Patch clamp

The patch clamp technique is an electrophysiological method that makes it possible to measure the current through single channel-proteins in the membrane of a cell.

The term “patch” refers to a small piece of membrane that is plucked from the cell via the patch pipette, which also serves to measure the currents.

During the measurement this membrane-patch is “clamped” to a defined potential. The necessary tiny current is in the range of pA and can be adjusted and measured precisely with the patch clamp amplifier.

Currents can be measured across a membrane patch, but also across the entire cell membrane. Using this method, it is possible to determine if (i.e.) ruminal epithelial cells express proteins for the conductance of NH4+ or acetate anions.

Image Credit:
PD Dr. Friederike Stumpff

Whole-Cell-Konfiguration

In this configuration, currents are measured across the entire cell membrane. Using a micromanipulator and under a microscope, a glass pipette is lowered onto the cell. By applying suction, a piece of membrane is sucked outwards and aligns with the walls of the pipette. If the suction is increased, the patch ruptures so that the pipette solution flows into the cell.

With a small silver wire, the potential of the cell can be measured or clamped to various levels via currents. The currents are then plotted over the voltage to determine in what way the conductance changes with the potential.

By variation of the bath solution, it is possible to determine what ions are conducted by the cell. For instance, it is possible to apply a solution containing ammonium ions or acetate, and see if these ions are conducted by the cell. At the institute of veterinary physiology, this approach is used to investigate both native cells and cells expressing particular channel proteins.

Image Credit:
PD Dr. Friederike Stumpff

Inside out configuration

In this configuration, a tiny piece of membrane is investigated.

Again, the pipette is lowered onto the cell. Via suction, the membrane is aligned with the pipette wall. However, now the entire pipette is moved upwards. With a bit of luck, the membrane patch will remain in the opening of the pipette so that its conductance to various ions can be investigated. Typically, channels can be seen to open and close, so that the current abruptly increases and decreases, forming a pattern of steps. The height of each one of these steps corresponds to the unitary conductance of a single channel.

In a next step, it is possible to investigate what happens when (i.e.) chloride is removed from the bath. If the current decreases, it is a chloride channel. Likewise, it is possible to see if the channel conducts anions such as iodide, flouride – or acetate, the anion of acidic acid.

Image Credit:
PD Dr. Friederike Stumpff

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