Patch clamp
Encyclopedia
Electrophysiology
Electrophysiology is the study of the electrical properties of biological cells and tissues. It involves measurements of voltage change or electric current on a wide variety of scales from single ion channel proteins to whole organs like the heart...
that allows the study of single or multiple ion channel
Ion channel
Ion channels are pore-forming proteins that help establish and control the small voltage gradient across the plasma membrane of cells by allowing the flow of ions down their electrochemical gradient. They are present in the membranes that surround all biological cells...
s in cells
Cell (biology)
The cell is the basic structural and functional unit of all known living organisms. It is the smallest unit of life that is classified as a living thing, and is often called the building block of life. The Alberts text discusses how the "cellular building blocks" move to shape developing embryos....
. The technique can be applied to a wide variety of cells, but is especially useful in the study of excitable cells such as neuron
Neuron
A neuron is an electrically excitable cell that processes and transmits information by electrical and chemical signaling. Chemical signaling occurs via synapses, specialized connections with other cells. Neurons connect to each other to form networks. Neurons are the core components of the nervous...
s, cardiomyocytes, muscle fibers and pancreatic
Pancreas
The pancreas is a gland organ in the digestive and endocrine system of vertebrates. It is both an endocrine gland producing several important hormones, including insulin, glucagon, and somatostatin, as well as a digestive organ, secreting pancreatic juice containing digestive enzymes that assist...
beta cell
Beta cell
Beta cells are a type of cell in the pancreas located in the so-called islets of Langerhans. They make up 65-80% of the cells in the islets.-Function:...
s. It can also be applied to the study of bacterial ion channels in specially prepared giant spheroplasts.
The patch clamp technique is a refinement of the voltage clamp
Voltage clamp
The voltage clamp is used by electrophysiologists to measure the ion currents across the membrane of excitable cells, such as neurons, while holding the membrane voltage at a set level. Cell membranes of excitable cells contain many different kinds of ion channels, some of which are voltage gated...
. Erwin Neher
Erwin Neher
Erwin Neher is a German biophysicist.Erwin Neher studied physics at the Technical University of Munich from 1963 to 1966. In 1966, He was awarded a Fulbright Scholarship to study in the US...
and Bert Sakmann
Bert Sakmann
-External links:*...
developed the patch clamp in the late 1970s and early 1980s. This discovery made it possible to record the currents of single ion channel
Ion channel
Ion channels are pore-forming proteins that help establish and control the small voltage gradient across the plasma membrane of cells by allowing the flow of ions down their electrochemical gradient. They are present in the membranes that surround all biological cells...
s for the first time, proving their involvement in fundamental cell processes such as action potential
Action potential
In physiology, an action potential is a short-lasting event in which the electrical membrane potential of a cell rapidly rises and falls, following a consistent trajectory. Action potentials occur in several types of animal cells, called excitable cells, which include neurons, muscle cells, and...
conduction. Neher and Sakmann received the Nobel Prize in Physiology or Medicine
Nobel Prize in Physiology or Medicine
The Nobel Prize in Physiology or Medicine administered by the Nobel Foundation, is awarded once a year for outstanding discoveries in the field of life science and medicine. It is one of five Nobel Prizes established in 1895 by Swedish chemist Alfred Nobel, the inventor of dynamite, in his will...
in 1991 for this work.
Basic technique
Electrode
An electrode is an electrical conductor used to make contact with a nonmetallic part of a circuit...
, a glass micropipette
Micropipette
A micropipette is one of two different instruments used in science laboratories.-History:The first micropipette was patented in 1960 by Dr Hanns Schmitz . The founder of the company Eppendorf, Dr...
that has an open tip diameter of about one micrometre
Micrometre
A micrometer , is by definition 1×10-6 of a meter .In plain English, it means one-millionth of a meter . Its unit symbol in the International System of Units is μm...
, a size enclosing a membrane surface area or "patch" that often contains just one or a few ion channel molecules. This type of electrode is distinct from the "sharp microelectrode" used to impale cells in traditional intracellular recordings, in that it is sealed onto the surface of the cell membrane
Cell membrane
The cell membrane or plasma membrane is a biological membrane that separates the interior of all cells from the outside environment. The cell membrane is selectively permeable to ions and organic molecules and controls the movement of substances in and out of cells. It basically protects the cell...
