Lipid and Stress Dependence of Amphotericin B Ion Selective Channels in Sterol-free Membranes

The idea that amphotericin B (AmB) may not require sterols to form ion selective channels has recently been criticized on the grounds that egg phospholipids commonly used in experiments may contain small amounts of sterol which associate with AmB to form AmB/sterol pore channel structures. It was recently shown in this laboratory that modest osmotic stress can enhance the formation of AmB channels in sterol-free egg phosphatidylcholine (eggPC) membranes. We have tested AmB's ability to form ion channels/defects in synthetic palmitoyl oleoyl (POPC), dieicosenyl (DEPC) and natural eggPC osmotically stressed large unilamellar vesicles (LUV) using pyranine fluorescence detected ion/H+ exchange. These sterol-free POPC LUV exhibit greatly increased sensitivity to cation selective AmB channel formation when osmotically stressed; even more than eggPC. Under these stressed conditions, AmB activity was observed at [AmB]/POPC ratios as low as 3.5x10(-4), corresponding to about 34 AmB molecules/vesicle. DEPC vesicles were almost completely unresponsive, demonstrating a strong bilayer thickness dependence. These results prove conclusively that AmB can form sterol-free channels and do so within therapeutic concentration ranges (>0.5-10x10(-6) M) in a stress-dependent manner. This phenomenon may allow us to use osmotic stress changes in simple model systems to spectroscopically isolate and characterize the thus-far elusive AmB channel forming aggregate. In addition, this stress dependence may be responsible for the potentiation of renal toxicity of AmB in the ascending branch of the loop of Henle which is under greatest osmotic stress.