Email: hfischer@chori.org
Phone: 510-450-7696

Email: billek@chori.org
Phone: 510-450-7699

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Techniques

Electrophysiological techniques are central for the investigation of ion transport mechanisms. Our laboratory has successfully applied electrophysiological approaches to CF research. By using the patch clamp and the Ussing chamber techniques our investigations opened up significant new avenues for CF research. In addition we use real-time fluorescent microscopic techniques to measure intracellular pH.

Measurement of transepithelial ion transport in Ussing chambers. This technique is used to measure ion transport by intact, isolated epithelial tissue grown in cell culture. It is named after the Danish physiologist Hans Ussing who invented it. We use it for CF drug screening in airway epithelial preparations, and also to verify results from single cell investigations. Ussing chamber measurements have the advantage that cells are investigated in a polarized epithelial setting, which allows for the identification of the localization of transport proteins to the apical or

basolateral membranes. This technique can alsocan also be used to investigate an apical (or basolateral) membrane preparation in isolation by specifically permeabilizing the other membrane. These measurements are quantitative, easy to learn, and quick.


pH stat technique. We use pH stat to measure epithelial acid or base secretion. It is based on the continuous titration of the epithelial bathing medium to a given pH, which then is used to quantify secretion of acid or base by the epithelium. These measurements are useful to characterize the effect of epithelial activity on the extracellular pH, for example, the pH of the

airway surface liquid. There are indications that the airway pH is one factor that controls the antibacterial activity of the airways. Using this technique, we have originally discovered that the airway epithelium secretes acid into the airway surface liquid (Fischer et al, 2002).


Patch clamp technique. Patch clamping is used to measure ion currents across a single cell or through a single ion channel in the cell membrane. This technique was derived from other intracellular microelectrode techniques in the early 1980s by a team of investigators honored by the Nobel prize in 1991. Cells are investigated in isolation on the stage of a microscope. A small glass pipette (~1 um inner

diameter) is attached onto a cell and used as an electrode. We use various patch clamp techniques, including whole cell and cell-attached recordings, and recordings from excised inside-out or outside-out membrane patches. In addition we use noise analytical techniques to investigate channel-types that are difficult to record with standard patch-clamp techniques. Patch-clamping is extremely versatile and we use it to investigate: 1) intracellular signaling cascades that regulate ion channel activity, 2) channel characteristics and kinetics, and 3) drug effects on ion channels.

Recordings of the nasal potential difference (nasal PD). This is a technique that is used to indirectly measure channel activity in people or in mice. Measurements are based on ionselective diffusion potentials across the nasal epithelium. The nose is used because it is easily accessible. It has been shown that the nasal PD is significantly changed in CF. We use these measurements as a diagnostic test for CF, and also for in vivo drug testing in mice.

Measurement of intracellular pH. Both intracellular buffers and acid and base transporters in the plasma membrane regulate the intracellular pH.  It can be measured by using the pH-sensitive fluorophore BCECF. When cells are loaded with BCECF and excited with light at 440 nm and 495 nm, the ratio of the emitted light

at 510 nm is related to the intracellular pH. Typically, the ratio image (in false colors) is used to visualize the intracellular pH. Using this technique we found out that the NADPH oxidase of the airways, DUOX1, generates intracellular acid, which is the secreted into the airway surface liquid. There, it likely contributes to the generation of H2O2 and bacterial killing. 

 

Revised: Wednesday, January 25, 2012 4:48 PM

 

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