance.35Inside-out recordingPatch-pipetteThe internal face of the lipidbi-layer faces the bath solution36 Inside-out recordingThere are two single-channel configurations that do away with the cell altogether by excising a patch of m

26、embrane from the cell and studying it in isolation. This provides the experimenter with ultimate control over the environment of the patch and any ion channels in it, both electrically and chemically. Outside and inside refer here to the extracellular and intracellular side of the membrane, respecti

27、vely, and out refers to the bath.37 Inside-out recordingThe inside-out excised patch is obtained from a cell-attached patch configuration, where the pipette is pulled away. The result is now a vesicle attached to the pipette tip. The vesicle can be destroyed by exposure to air, i.e. the pipette is b

28、riefly lifted above the bath. This leaves a patch with the cytosolic side facing the bath.3839 Inside-out recordingInside-out patches are ideal for studying the effects of cytosolic factors on channels. It is clear that inside-out patches are much harder to work with than outside-out patches. To obt

29、ain them involves an extra step (destruction of the vesicle), and the bath solution must be replaced with intracellular solution in each experiment.40 Outside-out recordingPatch-pipetteThe external face of the lipidbi-layer faces the bath solution41 Outside-out recordingThe outside-out patch is obta

30、ined by simply pulling away the patch pipette from a whole-cell configuration. The membrane will eventually break and, owing to the properties of the phospholipids, fold back on itself into a patch covering the pipette. Note that for a physiological situation the pipette solution should resemble the

31、 intracellular ionic environment because it is facing the intracellular side of the membrane.4243 Outside-out recordingOutside-out patches can be used to study the effects of extracellular factors on the channels, because the bath composition can be altered easily during recording.44The equivalent c

32、ircuit for the excised patch configurations45The equivalent circuit for the excised patch configurations The most important resistor, as in the cell-attached patch configuration,is the patch resistance Rpatch. The circuit is meant to monitor current through this resistor. The parallel leak resistor

33、Rleak should be very high to minimise short-circuiting of the patch current. Although the pipette resistance Rpipette should be as low as possible, in practice the primary consideration regarding the pipette tip size is the amount of membrane that the experimenter wishes to catch. The pipette capaci

34、tance Cpipette can be significant within the range of amplification and time resolution required for single-channel recording and must be compensated.46The equivalent circuit for the excised patch configurationsGreat care should be taken in applying sign conventions correctly, because these are the

35、reverse of the two excised patch configurations and easily confused: the holding potential as indicated on a patch clamp amplifier indicates the potential of the patch electrode with reference to ground (the bath). In the case of an outside-out excised patch, where the pipette faces the cytosolic si

36、de of the membrane, this is also the patch potential. Conversely, in inside-out patches the electrodes, and therefore the potentials, are reversed.4748 Preparations usedThe main factor in determining whether one can make a patch-clamp recording is how clean the surface of the cell is that is under i

37、nvestigation. If the membrane surface is clean then recordings can be made from almost any cell. The most popular preparations are: Cultured cells Acutely isolated cells Cells in thin slices of brain tissue Cells that have been transfected with cloned ion channels49 Determining the holding potential

38、The calculation of the membrane or holding potential (the potential of the patch or cell at which a recording is being made) depends on the type of patch recording being made. We shall denote the membrane potential as Vm, the commandpotential supplied by the patch amplifier as Vcmd, and the resting

39、membrane potential as RMP.For whole-cell and outside-out recordings: Vm = VcmdFor cell-attached patches: Vm = RMP - VcmdFor inside-out patches:Vm = -Vcmd50 An exampleLets assume we want to carry out a recording at -70 mV. For whole-cell and outside-out patches we would simply set Vcmd to -70 mV. In

40、inside-out recording we would set Vcmd to +70 mV.In cell attached recording we need to have an estimate of the RMP (lets say it is -55 mV). Therefore we would need to set Vcmd to +15 mV to obtain a transmembrane potential (across the patch of -70 mV).Lets now change the membrane potential to -40 mV.

41、 What Vcmd do we need to set in each case?51 Current conventionsPOSITIVE and NEGATIVE currentsElectrophysiologists have agreed on a convention to describe currents recorded in patch-clamp experiments. POSITIVE ions flowing OUT of the patch-pipette are measured as POSITIVE currents and are recorded a

42、s UPWARD deflections on the current trace. POSITIVE ions flowing INTO the patch-pipette are measured as NEGATIVE currents and are recorded as DOWNWARD deflections on the current trace.52Current conventions contdINWARD and OUTWARD currentsMovement of positive ions in the direction of the outside memb

43、rane surface to the inside membrane surface is an INWARD current. These are shown as DOWNWARD deflections.Movement of positive ions in the direction of the inside membrane surface to the outside membrane surface is an OUTWARD current. These are shown as UPWARD deflections.53Current conventions contd

44、In WHOLE-CELL and OUTSIDE-OUT patches the movement of positive ions from the external membrane surface to the internal membrane will mean that positive ions are moving into the patch pipette. Thus each time a channel opens to allow ion flow this will be recorded as a negative-going rectangular pulse

