Patch clamp electrophysiology is an experimental technique that reads into electrical events in cells. The electric potential between the cell membrane reveals the workings of cell membranes and ion channels, a group of proteins that detect and control the movement of ions in and out of the cells.[1]
The technique was developed through a collaboration between Erwin Neher and Bert Sakmann. It enabled them to discover ion channels and demonstrate how the channels detect and regulate ion movement in and out of the cells. Their works also shed light on the underlying causes of several diseases, earning them an equally shared 1991 Nobel Prize in Physiology or Medicine.[2]
The species of ions and their distributions in the cells and their surroundings are regulated tightly in animal cells. The entry and exit of the ions are monitored constantly. The ion gradients and membrane potential are maintained at a specific concentration and changed only in response to physiological events such as movement and cell-to-cell communication.[1]
The patch clamp technique combines the principles of intracellular recording and voltage clamps, two existing techniques that measure electric current in cells at the time.
Stemmed from experiments with frog muscles in the late 40s, intracellular recording uses two electrodes to measure the electric potential differences between the interior and exterior of the cell, termed membrane voltage or membrane potential.
It uses a small and sharp glass micropipette as one of the electrodes to pierce through the membrane. The small diameter reduces cell damage and provides high resistance, preventing the draining of the cytosol to the fluid in the micropipette.
The other electrode, the bath electrode, has no direct contact with the membrane. Rather, it is soaked in a bath containing a solution with appropriate electrolytes, completing the circuit. When the probing electrode penetrates the cell membrane, the potential difference between the two electrodes represents the membrane potential of the measuring cell.[1]
Originally developed for neurons and other excitable cells, the voltage clamp uses two microelectrodes to measure the membrane potential through an electronic feedback system.
During the measurement or clamping, the microelectrodes are in contact with the cell of interest. One of the electrodes provides a holding voltage, clamping the membrane potential at a designated holding potential controlled by an external device.[3]
The other electrode acts as the current electrode, supplying electric current into the clamped cells to compensate for deviation from the holding potential. Therefore, the amount of current supplied into the clamped cells reflects the membrane potential but in the opposite direction of current flow, as represented by the plus or minus sign.[1,3-4]
The technique is the basis of Alan Hodgkin and Andrew Huxley’s experiment with squid giant axons, which won them two thirds of the 1963 Nobel Prize in Physiology or Medicine.
Their use of voltage clamps on the squid nerve fiber demonstrates how the ion flux across the membrane in neurons gives rise to the action potential – the basis of nerve cell-to-cell communication and impulses in the muscles.[4]
Neher and Sakmann combined the better aspects of intracellular recording and voltage clamp. So that voltage clamp becomes applicable to a specific membrane region of smaller cells.
In patch clamp electrophysiology, a wide-bore glass micropipette is used as an electrode, instead of a sharp glass micropipette. The patch pipette is filled with a buffer with appropriate electrolytes, acting as a measuring electrode that directly probes the cell membrane.
To complete the circuit, the other electrode, called the ground or bath electrode, is immersed in a buffer of choice, which also serves as a reference voltage and maintains the holding potential.[1]
The wider patch pipette enables the electrode to measure the voltage and instantaneously supply electric current when the membrane potential deviates from the holding potential. The larger pipette also increases the adhesion between the membrane and electrode, lessens the resistance, and establishes a tight seal.[1,3]
The seal provides a stable electrode-membrane connection, allowing the membrane potential to be measured with minimum noise from the surrounding. When the patch pipette ruptures the membrane, the seal remains intact, preventing the leaking of cytosolic content that could affect the measurement.[1]
Since its first conception, patch clamp techniques have undergone modifications into several configurations, which can record an individual ion channel or obtain the electric current of the whole cell or tissue slices.
Considered the original patch clamp, the cell-attached patch clamp measures membrane potential from the electric current that passes an ion channel. In this configuration, the patch pipette forms a highly resistant seal with the membrane, called gigaseal, enabling pico (10-12) scale measurement of the membrane potential.[3]
The cell-attached patch configuration does not require the electrode to penetrate the membrane, leaving the cell intact during patch clamp. For this reason, this type resembles the native physiological state the most.[1]
In this configuration, the tissue or cells are immersed in the bath solution together with the bath electrode. To obtain the membrane potential, puncture the cell membrane with the patch pipette.
The disrupted membrane allowed the pipette buffer to directly contact the cytoplasm, resulting in membrane potential measurement, or an instant current injection that compensates for the voltage difference between the intracellular and extracellular environment.[1]
This type of patch clamp is most similar to intracellular recording and is used for the overall assessment of ion channels distributed in the whole cell or tissue slices.
It also permits the manipulation and control of the cytosolic environment through the pipette buffer. However, the whole-cell patch requires a highly skilled worker who can avoid the drainage of cytosol that could inadvertently remove essential cytosolic factors.[1,3]
Modified from a whole-cell patch clamp, the outside-out excised configuration measures an individual ion channel by rupturing the cell membrane.
Here, the patch pipette is pulled away after it makes contact with the membrane, causing the membrane phospholipids to aggregate around the patch pipette. This lets users test the effect of extracellular factors directly on the ion channel.
This configuration disrupts cellular cytoskeletons and requires skilled workers to manipulate the cell without completely letting the cytosol leak into the bath solution.[1]
Similar to the outside-out excised patch, the membrane is punctured by pulling the patch pipette away after its initial contact with the membrane. Unlike the outside-out type, the patch pipette is briefly removed from the bath solution, leaving the patch with the bath-facing cytosolic side.
The inside-out excised patch configuration necessitates a bath solution that resembles the cytosol to prevent cytosolic washout, making this patch clamp type suitable for testing the influence of a specific extracellular factor on the ion channels.
Like the outside-out excised patch, the inside-out one also destroys the cytoskeleton network and requires a skilled worker to handle the experiment.[1]
Another whole-cell patch modification, a perforated patch clamp records the whole-cell membrane potential.
The setup in the perforated patch is like the whole-cell patch clamp, except an antifungal such as amphotericin B and nystatin is added to the pipette solution. It acts as a membrane-perforating agent that only allows small ions to pass through the punctured membrane, blocking the loss of large cytosolic components.[1]
As a standalone technique, patch clamp allows scientists to investigate the function of ion channels, providing insights into animal physiology and neurobiology. It also unveils the mechanisms of several diseases.
For example, defective calcium ion channels can manifest in cardiovascular diseases or neuro-muscular disorders such as Lambert-Eatons disease. Epilepsy and cystic fibrosis result from malfunctioning sodium-potassium and chloride ion channels, respectively.[2]
In addition, patch clamp can combine with other experimental techniques to acquire more information on the ion channel function in various systems.
For instance,
Since its conception, patch clamp has become the cornerstone technique in electrophysiology. The technique has been modified into several configurations, which can be used as a stand-alone technique or together with other approaches to illustrate the roles and relevance of ion channels in animal physiology, neurobiology, and several diseases.
If you are looking for a patch clamp device that allows observation and imaging during the procedure, check out our Visual Patching Imagining Chamber!
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