Small Molecule Channels Harness Membrane Potential to Concentrate Potassium in trk1Δtrk2Δ Yeast

Abstract

Many protein ion channels harness membrane potential to move ions in opposition to their chemical gradient. Deficiencies of such proteins cause several human diseases, including cystic fibrosis, Bartter Syndrome Type II, and proximal renal tubular acidosis. Using yeast as a readily manipulated eukaryotic model system, we asked whether, in the context of a deficiency of such protein ion channel function in vivo, small molecule channels could similarly harness membrane potential to concentrate ions. In yeast, Trk potassium transporters use membrane potential to move potassium ions from a compartment of relatively low concentration outside cells (~15mM) to one of >10 times higher concentration inside (150-500mM). trk1Δtrk2Δ yeast are missing these potassium transporters and thus cannot concentrate potassium or grow in standard media. Here we show that potassium permeable, but not potassium selective, small molecule ion channels formed by the natural product amphotericin B can harness membrane potential to concentrate potassium in trk1Δtrk2Δ cells and thereby restore growth. This finding expands the list of potential human channelopathies that might be addressed by a molecular prosthetics approach..

Natamycin interferes with ergosterol-dependent lipid phases in model membranes

“…While natamycin does not form classical pores for ions, in contrast to nystatin and AmpB [ 3 , 7 , 25 , 26 , 57 ], its membrane disturbing effect could potentially increase membrane access of (small) polar molecules. To test this notion, we employed a fluorescence assay based on quenching of the fluorescent phosphatidylcholine analogue NBD-PC by dithionite [58] .…”

“…In a recent study, we demonstrated that natamycin interferes with ergosterol- dependent activity of the lysine transporter Lyp1 reconstituted into model membranes [5] . In contrast to other polyenes, such as nystatin or amphotericin B (AmpB), natamycin does not form pores in lipid bilayers, such that a collapse of ion gradients across the plasma membrane (PM) is unlikely [ 3 , 6 , 7 ]. Natamycin may also have other mechanisms of action, such as inhibiting the fusion of yeast vacuoles or disruption of calcium homeostasis followed by loss of membrane potential, as shown for its action on leishmania parasites [ 8 , 9 ].…”

Ergosterol mediates aggregation of natamycin in the yeast plasma membrane

“…Together, these results show that the PM is the target site for the polyene, and once the integrity of the cell membrane is compromised, natamycin enters the cell and stains internal compartments, such as the vacuole. Thus, even though natamycin is not known to form pores for ions in membranes 13,16,28 , it changes the membrane integrity and facilitates passage of small molecules, like PI. This finding is in accordance with our recent observation, that access of the aqueous quencher dithionite to the fluorescent phospholipid analogue NBD-PC is increased in the presence of natamycin in model membranes 18 .…”

“…On the other hand, for nystatin, we found an even more pronounced ergosterol dependence of binding to yeast cells using our label-free imaging approach 35 . Both, nystatin and AmpB form sterol-dependent ion channels in membranes, while natamycin does not, and for AmpB ergosterol-specific interactions in lipid bilayers have been demonstrated using NMR spectroscopy 13,59,60 . Such divergent findings indicate fundamental differences in the mechanism by which various polyene molecules attack lipid membranes.…”

“…These polyenes target ergosterol in the yeast PM, but their precise mode of action is often poorly understood. While AmpB and nystatin form ion pores in the membrane, natamycin kills yeast by a different but poorly defined mechanism 12,13 . Natamycin is often used to treat fungal keratitis but also to protect food products against fungal molds 14 .…”