45、 and will be an inward current.CURRENT CLAMPThe patch-clamp amplifier can also be used in CURRENT-CLAMP mode as opposed to VOLTAGE-CLAMP. In CC we measure the change in potential and keep the current fixed. CC is useful if we want to observe changes in membrane potential,rather than infer them from

46、voltage-clamp recordings.54HOWEVER beware!In CELL-ATTACHED and INSIDE-OUT patches the movement of positive ions from the external membrane surface to the internal membrane will mean that positive ions are moving out of the patch-pipette. Therefore each time a cation channel opens we will see this as

47、 a positive-going rectangular pulse.This is however defined as an inward current because of the direction of the ion movement. It is therefore common to INVERT such current traces so that they are illustrated as downward deflections.5556Inward or outward current?Whether we observe an inward or an ou

48、tward current dependson what potential we hold the cell (patch) at and whether thisis more positive or negative than the REVERSAL POTENTIALfor the current. If a single ion carries the current then the reversal potential can be calculated from the NERNST EQUATION.For a monovalent cation at 20 C then

49、the Nernst equation is:57The direction of the current is determined by the driving forcecurrent = (Vm - Er) conductanceVm - Er is the driving force where Vm is the membrane potential and Er is the reversal potential.Thus if Vm is more NEGATIVE than Er we will get an INWARD current i.e. corresponding

50、 to DEPOLARISATION of the cell in current-clamp. If Vm is more POSITIVE than Er we will get an OUTWARDcurrent i.e. corresponding to HYPERPOLARISATION of the cell in current-clamp.58 Example of an I/V plotThese currents were recorded in an outside-out patch and were evoked by the application of kaina

51、te to a patch containing many non-NMDA receptor-channels. The patch was held at the potentials indicated. Currents are inward a potentials negative to 0 mV and outward at potentials positive to 0 mV. Thus 0 mV is the reversal potential for this current. There are too many channels open simultaneousl

52、y to be able to observe the individual events.59For anions.For anions the movement of the ion is in the opposite direction than that for cations for the inward and outward currents. Thus movement of, say, chloride from the external membrane surface to the internal membrane surface is an an OUTWARD c

53、urrent. This will be HYPERPOLARISING. It would still be illustrated as an UPWARD deflection.Movement of chloride in the direction of internal membrane to external membrane surface is an INWARD current and is DEPOLARISING.The Nernst equation for a monovalent anion, at 20 C is given by:60Single-channe

54、l I/V plots are used to determine the conductance of an ion channelgchannel= I V= 1.6 10-12 A 50 10-3 V= 32 10-12 S= 32 pSFrom Hamill et al 198161Some channels display more than one conductance levelUnlike nicotinic ACh receptors, NMDA receptor show twoconductance levels.62Which configuration to use

55、?The type of information required from an experiment will determine the patch-clamp configuration that is chosen.For example:Whole-cell recording - measures the activity in entire cell e.g. if you want to record a synaptically evoked EPSC or if you want to study the average activity and modulation o

56、f a population of channels.63Glutamatergic EPSC recorded from a cerebellar granule cell (P12)AP5Control10 pA25 msData from Clark, Farrant & Cull-Candy (1997)Journal of Neuroscience 17, 107-11664Which configuration to use?Outside-out patch - commonly used to study ligand-gated ion channels as we can

57、apply agonist to the external surface of the membrane and hence gain access to the ligand bindingsite. 65Outside-out patches are used to study fast agonist activation of ligand-gated ion channelsGlutamate + glycineGlycine alonepiezoPatch-pipetteGlutamate + glycineGlycine alonepiezoPatch-pipetteGluta

58、mate + glycine Glycine alonepiezoPatch-pipette10 pA500 ms1 ms pulse of 1 mM glutamate66Which configuration to use?Cell-attached patches - used to study channels in their normal environment. Patch excision may result in modulation of channel properties. Patch excision will also result in the loss of

59、proteins/enzymes that may contribute to the activity of the channel.Cell-attached patch recording is the best method to demonstrate that diffusable messengers might modulate channel activity.67The presence of external Ca2+ results in inhibition of sustained K+ channel activityCa2+Ca2+Ca2+Ca2+Ca2+Ca2

60、+Ca2+Ca2+Ca2+Ca2+Ca2+Ca2+Ca2+Ca2+Ca2+Ca2+Ca2+Ca2+Ca2+Ca2+Ca2+Ca2+Ca2+Ca2+Ca2+Ca2+Ca2+Ca2+Ca2+Ca2+Ca2+Ca2+Ca2+Ca2+Ca2+Ca2+Ca2+Ca2+Ca2+Ca2+Ca2+Ca2+Ca2+Ca2+Ca2+Ca2+Ca2+Ca2+Ca2+Ca2+Ca2+Ca2+Ca2+Ca2+Ca2+Ca2+Ca2+Ca2+Ca2+Ca2+Ca2+Ca2+Ca2+Ca2+68It is thought that by removing external Ca2+ thiswill result in a