Description of the Patchclamp Technique

Successful patch clamping is the result of having mastered two technical aspects. First, one must be able to physically position the micropipette onto the cell surface without breaking through the membrane. Secondly, the solutions both within the pipette and in the superfusing medium should mimic the intra-and extra-cellular media as closely as possible. The following paragraphs introduce the basic elements ofpatch clamping that should enable the reader to understand the concepts involved. Due to space limitations, descriptions will be brief. Those interested in furthering their knowledge of the more practical aspects (pipette fabrication, micro-circuitry, electronics, voltage-clamp protocols) of patch clamping should consult other textbooks and monographs on these subjects (3, 9). To further understand the more practical applications ofthe patch-clamp technique, we suggest that the readers consult the extensive list of literature that has been published using this technique in different cell types.

3.2.1. Forming the Gigaohm Seal

The pipette is maneuvered toward the cells using a remote-controlled micromanipulator (Fig. 2A). When the pipette filled with electrolyte solution is immersed in the bathing medium, a junction potential is created which must be zeroed (i) prior to touching the cell membrane. As the pipette touches the cell, tip resistance is increased, making the square-wave voltage pulse seem smaller (ii). With the tip of the pipette firmly pressed down on the cell (near the center of the cell to satisfy the "space clamp" criterion), negative pressure is applied via the pipette holder (iii) and tip resistance increases into the gigaohm range. At this point, the voltage pulse should be seen as a flat line, with capacitance spikes apparent at its right and left edges.

3.2.2. Configurations and Their Applications

When a gigaohm seal has been achieved, the patch is said to be in the cell-attached configuration. Voltage pulses applied by the amplifier via the pipette allow to record the opening of single-channels located within the patched membrane area (Fig. 2B). In this configuration, one can control channel activity only via voltage or solution alterations, which is a serious limitation for experimental purposes. In order to control both the intra- and extracellular media, the membrane patch may be manipulated in a few ways. First, the pipette can be rapidly pulled up, without breaking the gigaohm seal, resulting in an inside-out configuration, where the outer face of the membrane is sealed within the pipette and the inner face is exposed to the bathing medium (iv). Secondly, an antibiotic ionophore (e.g., nystatin, amphotericin B) can be added to the pipette solution and allowed to diffuse toward the membrane. Gradually, embedded ionophore molecules permeabilize the membrane (perforated patch), allowing for diffusion of small molecules such as ions, but not of cytoplasmic proteins and large molecules (v). Thirdly, additional negative pressure or a strong and fast voltage surge ("ZAP") can be applied via the pipette to rupture the membrane patch, giving the standard whole-cell configuration (vi). In this mode, the user can easily control both the intra- and extra-cellular environments. Macroscopic currents recorded from cells in this configuration represent the cumulative activity of all the ion channels contained within the cell membrane (Fig. 2C). The outside-out configuration is achieved by rupturing the cell membrane, and then gently pulling up the pipette (vii), thereby stretching the membrane outside the pipette until it reseals itself (viii), forming a miniaturized cell. The latter two configurations require that cells be tightly attached to the bottom of the chamber.

Each of these patch-clamp configurations has its experimental applications. Single-channel current recordings from cell-attached patches provide vital information about the gating and kinetics of individual channel proteins under physiological conditions, and are also an invaluable tool in identifying closely related currents based on their single-channel permeability or ion conductance. The possibility of simultaneously recording the activity of multiple channels is the greatest advantage provide by the use of whole-cell configurations. Furthermore, it allows the researcher to measure currents from small conductance ion channels, currents which would be difficult to resolve under other circumstances. On the other hand, the large pipette volume also acts as a sink into which normally intracellular metabolic factors may dissolve during the experiment, potentially resulting in increased channel rundown.

