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Techniques Used in the Fischer/Illek Lab

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.

Measurement of transepithelial ion transport in Ussing chambers. This technique is used to measure ion transport by intact, isolated epithelial tissue. It is named after the Danish physiologist Hans Ussing who invented it. We use it for drug screening and to verify results from other techniques. These measurements are quantitative, easy to learn, and quick.

pH stat technique. This method is used 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 quantifies secretion of acid (or base) by the epithelium. Using this technique, we have recently discovered that the airway epithelium secretes acid into the ASL.

Figure 1 Patch clamping of single cells on the stage of a microscope

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. Cells are investigated in isolation on the stage of a microscope. A small glass pipette (~1 mm 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. We measure 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 for in vivo drug testing.

Current Projects
One current CF-related project is the role and regulation of basolateral chloride channels in salt secretion and absorption across airways. The primary transport process across the airways is active sodium absorption. Chloride follows passively according to its electrochemical driving forces. During transcellular chloride movement chloride has to cross the apical and the basolateral membrane. Previous work has focused on the apical membrane where CFTR is expressed (the chloride channel that is defective in CF). Transport across the basolateral membrane was unclear. We found that three physiologically and biophysically distinct chloride channels are present and functional, and they determine the overall rate of chloride transport by the epithelium.

Another project is the acid and base transport across the airway epithelium. Interestingly, the airway surface liquid (ASL) is slightly acidic (pH = 6.9) compared to other extracellular fluids (pH = 7.4) in normals. It has been hypothesized that ASL in both CF and asthma is even more acidic than normal. The source of the acidity is unknown. We hypothesized that the airway epithelium expresses apical proton transporters, which acidify the ASL in a regulated manner. In a study using the pH stat technique and patch clamping we found apical proton channels in airway epithelial cells, which are the major route for acid secretion into the ASL. Currently we investigate whether the proton channels are mis-regulated in asthma and CF. In addition we investigate the secretion of bicarbonate (which buffers protons in the ASL). Our results suggest that a disequilibrium between acid (proton) and base (bicarbonate) secretion into the ASL is an important factor in the pathogenesis of CF and asthma.


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