Principles behind the techniques - Channel-specific techniques:

Classical voltage clamp is performed with 2 intracellular “sharp” microelectrodes (tip diameter <0.1µm, resistance ~10 MegaOhm when filled with 3 M KCl), one for clamping the membrane potential to values according to various pulse protocols for activation, inactivation, etc., the other measuring the current conducted during the voltage steps. The advantage of this technique that is still used, particularly for oocytes, is its simplicity. The disadvantage is that the membrane of larger cells and oocytes may not be homogenously clamped to a certain value which is especially problematic if rapidly-activating ionic currents are studied.
 
Whole-cell recordings are performed with a glas pipette pulled as “patch clamp microelectrode” (Fig. 6 left; tip diameter of ~1 µm, resistance of ~1 MOhm when filled with 100 mM KCl). Once the seal is established, a brief suction is applied to the interior of the electrode in order to rupture the patch of membrane under the electrode tip. After this break-in, there will be a low-resistance pathway for current and diffusional flow between the electrode and the cell interior. The cell membrane is voltage-clamped at the pipette potential by virtue of this low-resistance pathway, and the electrode monitors the current flowing across the entire cell surface. The ionic composition of the cell cytoplasm rapidly equilibrates with the pipette contents, offering a pathway for the control of cellular constituents. Although whole-cell recording can produce highly accurate current recordings, two important limitations are associated with the technique. The first of these is correlated with the resistance in series with the membrane (Rs), which in whole-cell recording is the access resistance between the interior of the pipette and the cell cytoplasm (for details see Armstrong and Gilly, 1992). Another limitation of the whole-cell configuration is that important intracellular regulatory molecules, like cAMP, Ca2+, or GTP, can diffuse out of the cell through the patch electrode. Thus, the physiological regulation of these important second messenger substances is disrupted during whole-cell recording. The perforated-patch technique (Horn and Korn, 1992) provides a solution to this problem by making it possible to record macroscopic currents with a cell-intact recording configuration. This configuration is obtained by including a pore-forming antibiotic, like nystatin or amphotericin B in the pipette solution. After a seal is formed on the cell, the antibiotic channels insert in the patch of membrane under the electrode tip, thereby providing electrical continuity between the pipette and the cell interior.
In conclusion, the whole-cell mode measures the current through the total cell membrane superimposed with noise. This “macroscopic” current corresponds to the average of many simultaneously conducting channels and therefore resembles the intracellular recordings. Because of the simple and fast analysis, it is the configuration that is most frequently used.
 
Cell-attached, inside-out and outside-out modes allow the measurement of few channel proteins or even a single channel in the electrically isolated membrane patch, dependent on how many channels are embedded in the membrane patch. The various configurations are available to the investigator once the seal is established (see Fig. 3). The cell-attached mode is already made as soon as a GigaOhm seal between the patch electrode and the cell membrane is established (Fig. 6 left, same electrode as for whole-cell recordings, however filled with an “extracellular” solution). The background noise can be sufficiently attenuated so that the current flowing through a single ionic channel can be resolved (single-channel recordings) (Hamill et al., 1981). A special form is the bleb-attached mode that enables the measurement of adult native cells without enzymatic treatment (Fig. 6). Blebs (or blisters) can be formed, e.g. by stretching of skeletal muscle fibres in a high-calcium bath solution (Quasthoff et al., 1990; Lerche et al., 1995). The advantage is that the plasmamembrane is absolutely clean so that a GigaOhm seal can be relatively easily yielded.
 

Figure 6
Typical microelectrode suited for patch clamping (left panel, courtesy of Dr. Ebert) and its attachment to either a membrane bleb formed from a native human skeletal muscle fibre (middle panel) or a HEK cell (right panel). The upper left panel shows the glass microelectrode, the lower part the tip with a diameter of about 700 nm. The plasmalemmal bleb in the middle panel was formed by careful stretching of native muscle fibres at ³ 10-5 M [Ca2+] in the bathing solution. The membrane of native HEK and other cultured cells is so clean that GigaOhm seals can be easily produced without additional measures.
 

If the recording electrode is withdrawn from the cell after a cell-attached patch is formed, the patch of membrane can be excised from the cell with the inside surface of the membrane facing the bath solution; this is called the inside-out configuration. An outside-out patch can be formed by removing the electrode after entering the whole-cell configuration. One advantage of these cell-free patch recordings is that they provide more accurate control of the membrane potential. In the cell-attached mode, the membrane potential of the patch is equal to the resting potential of the cell minus the pipette potential. Since the resting potential may not be known with certainty or might vary during an experiment, there will be uncertainty in the value of the patch membrane potential. However, in the isolated patch configurations, the magnitude of the patch potential is equal to the pipette potential and is therefore known with precision. A second advantage is that the solution bathing one surface of the patch can rapidly be changed simply by changing the bath solution.
Once a recording mode is established the recordings can be performed with a typical patch clamp circuit which uses the electrode both to control the potential and to measure the currents conducted by the channels (Fig. 7).
 

Figure 7
Patch clamp set-ups. Upper panel: Classical whole-cell configuration and circuit using a glas micoelectrode. Lower panel: Patch clamp chip and circuit. This arrangement can be multiplied and automated for high-throughput screening. In addition, the arrangement allows the scientists to install a second technique, e.g. fluorescence or force microscopy, from the upside of the set-up.
 

back