Single Channel Versus Multi-Channel Electrophysiology: How Does it Work?

One may be familiar with one type of recording and be curious of the other. Some may use electrophysiology as a tool rather than a core technology and may have an interest in the advantages of each. Still others may work with a collaborating lab and have limited knowledge of the subject in general.

No matter the approach, its certainly useful to explore and summarize the differences between single-channel and multi-channel electrophysiology and the use of each in modern neuroscience applications.

Fundamental differences between single- and multi-channel electrophysiology

Multi-channel electrophysiological recordings are the sum total measurements of electrical activity across a cell membrane.

In contrast, single-channel recordings are the discrete measurements of individual channel activity across a cell membrane

As an analogy, multi-channel recordings are like a piano chord – a collection of notes that together produce a collective sound. Single-channel recordings are akin to a single note – the frequency and amplitude of which is reflective of single channel opening or closing events.

Before diving into further details regarding the distinction, a discussion of the types of membrane recording setups is warranted.

What is electrophysiology?

Excitable cells in the body express voltage differences (otherwise known as voltage potentials) across their respective cell membranes. Examples of excitable cells include cardiac myocytes (heart muscle), sensory neurons (nerves), motor neurons (muscle movement), and a host of other electrically active cell types.

Electrophysiology is the study of this electrical activity and the cellular factors or processes that are involved. Voltage sensitive channels open and close in response to electrical stimuli, and the resulting flux of ions across the membrane can be measured as electrical current. This is, of course, a gross simplification of electrophysiology and the intricate cell-specific mechanisms that are involved. Nonetheless, it will do for the current discussion (get it, current?).

Voltage clamp versus current clamp

In electrophysiological recording, one can “clamp” the membrane potential at a chosen value. Doing so makes it possible to measure the current across the membrane. Voltage-dependent channels conduct ions when activated or open, and cease to conduct when de-activated or closed. Perturbations of channel function via the actions of drugs, small molecules, or channel mutations can be examined using time- or concentration-dependent kinetic measurements.

The ion current or conductance can also be clamped, and changes in membrane voltage can be measured after injecting a current into the cell. This technique is important for understanding how voltage-gated channels activate in response to ion flux, action potentials, and neurotransmitters in the body.

Intracellular recording

In the intracellular recording technique, a fine microelectrode is injected into the cell and the voltage and/or current across the membrane is measured and compared with a reference or bath electrode. An amplifier magnifies the strength of the signal and a filter removes electrical noise.

Patch clamp recording

Patch clamp techniques differ from the above in that the electrode is attached to the extracellular side of the cell membrane using gentle suction, as opposed to being impaled into the intracellular compartment of the cell. The seal formed between the extracellular electrode and the membrane permits measurement of voltage and/or current across the “patch” of membrane contained within the glass electrode tip

Whole cell recording

In “whole cell” mode, the patch of membrane, as described above, can be removed through increased suction and total cell voltage and/or conductance can be measured.

Excised patch clamp recording

In the “excised patch” technique, the patch of membrane as described above is retained within the glass electrode tip and the remaining cell membrane is removed. This allows both sides of the membrane patch to be perturbated and subsequent changes in electrical activity measured with high sensitivity.

Artificial lipid bilayers

Another technique involves formation of an “artificial bilayer” across a small hole separating two vessels of buffer. Channels can be formed by addition of protein or vesicles to one of the compartments and changes in bilayer voltage and/or conductance (and the influence of exogenous chemicals or compounds) can be measured.

Multi-channel recordings

Multi-channel recordings, as in the case of intracellular or whole cell electrophysiology, are useful for study of the electrical activity of the cell. The channel activity of the cell in response to chemical or electrical stimuli can be fundamental in understanding the cellular response to drugs, neurotransmitters and action potentials, mechanosensation, chemical insults and toxicology, and a host of other processes.

Baseline currents

The transient or baseline currents across the cell membrane must be controlled and monitored throughout, in order to subtract this activity from actual measurements. Each technique, whether intracellular, whole-cell, or patch clamp, presents its own set of challenges for controlling background currents and the potential for membrane leakage during recording.

Cell models for recordings

To study distinct channel activity, the channel protein is typically overexpressed in the cell background. Many cell models exist for channel overexpression including a popular system for mammalian expression, African Clawed frog eggs or Xenopus oocytes. These unfertilized germ cells are harvested from the host and are conditioned to receive DNA or RNA necessary for channel overexpression. The oocytes are large enough to easily manipulate with injection tips and electrodes during the recording process.

Advantages of multi-channel recording

The conductance measured from overexpressing cells are a reflection of the channel activities. After subtraction of baseline from non expressing cells, detailed results can be analyzed. The kinetics of channel opening, closing, de-activation, block and unblock, and a host of other time and concentration dependent events can be closely examined.

Limitations of multi-channel recording

The measurements obtained are the collective activities of a population of channels. Therefore, individual channel attributes such as channel conductance, channel blockage, channel flickering, and other exclusive events cannot be determined by multi-channel means. Moreover, the conductance behavior of individual channels cannot be deciphered – is the conductance due to the open state and the physical dimensions of the channel or a result of rapid opening and closing of the channel? -- nor can the potential for polar effects and heterogeneity throughout the population of channels – some function normally while other exhibit aberrant behavior, etc.

Advantages of single-channel recording

Single-channel recording permits high-resolution measurement of unique channel events. One can see discrete jumps in conductance as the channel opens and a return to the former conductance when the channel closes. One can also see both the duration of channel opening and closing as well as the frequency of channel gating. Channel blockage due to exogenous compounds as well as potential de-activation due to channel over stimulation (a behavior of certain voltage-gated channels) can be observed.

Challenges and limitations of single-channel recording

Attached or excised patch clamp techniques can provide a platform for single channel recording. A challenge is balancing expression levels of the channel with capture of individual channels in a given patch – if expression is too high, single channels may be hard to isolate, if expression is too low, single channels may be difficult to locate. Commonly more than one channel will be present in a given recording, which is not necessarily detrimental to measurement experiments. With proper electrical resolution, one can see each discrete channel opening event, and the step-wise increases (or decreases) in conductance due to subsequent individual channel events.

Artificial bilayers can be very useful in tightly controlling channel entry into the membrane and hence the presence of single channels for recording. One can record the baseline and normalize measurements once the membrane is formed, then observe the entry or appearance of a single channel after addition to the bathing buffer.

The bilayer is artificial, however, and channel activities must be considered in the context of the membrane system chosen for the experiments and how this may differ from the physiological context. Moreover, the absence of cofactors or proteins which may interact and modulate channel activity is always a consideration, no matter the system or the recording modality.

A major limitation can also be the recording setup or rig and the electrical equipment used for measurement of channel activity. If the recording rig is not adequately isolated from vibrational or electrical noise, one runs the risk of not being able to observe single-channel events. The integrity of cell membrane or artificial bilayer can severely impact observations as well, as can the presence of contaminating chemicals and compounds.

For these reasons and more, single-channel recording must be performed in a tightly controlled environment free from the multitude of factors that can make analysis difficult.

All things considered, the two recording platforms can be complementary, and in synergy can offer exquisite insight into the behavior of channels underlying complex cellular processes.

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