Abstract
Cationic amphipathic drugs, such as amiodarone, interact preferentially with lipid membranes to exert their biological effect. In the yeast Saccharomyces cerevisiae, toxic levels of amiodarone trigger a rapid influx of Ca(2+) that can overwhelm cellular homeostasis and lead to cell death. To better understand the mechanistic basis of antifungal activity, we assessed the effect of the drug on membrane potential. We show that low concentrations of amiodarone (0.1-2 microm) elicit an immediate, dose-dependent hyperpolarization of the membrane. At higher doses (>3 microm), hyperpolarization is transient and is followed by depolarization, coincident with influx of Ca(2+) and H(+) and loss in cell viability. Proton and alkali metal cation transporters play reciprocal roles in membrane polarization, depending on the availability of glucose. Diminishment of membrane potential by glucose removal or addition of salts or in pma1, tok1Delta, ena1-4Delta, or nha1Delta mutants protected against drug toxicity, suggesting that initial hyperpolarization was important in the mechanism of antifungal activity. Furthermore, we show that the link between membrane hyperpolarization and drug toxicity is pH-dependent. We propose the existence of pH- and hyperpolarization-activated Ca(2+) channels in yeast, similar to those described in plant root hair and pollen tubes that are critical for cell elongation and growth. Our findings illustrate how membrane-active compounds can be effective microbicidals and may pave the way to developing membrane-selective agents.
Highlights
Broad range fungicidal effects at concentrations ranging from 5 to 15 M
We show that a polarized membrane is added to 3 ml of yeast cell suspension to a final probe concencritical for drug toxicity; the change in membrane tration of 0.2 M
Blocks Amiodarone Toxicity—We have shown that single or multiple gene deletions that decrease membrane potential have a protective effect against amiodarone toxicity
Summary
The staining curves (i.e. the dependgrown at 30 °C in standard synthetic complete (SC) medium ence of the emission intensity ratio (I580/I560) on the dura(Bio 101 Inc., Vista, CA) or YPD. Cytoplasmic pH Measurements— Cytoplasmic pH in yeast expressing pH-sensitive GFP, pHluorin, was determined by calculating the ratio of fluorescent intensity of emission at 520 nm with dual excitation at 410 and 485 nm, with calibration in media of defined pH, as described [18]. Amiodarone was injected to the desired concentration, and fluorescence was recorded on a BMG FLUOstar Optima multimode plate reader with accompanying BMG FLUOstar Optima version. Cells were resuspended in SC medium adjusted to the indicated pH with NaOH, or resus-. Pended in SC medium lacking glucose before amiodarone injection and fluorescence measurement
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
