Traces indicate the percentage of maximum ion efflux after Triton X-100 addition. a, Schematic for the 13C NMR HCO 3 − efflux experiment. b, 13C NMR spectra of H13CO 3 −-loaded POPC/10% cholesterol liposomes treated with AmB, C35deOAmB, or DMSO vehicle. NaH13CO 3 was loaded inside the liposomes and the intravesicular solution was buffered to pH 7.5, whereas the extravesicular solution was buffered to pH 7.3. Owing to this pH difference, intravesicular HCO 3 − displays a more downfield chemical shift relative to extravesicular HCO 3 −. Addition of AmB (1:4,000 AmB:POPC) produces an upfield 13C signal corresponding to extravesicular HCO 3 −, while the addition C35deOAmB or DMSO vehicle does not, demonstrating that AmB is able to facilitate HCO 3 − efflux. c, The percentage of efflux of HCO 3 − mediated by DMSO, AmB or C35deOAmB, quantified 10 min after addition to POPC liposomes (n = 3 biologically independent samples). Data from each run were normalized to the percentage of total ion release from 0 to 100%. After lysis of the liposome suspension, the integration of the signal corresponding to extravesicular HCO 3 − 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 HCO 3 −, Mn2+—which binds to HCO 3 − and quenches the observed 13C signal via paramagnetic relaxation enhancement—was added to the extravesicular solution. Because Mn2+ is impermeable to the POPC bilayer, Mn2+ can only affect the signal corresponding to HCO 3 − outside the liposomes. Addition of Mn2+ quenched the upfield signal produced with the addition of AmB but not the signal corresponding to intravesicular HCO 3 −, confirming that AmB causes efflux of HCO 3 −. e, To effect complete ion release, the POPC liposomes were lysed with Triton X-100 at the conclusion of the experiment. f, K+ efflux from POPC/10% cholesterol liposomes after addition of AmB equivalent to 1:1,000 AmB:lipid, or DMSO vehicle. g, Na+ efflux from POPC/10% cholesterol liposomes after addition of AmB equivalent to 1:1,000 AmB:lipid, or DMSO vehicle. h, Cl− efflux from POPC/10% cholesterol liposomes after addition of AmB equivalent to 1:1,000 AmB:lipid, or DMSO vehicle. i, HCO 3 − efflux from POPC/10% cholesterol liposomes after addition of AmB equivalent to 1:1,000 AmB:lipid, or DMSO vehicle. Kinetics of efflux were measured using rapid-injection NMR to add AmB to liposomes. j, H+ efflux from POPC/10% cholesterol liposomes after addition of AmB equivalent to 1:1,000 AmB:lipid, or DMSO vehicle. b, d–j, Panels show a representative spectrum or graph from at least three independent experiments. In all panels, measurements were taken from distinct samples. In c, the graph depicts mean ± s.e.m. Source data