Single cell patch clamp method

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

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. 

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. 

While an array of analytical techniques has been developed to study various aspects of cells (please see Cell Assays in Microfluidics), this entiy is focused on chemical cytometry. As mentioned above, chemical cytometry refers to methods in which intracellular constituents of a single cell are analyzed by means of a chemical separation. Such methods typically employ capillary electrophoresis for separations, combined with laser-induced fluorescence (LIF) or amperometry for detection. We refer readers to other entries for detailed descriptions of other methods used to evaluate the contents of cells ( Patch Clamp Measurements On-Chip, Mass Spectrometry on a Chip). 

Ion channels regulate ion currents and electrochemical potentials across plasma membranes, which are responsible for excitabiUty of nerve, muscle and other type of cells. Single channel current recording using the patch clamp method has revealed electric properties of many kinds of channels at a single... 

Selected entries from Methods in Enzymology [vol, page(s)] Analysis, software for, 207, 717 barrier models, 207, 818 closed and open time estimation, 207, 755 data acquisition, 207, 747 modal behavior analysis, 207, 757 multiple channel problem, 207, 756 single-channel [extraction of kinetic information, 207, 765 measurement in tissue slices, 207, 220] synaptic, resolution improvement in patch clamp recording, 207, 216 whole-cell recording in calcium channel, 207, 181 fluctuation analysis, 207, 192. 

Despite the predominant use of fluorescence and/or patch clamp techniques in single cell measurements, there has been a steady increase in the demand for new electroanalytical tools applicable to single cell studies [4]. Traditionally, such methods have been confined to the development and production of hand crafted sensors including the aforementioned glass capillaries [1-3] for patch clamping, as well as conical microelectrodes for scanning electrochemical microscopy (SECM) [5, 6] and carbon fiber microelectrodes to measure for example, the release of neurotransmitter from single neurons [7, 8]. 

As an example, we consider the oldest and most powerful method for single-molecule kinetic analysis, the patch clamp analysis of ion-channel proteins. Ion channels regulate the flow of simple ions such as Na, K, and Ca across cell membranes. Movement of ions is equivalent to electrical current, and we have very sensitive methods for detecting electrical currents. If we take a small glass pipette and insert it into a membrane just right, we can electrically insulate the patch of membrane inside the pipette from the rest of the world. If done properly, a single ion channel will be in the patch, and we can watch... 

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