Patch clamp technique methods

Researchers at the MoneU Center (Philadelphia, Pennsylvania) are using a variety of electrophysical and biochemical techniques to characterize the ionic currents produced in taste and olfactory receptor cells by chemical stimuli. These studies are concerned with the identification and pharmacology of the active ion channels and mode of production. One of the techniques employed by the MoneU researchers is that of "patch clamp." This method aUows for the study of the electrical properties of smaU patches of the ceU membrane. The program at MoneU has determined that odors stimulate intraceUular enzymes to produce cycUc adenosine 3, 5 -monophosphate (cAMP). This production of cAMP promotes opening of the ion channel, aUowing cations to enter and excite the ceU. MoneU s future studies wiU focus on the connection of cAMP, and the production of the electrical response to the brain. The patch clamp technique also may be a method to study the specificity of receptor ceUs to different odors, as weU as the adaptation to prolonged stimulation (3). 

Precise kinetic electroanalytical data permit to describe quantitatively the kinetics of the whole process with a precision that has never been achieved before by patch-clamp techniques or spectroscopic near-field methods. This enables to investigate finely these events and to identify the exact physicochemical nature of all the individual physicochemical and biological factors which concur to produce vesicular release. 

The field of ion-channel research has met with intense interest during the past ten years. One reason for this development has been the advent of new methods such as the patch clamp technique invented by Sakmann and Neher [1] and new approaches to the cloning of the complete amino acid sequencing of ion channels as it was introduced by Numa and collaborators [2]. 

Cahalan, M. and Neher, E. Patch clamp techniques an overview. Methods Enzymol. 207 3-14,1992. 

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. 

Hamill OP (1993) Cell-free patch clamp. In Kettenmann H, Grantyn R (eds) Practical Electrophysiological Methods. Wiley and Sons, New York, pp 284-288 Hamill OP, Marty A, Neher E, Sakmann B, Sigworth FJ (1981) Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pfltiger s Arch 391 85-100... 

The patch clamp technique can be applied to cultured kidney cells (Merot et al. 1988), to freshly isolated kidney cells (Hoyer and Gogelein 1991) or to cells of isolated perfused kidney tubules (Gogelein and Greger 1984). The latter method shall be described in more detail. 

Current variants of this technique make possible the application of solution on the exterior and interior of whole cells and on the membrane patches torn from the cell (outside-out or inside-out)—every thinkable configuration of solution and ion channel orientation craved by the ion channel researcher. Usually, primary cultured cells or cell lines are preferred as they reveal a relatively clean surface membrane (44) and require no enzymatic treatment that damages the plasmamembrane. The patch clamp technique is now the gold standard measurement for characterizing and studying ion channels and is one of the most important methods applied to physiology. 

In the whole cell model, a gigaseal is formed as the pipette is attached to the cell, and then a more dynamic suction is applied, which causes the interior of the cell to be sucked into the pipette tip (Fig. 3a). This action allows current and conductance of the entire cell to be measured. Therefore, the whole cell model measures changes caused by many ion channels on the entire cell membrane. Additionally, the liquid content of the cell will mix and equilibrate with the solution in the pipette, which allows pharmacological agents to be administered into the cell. Of the patch clamp techniques, the whole cell method is the most common and can be used to determine how pharmacological agents affect the total conductance of neurons. 

The patch-clamp technique requires very sophisticated equipment (Figure 16.22). Sakmann and Neher (Figure 16.1) received the Nobel Prize in Physiology or Medicine in 1991 for the development of this method. A detailed description of the electrophysiological... 

Jiang et al. reported that Ginsenosides-Re, -Rgi, -Rg2 and-Rh had both calcium channel blockade and anti-ffee-radical actions, -Rf had blockade action on L type channel in cultured myocardiocytes of rats with the cell attached configuration of patch-clamp technique and electron spin resonance method[21]. Ginsenoside-Re could coordinately promote the colony formation and increase H-TdR incorporation of bone marrow cell[22]. 

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]. 

Following these studies, improvements in the methods led to experiments being performed that employed a patch-clamp technique and a membrane of only a few micrometers in diameter. In this case, only one pore was opened at any given time, at most, and a birth-and-death process could interpret the experimental results quite closely such that the kinetics of opening and closing of the pore can be evaluated [26],... 

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