Patch-Clamp Method

Electrophysiological Experiments. Guinea pig myocardial cells prepared as described previously 24) were superfused at 37 C with a Tyrode solution. Electrical properties of the myocytes were examined by the patch-clamp methods (25) using fire-polished pipettes. The current was measured by means of a patch-clamp amplifier, stored on the tape through a digital PCM data recording system, and analyzed with a computer. 

A circuit that uses a differential amplifier to maintain constant membrane potential by electronically balancing the ion channel current. This method allows the experimenter to analyze action potentials of excitable membranes resulting from an initial transient rise in sodium ion permeability followed by a transient rise in potassium ion permeability The technique is especially valuable for studying kinetic properties of voltage-gated channels as well as voltage-dependent channels. See Membrane Potential Patch Clamp Methods... 

MEMBRANE POTENTIAL PATCH CLAMP METHOD Voltammetric sensors,... 

In a record obtained by the patch clamp technique, the channel is closed for much of the time (i.e. no current flows across the patch of membrane that contains it), but at irregular intervals the channel opens for a short time, producing a pulse of current. Successive current pulses are always of much the same size in any one experiment, suggesting that the channel is either open or closed, and not half open (there are exceptions to this rule). The durations of the pulses, however, and the intervals between them, vary in an apparently random fashion from one pulse to the next. Hence the openings and closings of channels are stochastic events. This means that, as with many other molecular processes, we can predict when they will occur only in terms of statistical probabilities. But one of the most useful features of the patch clamp method is that it allows observation of these stochastic changes in single ion channels as they actually happen individual protein molecules can be observed in action. 

Ions and small molecules may be transported across cell membranes or lipid bilayers by artificial methods that employ either a carrier or channel mechanism. The former mechanism is worthy of brief investigation as it has several ramifications in the design of selectivity filters in artificial transmembrane channels. To date there are few examples where transmembrane studies have been carried out on artificial transporters. The channel mechanism is much more amenable to analysis by traditional biological techniques, such as planar bilayer and patch clamp methods, so perhaps it is not surprising that more work has been done to model transmembrane channels. 

Fig. 2 Configuration of the different patch clamp methods. The arrows indicate the direction of suction...

Schlatter (1993) recorded membrane voltages of macula densa cells with the fast or slow whole-cell patch-clamp method. The effects of diuretics and the conductance properties of these cells were examined. 

The inhibitory potencies of three of these molecules agreed with published values for murine Kv 3 (Table 4.1). The one exception was ShK that appeared to be 10-fold less potent than both published values and our own in-house values obtained by manual patch-clamp methods. It has been reported that ShK has a slow on-rate (t= 20 min) for block of Kv 3 (Middleton et al. 2003). This phenomenon may contribute to the reduced potency of ShK in our automated electrophysiology assay, because our protocol included a compound incubation time of only 5 to 10 min. Longer compound incubation times may improve the potency of ShK but would be associated with greater run-down in the K+ current amplitude. We also tested the A LI-selective blocker, dendrotoxin, and not surprisingly it did not inhibit the Kv. 3 current at concentrations up to 167 nM, which is well above its IC50 value for Kv 1.1 in our hands (17 pM data not shown). 

The frog oocytes expressing the mutant channels were studied using the patch-clamp method, which allows the characterization of single channels on intact cells. ... 

Figure 3 Schematic of a) whole cell, b) outside-out, and c) inside-out patch clamp methods.

In the outside-out model, the pipette is attached to the entire cell as in the whole cell model, followed by a sharp pull that causes the cell membrane to break and reseal with the pipette tip (Fig. 3b). With the extracellular region exposed, channel activity as a response to different external stimuli can be probed. This configuration is less common than the inside-out method. Using an outside-out method, single-channel opening activity has been recorded while various neurotransmitters were released. For example, this patch clamp method was used as a detector for capillary electrophoresis separations of GABA, glutamate, and NMDA (7). 

Furthermore, the activity of a channel in its native membrane environinent. even in an intact cell, can be directly observed. Patch-clamp methods pro vided one ot the first views ot single biomolecules in action. Subsequently, other methods for observing single molecules were invented, opening new vistas on biochemistry at its most fundamental level. 

With the existence of ion channels firmly established by patch-clamp methods.. scientists sought to identify the molecules that form ion channels. The Na channel was first purified from the electric organ oi electric eel, which is a rich source of the protein forming this channel. That protein was purified on the basis of its ability to bind a specific neurotoxin. Tetrodotoxin, an organic compound isolated from the puffer fish, binds to channels with great avidity (K, 1 nM). The lethal dose of this poison for an adult... 

Currently, it is standard procedure to develop ion channel-specific antibodies for immunocytochemistry, to perform Western and Northern blot analyses, ion channel in situ hybridization, or reverse transcription polymerase chain reaction (RT-PCR). The introduction of the single-cell RT-PCR in combination with the patch-clamp method in the 1990s made it possible to identify gene transcripts and to correlate them with functional data for the same individual cell. Finally, one of the most powerful cell biological techniques in the study of ion channels is based on artificial expression systems such as microinjection of mRNA encoding channel subunits into Xenopus oocytes and selective expression of native ion channels or with different subunit composition (e.g., Ky channel subunits). Because the Xenopus oocytes are large, they are a perfect model to study artificially expressed channels. Another good model for artificial ion channel expression is the Chinese hamster ovary (CHO) cell line. 

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