Download 6-9_IonChannelsPatchClamp_TasiBenedekJozsef

2016/10/20
Presentation 6/9
Tasi Benedek József
The Patch clamp technique
The „patch clamp” is a technique in electrophysiology that allows the study of single or
multiple ion channels in a wide variety of cells. It is technically a refinement of the Voltage
clamp, an experimental method which measures the ion currents through cell membranes. The
patch clamp was developed by Erwin Neher and Bert Sakmann in the late ’70s and early ’80s.
The discovery improved the understanding of nerve activity (like the action potential, etc), and
for this the researchers were awarded the Nobel Prize in Physiology or Medicine in 1991.
The recording technique uses glass micropipettes (called „patch pipette”, which are
filled with some kind of solution depending on the examination) as electrodes; one for recording
and one in the bath around the cell as a ground reference. The tip of the recording electrode is
sealed onto the surface of the cell membrane. It forms a resistance in the 10-100 gigaohms
range, a so-called „gigaseal”, which makes it possible to electronically isolate the currents
measured across the membrane patch.
The amplifiers are either differential, or use voltage clamp circuitry for keeping the
voltage constant. The patch pipette is compared to the ground electrode while current is injected
into the system to maintain a constant voltage. The amount of current needed to clamp the
voltage is the opposite of the current running through the membrane. The process can be done
in „reverse”, meaning that we can clamp the current and observe the changes in the membrane
voltage. There are automated patch clamp systems too, but this is a rather newfound technology.
There are several variations of this technique, each used for different purposes:
For the cell-attached patch, the pipette is sealed onto the cell while the membrane
remains intact. This way we can record the current through the single/few ion channels inside
the patch.
The inside-out method has a patch of the membrane attached to the pipette, detached
from the rest of the cell. This allows the manipulation of the chemical composition of what the
surface is exposed to, since we can access the cytosolic surface of the membrane.
If more suction is applied during the cell-attached method, the membrane patch is
ruptured, providing access to the intracellular space. This way the resistance is lower, and thus
the electrical access to the inside of the cell is better. This is called the whole-cell patch.
The outside-out technique resembles the inside-out method, but it places the external
surface of the cell membrane on the outside of the patch. This state can be reached starting with
a whole-cell configuration, and then by withdrawing the electrode, thus tearing a bulb of
membrane from the cell, which then reform sas a convex membrane on the end of the pipette.
The perforated patch is very similar to the whole-cell configuration, but instead of
rupturing the membrane with extra suction, the solution inside the electrode contains a small
quantity of antibiotic agents, which forms small pores in the membrane by diffusion. This
allows for a more regulated access to the cell interior.
The loose patch employs a loose seal with low electical resistance instead of the tight
gigaseal. It is achieved by moving the pipette close, but not too close to the membrane, and has
one huge advantage over the other methods: the pipette can be removed from the surface
without damaging it, allowing repeated measurements on a single cell.
Sources:
https://en.wikipedia.org/wiki/Patch_clamp
http://www.nobelprize.org/nobel_prizes/medicine/laureates/1991/