Figure 2. Patch clamp electrophysiology. A: Formation of the gigaohm seal (described in the text) results in either the cell-attached (iii) or whole-cell (vi) patch clamp configurations. Whole-cell access can also be achieved using ionophores (v) that selectively permeabilize the membrane. Inside-out (iv) and outside-out (viii) configurations are variants of the cell-attached and whole-cell configurations, respectively, achieved by pulling the patch pipette away from the cell surface while maintaining the gigaohm seal. (Reproduced with permission from Ref. 3) Current measurements from cell-attached (B) and whole-cell (C)provide information on channel gating properties and kinetics. Openings of sarcolemmal channels contained within the pipette mouth can be observed in the cell-attached mode. The single-channel openings are characterized by their amplitude (Ba), open (Bb) and closed (Be) time durations, and open probability (Bd). Single-channel conductance, a value unique for each channel type, can be derived from the current-voltage relationship generated from these recordings. Whole-cell currents, representing the summation of all currents from channels on the membrane, are distinguished by their amplitude (Ca), and their activation (Cb), inactivation (Cc), and deactivation (Cd) kinetics.

Figure 2. Patch clamp electrophysiology. A: Formation of the gigaohm seal (described in the text) results in either the cell-attached (iii) or whole-cell (vi) patch clamp configurations. Whole-cell access can also be achieved using ionophores (v) that selectively permeabilize the membrane. Inside-out (iv) and outside-out (viii) configurations are variants of the cell-attached and whole-cell configurations, respectively, achieved by pulling the patch pipette away from the cell surface while maintaining the gigaohm seal. (Reproduced with permission from Ref. 3) Current measurements from cell-attached (B) and whole-cell (C)provide information on channel gating properties and kinetics. Openings of sarcolemmal channels contained within the pipette mouth can be observed in the cell-attached mode. The single-channel openings are characterized by their amplitude (Ba), open (Bb) and closed (Be) time durations, and open probability (Bd). Single-channel conductance, a value unique for each channel type, can be derived from the current-voltage relationship generated from these recordings. Whole-cell currents, representing the summation of all currents from channels on the membrane, are distinguished by their amplitude (Ca), and their activation (Cb), inactivation (Cc), and deactivation (Cd) kinetics.

The inside-out and outside-out excised-patch configurations allow for the precise control of the cellular environment, i.e. the user controls the ionic compositions on either side of the membrane. This allows for a more precise understanding of the permeation processes (pore selectivity and conductance), gating properties (channel opening and closure), and metabolic regulation (by cytosolic or extra-cellular proteins or pharmacological tools). The flexibility of the patch-clamp configurations provides the researcher with a selection of tools available to study current activity.

3.2.3. Troubleshooting and Limitations

Because we can only speculate as to the exact composition of the intra- and extracellular media, the ionic composition of the solutions used in patch clamp experiments becomes a determining factor in isolating and identifying currents. Currents can be identified using ion-selective solutions. For example, in studying outwardly rectifying K+ channels in vascular smooth muscle cells, the pipette solution may be Na+- and Ca2+ free to minimize the activity of Na+ and Ca2+ channels. Selective pharmacological channel attenuation is another approach used by electrophysiologists to dissociate currents. For example, to identify voltage-dependent K+ without the interference of other K+ channels, one might include ATP and EGTA (a Ca2+ chelator) to the pipette media to attenuate the activity of ATP-sensitive and Ca2+-activated K+ channels, respectively. Finally, the control of the transmembrane potential offered by the patch-clamp technique allows the user to selectively regulate channel activation by modifying the holding potential of the patch. For example, Na+ channel activity can be minimized by using a relatively positive holding potential of -40 mV. Similarly, using Ca2+-channel selective solutions, a depolarizing pulse to 0 mV from a holding potential of -70 mV will select for the activation of L- type voltage-dependent Ca2+ channels, while a pulse to -20 mV from a holding potential of -100 mV will select for T-type voltage-dependent Ca2+channels.

3.3. Voltage- and Current-clamp Modes

Most commercially available patch clamp amplifiers allow the user to select between the voltage-clamp and current-clamp modes. Measurement of ion currents, as described insofar, is done in the voltage-clamp mode. In this mode, the user defines the desired voltage to be clamped by the amplifier. In this mode, the amplifier measures the membrane potential, then injects or removes electrons (current) to deflect the membrane potential toward the desired (clamp) potential. The amplitude of the current that is applied by the amplifier is measured by the acquisition software and is assumed to be equal to, but opposite in direction to, the amount of current generated by the channel proteins at that membrane voltage. This mode is useful to study the kinetics of ion channels or of a specific ion channel at any specified voltage.

In the current-clamp mode, the amplifier simply reports membrane potential while applying (clamping) a desired current to the cell membrane. This mode is useful to study natural responses of a cell to a specific stimulus.

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