Structural Biochemistry/Membrane Proteins/Ion Channels/Patch Clamp
To measure what's happening in or on a single, living cell, scientists use a technique called the patch clamp which requires an extremely fine pipet held tightly against the cell membrane. The technique was introduced by Erwin Neher and Bert Sakmann in 1976. This powerful technique enables the measurement of the ion conductance through a small patch of cell membrane. The stepwise changes in membrane conductance are observed. These changes correspond to the opening and closing of individual ion channels. In this technique, a clean glass pipette with a tip diameter of about 1 micro m is pressed against an intact cell to form a seal. The slight suction from the pipette leads to the formation of a very tight seal. Therefore, the resistance between the inside of the pipette and the bathing solution is many gigaohms. Thus, a gigaohm seal known as a gigaseal ensures that an electric current flowing through the pipette is identical with the current flowing through the membrane covered by the pipette. The gigaseal makes possible high - resolution current measurement while a known voltage is applied across the membrane. A time resolution of microseconds monitors the flow of ions through a single channel and transition between the open and the closed states of a channel.
Patch clamp is a powerful technique in electrophysiology for studying ion channels in cells. This technique is very useful in the study of neutrons, muscle fibers, pancreatic beta cells, etc. Patch clamp can be applied and used to study bacterial ion channels. In the late 1970s and early 1980s, Erwin Neher and Bert Sakmann developed the patch clamp technique and were awarded the Nobel Prize in Medicine in 1991. Their discovery made it possible to record and measure currents of single ion channels.
Patch - clamp modes
The patch - clamp technique for monitoring channel activity is highly versatile. A high - resistance seal (gigaseal) is formed between the pipette and a small patch of plasma membrane. This configuration is called cell attached mode. The breaking of membrane patch by increased suction produces a low - resistance pathway between the pipette and the interior of the cell. The activity of the channels in the entire plasma membrane can be monitored in this whole - cell mode. To prepare a membrane in the excised - patch mode, the pipette is pulled away from the cell. A piece of plasma membrane with its cytoplasmic side now facing the medium is monitored by the patch pipette.
Depending on what is wanted to be study, variations of the patch clamp can be applied.
Cell-attached (on-cell patch)
The pipette is pressed against the cell to form a gigaseal by sucking a small area of the membrane to break both the membrane and cytoplasm off the cell. This leaves the patch of membrane attached to the pipette and exposes the interior of the membrane to the external solution. A voltage is applied across the patch, allowing experimenters to see recordings of discrete current steps through single ion channels without disrupting the cell’s interior.
This is an excised patch technique because the patch is excised or removed from the main body of the cell. The internal membrane surface faces the bath solution (the pipette solution). The intracellular space of the membrane is exposed to the bath to allow testing of various intracellular channel modulators.
Whole-cell recording (whole-cell patch)
Fresh glass pipette with a tip diameter of only a few micrometers is pressed on the cell membrane gently to form a gigaseal. Suction is applied to break the membrane and the cytoplasm. The pipette solution starts to mix in to make the cell have a similar ionic environment as the saline filling the pipette uses.
Using the whole-cell mode, the pipette is pulled away from the cell which excises a patch with the extracellular side of the membrane facing the bath solution (the pipette filling solution). The patch is superfused on the extracellular side with the solution. The solution contains agonists (i.e. GABA or glutamate) which activate the receptor channels in the patch.
Once the whole-cell is established, instead of using suction to rupture the patch membrane, an electrode solution that contains antibiotics diffuses into the membrane patch forming small perforations that provides electrical access to the intracellular side of the cell.
This employs a loose seal rather than a tight gigaseal. An advantage of loose seal is that the pipette can be removed from the membrane without rupturing the membrane, the membrane will still remain intact. This allows repeated measurements on the same cell without destroying the membrane’s integrity. A disadvantage is the resistance between the pipette and the membrane is reduced, thus allowing current to leak through the seal.