In the late 1970s, Bert Sakmann and Erwin Neher developed a patch clamp technique that accurately measured single channel activity [3
]. Their work led to the 1991 Nobel Prize in Physiology or Medicine.
The conventional patch clamp experiment, which consists of manual electrophysiology, uses glass microelectrodes that press against the cell surface to form a tight interaction with a giga-ohm (GΩ) seal resistance between the cell membrane and the rim of glass microelectrode. The current that flows across the plasma membrane can be directly measured by controlling the voltage applied to the membrane. This manual technique allows for a high degree of flexibility in recording configurations, including cell-attached patches, whole-cell patches, inside-out patches, and outside-out patches.
The cell-attached method, or on-cell patch, is often used to study ligand-gated ion channels, channels that are modulated by metabotropic receptors, or neurotransmitters. Compounds that will directly contact the external surface of the membrane are usually included in the pipette solution (Fig. ). This contact allows the concentration-response curves of the compound on ion channels can be accurately determined. The main disadvantage of this method is that only one compound concentration in a concentration-response curve can be measured per patch.
Fig. (1) Schematic representations of four patch clamp configurations. (A) Cell-attached patch or on-cell patch. The electrode is tightly
sealed to the patch of the membrane and the cell remains intact. (B) Whole cell patch. The tip of a micropipette is placed (more ...)
The whole cell method records the currents of multiple channels at once through a V-clamp and measures the membrane potential variation under the current clamp (Fig. ). The advantage of this method is that it allows better electrical access to the inside of a cell because the larger opening tip of the electrode provides lower resistance. The disadvantage is that intracellular contents may be dialyzed by a large volume of the pipette solution after 10 minutes of recording.
The inside-out patch is particularly useful for accessing the environment of the intracellular surfaces of the ion channels (Fig. ). Using this method, the channels that are activated by the intracellular ligands can be studied and the concentration response curves of the ligands can be established.
The outside-out patch can be used to examine the properties of an ion channel that is isolated from the cell (Fig. ). Compared with the cell-attached method, it is more convenient to perfuse the same patch with different solutions, and an entire concentration-response curve of a compound can be obtained in a single patch when the ion channels are activated from the extracellular face.
The loose patch clamp is different from the patch clamps discussed above. This type of clamp forms a loose-seal interaction between the cell membrane and the rim of the glass microelectrode. A significant advantage of this technique is that the pipette can be used repeatedly and the cell membrane remains intact. Thus, the loose patch clamp allows the recording of measurements in different locations on the plasma membrane of the same cell without destroying the cell during the each measurement. The major disadvantage of this method is the larger leakage that occurs due to the loose seal.
The manual patch clamp has long being considered the gold standard for investigations of ion channel properties and compound activities because it usually generates high-quality data, but the experimental procedures are complicated and time consuming. Therefore, the screening throughput for the compounds is very limited. To address this bottleneck, several automated patch clamp systems have been developed and introduced to the research market.