Perforated patch clamping

Inside-out: cell-attached configuration is achieved and the pipette is withdrawn while the gigaseal is maintained, rupturing the cell. The inside cytoplasmic side of the membrane faces the bath fluid.

Whole-cell: the cell-attached configuration is achieved and vigorous suction is applied to the pipette, causing the patch to break. The cytoplasm and the pipette solution are subsequently in direct contact. After a short time, diffusion of cytoplasmic constituents molecules and cell organelles leads to identical unphysiological chemical composition of the fluids in the cell and in the pipette.

The activity of all membrane ion channels is measured in this configuration. Outside-out: the whole-cell configuration is achieved and the pipette is gently withdrawn.

Abstract Electrophysiology is an essential tool aiding the study of the functions and dysfunctions of electrically excitable cells and their networks. The patch clamp method is a refined electrophysiological technique that can directly measure the membrane … more. Related articles Based on techniques. Joris , , Springer Protocols. Ellender , , Springer Protocols. Rosholm et al. See more. This is an ideal technique to study channels that are activated by intracellular ligands, while the experimenter controls the contents of exposing solution.

While in cell-attached patch configuration, the experimenter applies a short, strong negative pressure in the pipette, sometimes together with a short pulse of high voltage. It enables either whole cell currents flowing through the membrane or the membrane potential to be recorded.

For example, voltage-gated ion channels, such as voltage-gated potassium or sodium channels can be assessed. In conjunction with pharmacology, this provides an essential method to characterise and describe the behaviour of various ion channels under controlled conditions. A problem with whole cell recordings can be caused by cell dialysis. This is where intracellular messengers, such as ATP, decrease in concentration because some it flows into the pipette. Here, the whole-cell method is applied and then the pipette is retracted after the rupture of the membrane.

This causes part of the membrane to detach from the cell and reform into a small, vesicular structure, with the outside of the membrane remaining exposed to the bath solution. This allows the researcher to investigate the properties of single ion channels when they are isolated from the cell. It is easier for the researcher to change the external solution that the patch is exposed to, allowing a dose-response curve to be obtained.

However, it is a more complicated method than those previously described, with more steps that could fail. The perforated patch method can be used to prevent cell dialysis. Rather than rupturing the membrane when in the whole-cell configuration, pore-forming agents are used to permeabilise the patch of the membrane that is in contact with the micropipette. These agents are added to the pipette solution, and the procedure for the whole-cell configuration is carried out, without rupturing the membrane.

This minimises disruption to the cell, allowing recordings to be taken over a longer period of time. However, due to the increased access resistance, there may be a lower signal-to-noise ratio. Perforated-cell patch clamp is used to measure the sum of activity of ion channels in the membrane of a single cell.

It is ideal for recording whole cell currents without disrupting second messenger signalling cascades. This allows very little ion exchange between the intracellular solution and the electrolyte in the pipette. The technique allows the direct recording of electrical activity generated by the neuron, including the membrane potential, resistance, time constant, synaptic potentials, synaptic currents and action potentials.

Although this technique is relatively difficult to perform compared to patch clamp , sharp electrode recordings allow experiments to be conducted for longer periods, without the common problems of dialysis and run-down that affect neurons recorded using patch clamp.

In every experiment, there is a resistance across the membrane and the recording electrode or access resistance that is in series Rs. When a current I flows across the membrane this resistance leads to an inconsistency, or error, between the measured membrane potential V m and the true potential difference across the membrane.

The measured membrane potential is the command potential controlled by the amplifier. Perforated Patch-Clamp Technique. This process is experimental and the keywords may be updated as the learning algorithm improves. This is a preview of subscription content, log in to check access. Abe, Y. Google Scholar. Ahn, S. Pflugers Arch. CrossRef Google Scholar. Akaike, N. Andersen, O. Bekar, L. Glia 26 2 , — Cass, A. Nature , 47— Dean, J. Neuroscience 80 1 , 21—