Patch clamp is an electrophysiology method used to measure the very small electric currents that flow through the ion channels in the membrane of a single cell. Erwin Neher and Bert Sakmann, who developed the method, received the 1991 Nobel Prize in Physiology or Medicine for their discoveries concerning the function of single ion channels. Today it is one of the basic measuring tools for studying molecules that act on ion channels, venom peptides included.
A large share of the peptides in scorpion venom bind to potassium, sodium or chloride channels. Which channel a peptide affects, how strongly and how selectively, is determined largely by patch clamp measurements. This article outlines the basic principle of the method, the recording configurations used and how a measurement is read.
The basic principle
A glass micropipette with a tip about a micrometre across is filled with a conducting solution and brought into contact with the cell membrane. When gentle suction is applied, a very tight contact forms between the glass and the membrane. The resistance of this contact is in the gigaohm range, that is billions of ohms, which is why it is called a “gigaseal”. With insulation this good, currents in the picoampere range flowing through the small patch of membrane at the pipette tip can be distinguished from noise.
Measurements are mostly made in voltage clamp mode: the amplifier holds the membrane voltage at a value set by the researcher and records the current needed to do so. The recorded current is the current flowing through the ion channels that are open at that moment. In current clamp mode, by contrast, the current is held constant and changes in membrane voltage are recorded; this mode is used mainly to study the electrical behaviour of nerve cells.
The composition of the solutions inside and outside the cell, the temperature and the voltage steps applied are recorded with each measurement. Results from different laboratories can only be compared when these conditions are known.
Four basic recording configurations
| Configuration | How it is set up | What is measured |
|---|---|---|
| Cell-attached | The pipette seals onto the membrane, which stays intact | Single-channel currents of the few channels under the pipette |
| Whole-cell | The membrane under the pipette is ruptured and the pipette opens into the cell | The total current of all channels in the cell |
| Inside-out | A patch of membrane is pulled off, with its inner face towards the bath solution | The effect of substances applied to the intracellular side |
| Outside-out | A small membrane patch is obtained by pulling back from the whole-cell configuration | The effect of substances applied to the outer face, such as toxins |
Because venom peptides usually bind to channels from the outer face of the cell, the whole-cell configuration is used most often in screening studies: the peptide is added to the solution surrounding the cell and the change in current is followed.
How are venom peptides tested?
The channel to be studied is usually produced in a cell line into which its gene has been introduced, so that most of the measured current comes from a single channel type. The following steps are then taken:
- A stable baseline of the channel current is recorded before the peptide is added.
- The peptide is applied at increasing concentrations, and at each concentration the reduction or change in current is measured.
- The peptide is washed out to see whether the effect is reversible.
- From the concentration–response curve, the concentration that halves the current (IC50) is calculated.
- The same measurements are repeated on other channel subtypes to determine selectivity.
Selectivity is expressed as the ratio between the IC50 values of the same peptide on different channels; the larger the ratio, the more clearly the peptide distinguishes its target channel from the others.
Studies on Kv1 channels are a typical example of this approach: by comparing the IC50 values of an α-KTx peptide on Kv1.1, Kv1.2 and Kv1.3, researchers learn which channel it is more selective for. With sodium channel toxins, the focus is less on a reduction of current and more on changes in how the channel opens and closes; the features of this family are described in the article on sodium channel toxins.
Automated patch clamp and limitations
Classical patch clamp requires an experienced researcher working on cells one by one under a microscope, and only a limited number of recordings can be made per day. Automated patch clamp systems use planar chips with small holes instead of pipettes and measure many cells at the same time, which speeds up the screening of large numbers of molecules.
Some limitations should be kept in mind when interpreting results: a channel produced in a cell line may behave differently from the native channel in tissue, and knowing the purity and concentration of the peptide accurately has a direct effect on the IC50 value. For this reason, measurements made with separated peptides of confirmed identity, rather than with crude venom, give more reliable results. For the analytical profile of crude venom, see our analysis results page.
