The Patch Clamp Method
Published on Nov 18, 2015
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The Patch Clamp Method
By: Michelle Szklaruk, Lisa Shah and Ellie Ilieva
Historical Developments:
- Jan Swammerdam
- Luigi Galvani
- Hodgkin and Huxley
- Graham
- Cole and Marmont
- Sakmann and Neher
1. Earliest experiments in electrophysiology
2. The first experimental evidence of electrical activity by using metal wires in frog muscle
3. The first intracellular measurement of action potential in the giant squid axon
4. Impaling micro pipettes developed by skeletal muscle fibers
5. Voltage clamp technique
6. The Patch Clam technique
2. The first experimental evidence of electrical activity by using metal wires in frog muscle
3. The first intracellular measurement of action potential in the giant squid axon
4. Impaling micro pipettes developed by skeletal muscle fibers
5. Voltage clamp technique
6. The Patch Clam technique
Voltage Clamp
- Patch clamp technique is a refinement of voltage clamp technique
Voltage clamp, as invented by KC Cole, had a goal to keep the voltage constant. To do this you need one electrode to measure the voltage and another electrode to pass current, positive or negative to stop the voltage from changing. Patch clamping is voltage clamping but adapted so you only need one electrode.
Erwin Neher and Bert Sakman
Erwin Neher and Bert Sakmann developed the patch clamp in the late 1970's and early 1980's. This discovery made it possible to record the currents of single ion channels for the first time, proving their involvement in fundamental cell processes such as action potential conduction. Both received the Nobel Prize in Physiology or Medicine in 1991 for this work
What is it?
The patch clamp method is a laboratory technique in electrophysiology that allows the study of single or multiple ion channels in cells. Can be used in a variety of cells but are especially useful in studying excitable cells such as neurons and muscle fibers. The principle is to isolate a patch of membrane electrically and record current flow in the patch.
How does it work?
To record the current, we need to apply an electrical potential across the membrane patch. We use a glass pipette, a recording electrode, that has an open tip that is very small in diameter, about one mircometer, and is filled with electrolyte solution. The micro pipette is pressed against a tiny area or patch of a cell membrane and light suction is applied to help in the formation of a high resistance seal between the glass and the cell membrane. After the application of a small amount of suction, the seal between pipette and membrane becomes so tight that no ions can flow between the pipette and the membrane.
Patch Clamp Circuit
The interior of the pipette is filled with either high Sodium chloride or potassium chloride solution. A chloried silver wire connects the pipette solution to the head stage of an electronical amplifier. The amplifier is connected in the circuit so that the current flowing through the ion channel is measured as a voltage drop. A second choride silver wire is inserted into the bath around the cell and serves as a ground electrode. The investigator can change the composition of this bath or add drugs to study the ion channels under different condition.
There are 6 different variations of the patch clamp that we will briefly talk about.
There are 6 different variations of the patch clamp that we will briefly talk about.
Cell Attached or On-Cell Patch
The electrode is sealed to the patch of the membrane and the cell remains intact. Allows for the recording of currents through single ion channels in that patch of membrane, without disrupting the interior of the cell.
Cell Attached or On-Cell Patch
The electrode is sealed to the patch of the membrane and the cell remains intact. Allows for the recording of currents through single ion channels in that patch of membrane, without disrupting the interior of the cell.
Inside-Out Patch
After the cell membrane is formed, the micro pipette is quickly withdrawn from the cell. Ripping the patch of membrane off the cell, leaving the patch of membrane attached to the micro pipette and exposing the intracellular surface. Can be used when the experimentor wants to manipulate the environment at the intracellular surface of ion channels.
Whole-Cell Patch
Involve recording currents through multiple channels at once, over the membrane of the entire cell. The electrode is left in place on the cell but more suction is applied to rupture the membrane patch..Adv: larger opening at the tip of the patch clamp electrode provides lower resistance and better electrical access to inside of the cell. Dis: The volume of the electrode is larger than the cell, so the soluble contents of the cell's interior will slowly be replaced by the contents of the electrode.
Outside-Out Patch
After the whole-cell patch is formed, the electrode can be slowly withdrawn from the cell, allowing an irregular bulge "bleb" out of the cell. When the electrode is pulled far enough away, this bleb will detach from the cell and reform as a convex membrane on the end of the electrode with the original outside of the membrane facing outward from the electrode. This patching gives the experimenter the opportunity to examine the properties of an ion channel when its isolated from the cell.
Perforated Patch
The experimenter forms the cell membrane seal, but doesn't use suction to rupture the patch membrane. Instead the electrode solution contains small amounts of anti fungal or antibiotic. As the antibiotic molecules diffuse into the membrane patch, they form small perforations in the membrane that provide electrical access to the cell interior.
Loose Patch
It employs a loose seal rather than a tight one
adv: The pipette can be repeatedly removed from the membrane, keeping the membrane intact and allows for repeated measurements in different locations on the same cell without destroying the membrane.
Dis: The resistance between the pipette and the membrane is greatly reduced, allowing the current to leak through the seal.
adv: The pipette can be repeatedly removed from the membrane, keeping the membrane intact and allows for repeated measurements in different locations on the same cell without destroying the membrane.
Dis: The resistance between the pipette and the membrane is greatly reduced, allowing the current to leak through the seal.
Limitations
- Money
- Time
- Samples
- Membrane distortion
- Need skills
1.Cost of process is expensive
2.Time consuming
3.Lot of samples required
4.Chance of membrane distortion
5. Requires strong background in ionic channel biophysics. You need skillful training performance and recording
2.Time consuming
3.Lot of samples required
4.Chance of membrane distortion
5. Requires strong background in ionic channel biophysics. You need skillful training performance and recording
Why use it?
- Low-noise recordings of currents
- Access to the inside of the cell
- Seal impermeable to ion flow
- One measurement
2. You can insert an electrode into the cell or you can change the intracellular fluid
3. High electrical resistance
4. Allows one to measure current through ion channels versus voltage, time and temperature.
3. High electrical resistance
4. Allows one to measure current through ion channels versus voltage, time and temperature.
Untitled Slide
Patch clamp technique applies to cellular and molecular biology because it provides access to the inside of the cell.
-identify multiple types of calcium channels
-measure the effect of potassium channel openers
It allows us to measure current through ion channels.
-identify multiple types of calcium channels
-measure the effect of potassium channel openers
It allows us to measure current through ion channels.
Conclusion
- It is highly modified and a successful technique
- Still being developed to obtain better accurate and efficient information
References
- Wyllie DJA(2007) Single-channel recording. In Patch-Clamp Analysis- Advanced Techniques 2nd Edition. Ed. Walz W. Humana Press Totowa, New Jersey USA
- Sakmann B(1992) Elementary steps in synaptic transmission revealed by currents through ingle ion channels. Neuron 8
- Aidley DJ & Stanfield PR (1996) Ion Channels - Molecules in Action Cambridge University Press
