Whole cell patch clamp

The single electrode patch clamp technique allows direct evaluation of the magnitude and the electrical properties of currents under voltage clamp conditions (Hamill et al., 1981; Sakmann and Neher, 1984). In its whole cell configuration, this method has been used to study volume-sensitive channels under well-defined (ionic) conditions in numerous cell models, leading to the identification of K+ channels involved and to a detailed electrical characterization of VRAC (see earlier discussion). Although whole cell patch clamping is a well-established technique, widely used by many different research groups around the world, and almost all of the equipment and software needed is available commercially, investigators who are planning to introduce this technique in their own laboratory are strongly advised to get into contact with a laboratory specialized in membrane electrophysiology. This is because of the special requirements needed to isolate the setup from environmental electrical noise and mechanical vibrations. Because a high-resistance seal between the pipette and the cell membrane can be obtained easily when the cell has a smooth surface (Hamill et al., 1981; Neher, 1981), it is much easier to study cultured cells at low densities than using fully differentiated enterocytes containing numerous microvilli and an apical mucous layer. As a consequence, isolated villi and crypts, as well as stripped intact intestinal epithelium, are less suitable for patch clamp analysis. In combination with fluorescence microscopy, patch clamping is very useful to study channel activation in cells transfected with fluorescently tagged vectors, making it possible to identify cells with high expression levels. Notably, in contrast to the isotope efflux assays and short circuit measurements (described later), the RVD is not functional under whole cell patch clamp conditions and the cell swelling-induced currents last as long as the bathing solution remains hypotonic with respect to the pipette solution (see Nilius and Droogmans, 2003).

Whole cell recordings can be obtained after disruption of the membrane patch directly under the pipette by mild negative pressure, creating a direct access between the cell interior and the microelectrode. Although this allows us to control the intracellular composition, it could also result in a dilution of essential cellular components and lead to a run down of the currents. To avoid diffusion of small molecules, but not ions, the perforated patch configuration (Levitan and Kramer, 1990) can be used, in which the membrane patch under the pipette is not disrupted but is instead permea-bilized using pore-forming antibiotics (nystatin, amphothericin).

To investigate VRAC activation in Intestine 407 cells, we use a bathing isotonic solution composed of 110 mMCsCl, 5 mMMgSO4,3.5 mMsodium gluconate, 12 mMHEPES, 8 mMTris-HCl, and 100 mMmannitol at pH 7.4 (Van der Wijk et al., 1999). The intracellular pipette solution contains 110 mM CsCl, 2 mM MgSO4, 25 mM HEPES, 1 mM EGTA, 1 mM Na2ATP, and 50 mM mannitol, pH 7.4. Patch pipettes are pulled from borosilicate glass (Clark Electromedical Instruments, Pangbourne, Berks, UK) and heat polished and have a resistance of 2 to 3 MQ. Whole cell current recordings are made using a RK-300 amplifier (Bio-Logic, Claix, France) and digitized using a Digidata 1200 AD converter (Axon Instruments Inc., Foster City, CA).

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