Single Cell Electrophysiology Techniques

Second post for PHYL3001! Here we will learn about the different methods used to measure membrane potential and voltage across the membrane.

In vitro (cell free) systems- artificial membranes

Artificial membranes are a useful way of finding out how altering the ionic composition on either side can affect the membrane potential. These thin membranes, which can contain membrane proteins, are "painted" across a small diameter hole. The solutions on either side of the artificial membrane can have their composition altered, and their voltage measured by the use of Ag/AgCl electrodes.

In vivo systems- glass micropipette electrodes

Glass micropipette electrodes, which can be poked through a cell membrane without damaging it, can be used to measure membrane potentials in vivo. These cone-shaped electrodes, which have a small opening at the bottom, are filled with a solution such as KCl and are connected to a pre-amplifier by an Ag/AgCl wire. A second wire connected to the pre-amplifier, called the reference wire, sits in the bathing medium surrounding the cell. This allows the potential difference between the inside and outside of the cell to be measured.

While glass micropipettes can be useful in helping us record the inside of the cell, they do have disadvantages. They have a high electrical resistance, resulting in lots of noise and interference. Special electronics are also required due to the high resistance.

How are ionic currents passing through membranes measured?

Ionic currents passing through the membrane can be measured by means of the voltage clamp method. Two electrodes are used in this method: one to measure the membrane potential and the other to inject a current into the cell. The electrode measuring membrane potential feeds back to a feedback amplifier, which adjusts the current to keep the membrane potential constant.

The voltage clamp method relies on the logic that to keep the membrane potential constant, the current going into the cell must be equal to the current going out of the cell. Hence, when we adjust the current to keep the membrane potential the same, we are really adjusting the inward current to be equal to the outward current. In this way, we can measure the outward membrane current. (Or at least that's my understanding of how this works :P)

How is single channel behaviour measured?

The behaviour of single channels can be measured by use of the patch clamp technique, which uses blunt glass micropipettes. Unlike those used in the electrode technique above, these pipettes do not impale the membrane. Instead, they suck up a small portion, such that only a small number of ion channels are contained within the "sucked up" part. The current in this small section can be measured.

There are a few variations on the patch clamp method. If a strong suction is used, the cell membrane can rupture, allowing the pipette to directly access the cytoplasm. The pipette can then be retracted and the broken ends of the membrane allowed to anneal, forming a loop of membrane in an "outside-out configuration." (If the pipette is retracted when using normal suction as above, then an "inside-out configuration" is created instead.)