Use of microphysiometry for analysis of heterologous ion channels expressed in yeast

Abstract

Measurement of extracellular acidification rates by microphysiometry provides a means to analyze the function of ion channels expressed in yeast cells. These measurements depend on the proton pumping action of the H(+)-ATPase, a central component of the yeast plasma membrane. We used microphysiometry to analyze the activity of two ion channels expressed in yeast. In one example, an inwardly rectifying K+ channel, gpIRK1, provides a potassium uptake function when expressed in a potassium transporter-defective yeast strain. Rates of acidification in gpIRK1-expressing cells directly reflect channel function. Addition of cesium, an inhibitor of gpIRK1 activity, results in an immediate reduction in acidification rates. In a second example, expression of a nonselective cation channel, the influenza virus M2 protein, is believed to interfere with the maintenance of the electrochemical proton gradient by the H(+)-ATPase. In cells expressing the M2 channel, addition of inhibitors increases the rate of proton extrusion. Moreover, functional differences between two M2 inhibitors, amantadine and BL-1743, are distinguished by the microphysiometer. This application demonstrates the utility of the microphysiometer for functional studies of ion channels; it is adaptable to a screening process for compounds that modulate ion channel activity.

Novel Neuroprotective K+ Channel Inhibitor Identified by High-Throughput Screening in Yeast

“…In this study, we developed a novel assay for discovery of K ϩ channel modulators by screening chemical libraries in yeast. The use of yeast expressing a mammalian K ϩ channel for K ϩ channel drug discovery has been suggested previously (Hahnenberger and Kurtz, 1997); however, based on measurement of medium acidification, that approach was successful only for confirming a few model inhibitors, not for discovery of new drugs (Hahnenberger et al, 1996). Our assay monitors optical density of yeast culture growth as a reporter of activity of mammalian channel Kir2.1.…”

Cytosensor techniques for examining signal transduction of neurohormones

“…The same study also demonstrated that activation of the nonselective viral cation channel N2 also induced an ECAR in recombinant cells and that this ECAR was blocked by amantadine and BL-1743, two M2 inhibitors (Hahnenberger et al 1996). However, care must be taken when interpreting studies using ion channel blockers in microphysiometry as some of these compounds also have direct effects on cell metabolism, as recently reported that the potassium channel blocker clofilium (Rabinowitz et al 1997).…”

“…In modified yeast cells, the rate of acidification directly reflects the activity of the inwardly rectifying potassium channel, gpIRK1 (Hahnenberger et al 1996). The same study also demonstrated that activation of the nonselective viral cation channel N2 also induced an ECAR in recombinant cells and that this ECAR was blocked by amantadine and BL-1743, two M2 inhibitors (Hahnenberger et al 1996).…”

Alternative Experimental Models for Studying Influenza Proteins, Host–Virus Interactions and Anti-Influenza Drugs

“…Hence, yeast can also be used to study subcellular localization of the viral protein and its impact on cellular function. Expression of the M2 ion channel in yeast affected growth [130] and the use of a reversible M2 channel inhibitor, BL-1743, caused an increase in transmembrane proton flux due to the re-establishment of the proton gradient by M2. Interestingly, when the influenza PA-X protein was expressed in yeast, 29 unintended mutations appeared in PA-X.…”

“…The above studies demonstrate the efficiency of yeast for high-throughput drug screening in living cells, increasing the number of potential drugs in the pipeline. The same yeast systems could be used to study already known inhibitors and to identify mutations in the viral proteins that cause drug resistance (see in Hahnenberger et al, [130] and Kurtz et al, [134]).…”