Extended Data Figure 1 |. AmB can transport potassium, sodium, chloride, protons and bicarbonate across a lipid membrane.

Traces indicate percent of maximum ion efflux after Triton-X addition. a) Schematic for the ¹³C-NMR bicarbonate efflux experiment. (b) ¹³C-NMR spectra of H¹³CO₃⁻-loaded POPC/10% cholesterol liposomes treated with AmB, C35deOAmB, or DMSO vehicle. NaH¹³CO₃ was loaded inside the liposomes and the intravesicular solution was buffered to pH 7.5, while the extravesicular solution was buffered to pH 7.3. Due to this pH difference, intravesicular HCO₃⁻ displays a more downfield chemical shift relative to extravesicular HCO₃⁻. Addition of AmB (1:4,000 AmB:POPC) produces an upfield ¹³C signal corresponding to extravesicular HCO₃⁻, while the addition C35deOAmB or DMSO vehicle does not, demonstrating that AmB is able to facilitate HCO₃⁻ efflux. (c) Percent efflux of bicarbonate mediated by DMSO, AmB, or C35deOAmB quantified 10 minutes after addition to POPC liposomes (n = 3 biologically independent samples). The data from each run was normalized to the percent of total ion release from 0 to 100%. After lysis of the liposome suspension, the integration of the signal corresponding to extravesicular HCO₃⁻ relative to the integration of a ¹³C glucose standard was scaled to correspond to 100% efflux. (d) To confirm that the upfield signal corresponds to extravesicular HCO₃⁻, Mn²⁺, which binds to HCO₃⁻ and quenches the observed ¹³C signal via paramagnetic relaxation enhancement (PRE), was added to the extravesicular solution. Because Mn²⁺ is impermeable to the POPC bilayer, Mn²⁺ can only affect the signal corresponding to HCO₃⁻ outside of the liposomes. Addition of Mn²⁺ quenched the upfield signal produced with the addition of AmB but not the signal corresponding to intravesicular HCO₃⁻ confirming that AmB causes efflux of HCO₃⁻. (e) To effect complete ion release, the POPC liposomes were lysed with Triton X-100 at the conclusion of the experiment. (f) Potassium efflux from POPC/10% cholesterol liposomes after addition of [AmB] equivalent to 1:1000 AmB:lipid, or DMSO vehicle. (g) Sodium efflux from POPC/10% cholesterol liposomes after addition of [AmB] equivalent to 1:1000 AmB:lipid, or DMSO vehicle. (h) Chloride efflux from POPC/10% cholesterol liposomes after addition of [AmB] equivalent to 1:1000 AmB:lipid, or DMSO vehicle. (i) Bicarbonate efflux from POPC/10% cholesterol liposomes after addition of [AmB] equivalent to 1:1000 AmB:lipid, or DMSO vehicle. Kinetics of efflux were measuring using rapid injection NMR to add AmB to liposomes. (j) Proton efflux from POPC/10% cholesterol liposomes after addition of [AmB] equivalent to 1:1000 AmB:lipid, or DMSO vehicle. In (b,d-j), a representative spectrum or graph from at least three independent experiments is shown. In all panels, measurements were taken from distinct samples. In panel (c), graph depicts mean ± SEM.