, rather than inserted through it. In some experiments, the micropipette tip is heated in a microforge to produce a smooth surface that assists in forming a high resistance
Electrical resistance
The electrical resistance of an electrical element is the opposition to the passage of an electric current through that element; the inverse quantity is electrical conductance, the ease at which an electric current passes. Electrical resistance shares some conceptual parallels with the mechanical...
seal with the cell membrane. The interior of the pipette is filled with a solution matching the ionic composition of the bath solution, as in the case of cell-attached recording, or the cytoplasm
Cytoplasm
The cytoplasm is a small gel-like substance residing between the cell membrane holding all the cell's internal sub-structures , except for the nucleus. All the contents of the cells of prokaryote organisms are contained within the cytoplasm...
for whole-cell recording. A chlorided silver wire is placed in contact with this solution and conducts electric current to the amplifier. The investigator can change the composition of this solution or add drugs to study the ion channels under different conditions. The micropipette is pressed against a cell membrane and suction is applied to assist in the formation of a high resistance seal between the glass and the cell membrane (a "gigaohm seal" or "gigaseal," since the electrical resistance of that seal is in excess of a gigaohm). The high resistance of this seal makes it possible to electronically isolate the currents measured across the membrane patch with little competing noise
Electronic noise
Electronic noise is a random fluctuation in an electrical signal, a characteristic of all electronic circuits. Noise generated by electronic devices varies greatly, as it can be produced by several different effects...
, as well as providing some mechanical stability to the recording.
Unlike traditional two-electrode voltage clamp
Voltage clamp
The voltage clamp is used by electrophysiologists to measure the ion currents across the membrane of excitable cells, such as neurons, while holding the membrane voltage at a set level. Cell membranes of excitable cells contain many different kinds of ion channels, some of which are voltage gated...
recordings, patch clamp recording uses a single electrode to record currents. Many patch clamp amplifiers do not use true voltage clamp circuitry but instead are differential amplifier
Differential amplifier
A differential amplifier is a type of electronic amplifier that amplifies the difference between two voltages but does not amplify the particular voltages.- Theory :Many electronic devices use differential amplifiers internally....
s that use the bath electrode to set the zero current level. This allows a researcher to keep the voltage constant while observing changes in current
Electric current
Electric current is a flow of electric charge through a medium.This charge is typically carried by moving electrons in a conductor such as wire...
. Alternatively, the cell can be current clamped in whole-cell mode, keeping current constant while observing changes in membrane voltage
Membrane potential
Membrane potential is the difference in electrical potential between the interior and exterior of a biological cell. All animal cells are surrounded by a plasma membrane composed of a lipid bilayer with a variety of types of proteins embedded in it...
.
Variations
Several variations of the basic technique can be applied, depending on what the researcher wants to study. The inside-out and outside-out techniques are called "excised patch" techniques, because the patch is excised (removed) from the main body of the cell. Cell-attached and both excised patch techniques are used to study the behavior of individual ion channels in the section of membrane attached to the electrode.Whole-cell patch and perforated patch allow the researcher to study the electrical behavior of the entire cell, instead of single channel currents. The whole-cell patch, which allows for low resistance electrical access to the inside of a cell, has now largely replaced high resistance microelectrode recording techniques to record currents across the entire cell membrane.
Cell-attached or on-cell patch
The electrode is sealed to the patch of membrane, and the cell remains intact. This allows for the recording of currents through single ion channels in that patch of membrane, without disrupting the interior of the cell. For ligand-gated ion channels or channels that are modulated by metabotropic receptors, the neurotransmitterNeurotransmitter
Neurotransmitters are endogenous chemicals that transmit signals from a neuron to a target cell across a synapse. Neurotransmitters are packaged into synaptic vesicles clustered beneath the membrane on the presynaptic side of a synapse, and are released into the synaptic cleft, where they bind to...
or drug being studied is usually included in the pipette solution, where it can contact what had been the external surface of the membrane. While the resulting channel activity can be attributed to the drug being used, it is usually not possible to then change the drug concentration. The technique is thus limited to one point in a dose response curve
Dose-response relationship
The dose-response relationship, or exposure-response relationship, describes the change in effect on an organism caused by differing levels of exposure to a stressor after a certain exposure time...
per patch. Usually, the dose response is accomplished using several cells and patches. However, voltage-gated ion channel
Voltage-gated ion channel
Voltage-gated ion channels are a class of transmembrane ion channels that are activated by changes in electrical potential difference near the channel; these types of ion channels are especially critical in neurons, but are common in many types of cells....
s can be clamped at different membrane potentials using the same patch. This results in graded channel activation, and a complete I-V (current-voltage) curve
Ohm's law
Ohm's law states that the current through a conductor between two points is directly proportional to the potential difference across the two points...
can be established with only one patch.
