Na+, K+ and Cl- selectivity of the permeability pathways induced through sterol-containing membrane vesicles by amphotericin B and other polyene antibiotics.

Membrane diffusion potentials induced by amphotericin B (AmB), amphotericin B methyl ester (AmE), N-fructosyl AmB (N FruAmB) and vacidin, an aromatic polyene antibiotic, in ergosterol- or cholesterol-containing egg yolk phosphatidylcholine large unilamellar vesicles (LUV), were measured in various media, in order to determine the relative selectivity of Na+, K+, Cl- and other ions in these environments. Changes in the membrane potential were followed by fluorescence changes of 3,3'-dipropylthiadicarbocyanine (diS-C3-(5)). Subtle changes in intercationic selectivity were monitored by measuring biionic potentials, using the fluorescent pH sensitive probe pyranine. In all the cases studied, the intercationic selectivity of the permeability pathways induced by the four antibiotics was weak compared to that of specific biological channels, though distinct differences were noted. With AmB the selectivity appeared to be concentration dependent. Above 5 x 10(-7) M, the sequence determined for sterol-free small unilamellar vesicles (SUV) and cholesterol-containing SUV and LUV, Na+ > K+ > Rb+ > or = Cs+ > Li+ (sulfate salts), corresponded closely to Eisenman selectivity sequence number VII. At 5 x 10(-7) M and below the selectivity switched from Na+ > K+ to K+ > Na+. In contrast, Li+ was the most permeant ion for AmB channels in the presence of ergosterol. The selectivity between Na+ or K+ vs. Cl- varied with the antibiotic. It was very strong with vacidin at concentrations below 5 x 10(-7) M, smaller with AmB, nil with AmE and N FruAmB. The selectivities observed were antibiotic, concentration and time dependent, which confirms the existence of different types of channels.

A kinetic method for measuring functional delivery of amphotericin B by drug delivery systems.

The human toxicity of amphotericin B can be considerably reduced by associating the drug with liposomes of varying lipid compositions. Some lipid compositions are much more effective than others. We show that a simple kinetic fluorescence assay using pyranine as an indirect probe of amphotericin-induced K+ currents may be used to study different liposomal drug delivery systems in vitro. We find that lipid mixtures composed of DMPC/DMPG/amphotericin at a 7:3:1 mole ratio show very slow functional delivery with a preference for ergosterol over cholesterol-containing membrane vesicles. On the other hand, amphotericin delivered from egg phosphatidylcholine liposomes lead to 100-fold increases in K+ leakage at one-fifth the amphotericin concentration of the 7:3:1 system. The egg phosphatidylcholine system as well as micellar amphotericin also show a slight selectivity towards cholesterol-containing vesicles over ergosterol. These results are consistent with previous clinical and in vitro cellular studies and this technique may prove valuable in screening of other delivery systems.

Potassium-selective amphotericin B channels are predominant in vesicles regardless of sidedness.

Amphotericin B (AmB) is a membrane-active antibiotic which has been shown to increase ion and small molecule permeability in a variety of model and biological membrane systems. A major mechanistic model, based on BLM systems, proposes that amphotericin forms barrellike pores with cholesterol which are cation selective when added to one side of the membrane and anion selective when added to both sides. We have tested this hypothesis on small and reverse-phase large unilamellar vesicles (SUV and REV) with and without cholesterol. The method used to measure K+, Cl-, and net ion currents is based on ion/H+ exchange detected by the entrapped pH probe pyranine. We find that AmB forms channels which have net selectivity for K+ over Cl- regardless of sidedness or sterol content in SUV. REV with 10% cholesterol also show net K+ selectivity with double-sided addition. Differences are noted between cholesterol- and non-sterol-containing vesicles consistent with at least two separate modes of action: (1) cholesterol-containing SUV form some larger diameter pores which allow the passage of larger ions especially when added to both sides; (2) SUV without sterol form pores which are still K+ over Cl- selective, but larger ions do not pass. The latter mode of action precludes a sterol/pore type of model but not necessarily a barrellike model consisting only of amphotericin molecules.(ABSTRACT TRUNCATED AT 250 WORDS)