Inside-out patch
After the gigaseal is formed, the micropipette is quickly withdrawn from the cell, thus ripping the patch of membrane off the cell, leaving the patch of membrane attached to the micropipette, and exposing the intracellularIntracellular
Not to be confused with intercellular, meaning "between cells".In cell biology, molecular biology and related fields, the word intracellular means "inside the cell".It is used in contrast to extracellular...
surface of the membrane to the external media. This is useful when an experimenter wishes to manipulate the environment at the intracellular surface of ion channels. For example, channels that are activated by intracellular ligands
Second messenger system
Second messengers are molecules that relay signals from receptors on the cell surface to target molecules inside the cell, in the cytoplasm or nucleus. They relay the signals of hormones like epinephrine , growth factors, and others, and cause some kind of change in the activity of the cell...
can then be studied through a range of ligand concentrations.
Whole-cell recording or whole-cell patch
Whole-cell recordings, in contrast, 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, thus providing access to the intracellular space of the cell. The advantage of whole-cell patch clamp recording over sharp microelectrode recording is that the larger opening at the tip of the patch clamp electrode provides lower resistance and thus better electrical access to the inside of the cell. A disadvantage of this technique is that 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. This is referred to as the electrode "dialyzing"Dialysis (biochemistry)
In biochemistry, dialysis is the process of separating molecules in solution by the difference in their rates of diffusion through a semipermeable membrane, such as dialysis tubing....
the cell's contents. Thus, any properties of the cell that depend on soluble intracellular contents will be altered. The pipette solution used usually approximates the high-potassium environment of the interior of the cell. Generally speaking, there is a period at the beginning of a whole-cell recording, lasting approximately 10 minutes, when one can take measurements before the cell has been dialyzed.
Outside-out patch
After the whole-cell patch is formed, the electrode can be slowly withdrawn from the cell, allowing a bulb of membrane to blebBleb (cell biology)
In cell biology, a bleb is an irregular bulge in the plasma membrane of a cell caused by localized decoupling of the cytoskeleton from the plasma membrane...
out from 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 (like a ball open at the electrode tip), with the original outside of the membrane facing outward from the electrode. Single channel recordings are possible in this conformation if the bleb of membrane is small enough. Outside-out patching gives the experimenter the opportunity to examine the properties of an ion channel when it is isolated from the cell, and exposed to different solutions on the extracellular
Extracellular
In cell biology, molecular biology and related fields, the word extracellular means "outside the cell". This space is usually taken to be outside the plasma membranes, and occupied by fluid...
surface of the membrane. The experimenter can perfuse the same patch with different solutions, and if the channel is activated from the extracellular face, a dose-response curve can then be obtained. This is the distinct advantage of the outside-out patch relative to the cell-attached method. However, it is more difficult to accomplish, as more steps are involved in the patching process.
Perforated patch
In this variation of whole-cell recording, the experimenter forms the gigohm seal, but does not use suction to rupture the patch membrane. Instead, the electrode solution contains small amounts of an antifungal or antibiotic agent, such as amphothericin-B, nystatinNystatin
Nystatin is a polyene antifungal medication to which many molds and yeast infections are sensitive, including Candida. Due to its toxicity profile, there are currently no injectable formulations of this drug on the US market...
, or gramicidin
Gramicidin
Gramicidin is a heterogeneous mixture of six antibiotic compounds, gramicidins A, B and C, making up 80%, 6%,and 14% respectively, all of which are obtained from the soil bacterial species Bacillus brevis and called collectively gramicidin D. Gramicidin D are linear pentadecapeptides; that is...
. As the antibiotic molecules diffuse into the membrane patch, they form small perforations in the membrane, providing electrical access to the cell interior. This has the advantage of reducing the dialysis of the cell that occurs in whole-cell recordings, but also has several disadvantages. First, the access resistance is higher, relative to whole-cell, due to the partial membrane occupying the tip of the electrode (access resistance being the sum of the electrode resistance and the resistance at the electrode-cell junction). This will decrease electrical access and thus decrease current resolution, increase recording noise, and magnify any series resistance error. Second, it can take a significant amount of time for the antibiotic to perforate the membrane (10–30 minutes, though this can be reduced with properly shaped electrodes). Third, the membrane under the electrode tip is weakened by the perforations formed by the antibiotic and can rupture. If the patch ruptures, the recording is then in whole-cell mode, with antibiotic contaminating the inside of the cell.