Amphotericin B channels in the bacterial membrane: role of sterol and temperature

Abstract

Amphotericin B is an antibiotic that forms ion channels in the membrane of a host cell. The change in permeability produced by these channels is greatly improved by sterols; nevertheless, the single channel conductivity remains invariant. Hence, it is proposed that sterols do not act directly, but rather through the modulation of the membrane phase. We look at the formation of these channels in the bacterial membrane to determine the mechanism of its known antibiotic resistance. We found that channels can indeed be formed in this membrane, but a substantial amount of amphotericin B is required. We also study the effects of the antibiotic concentration needed for channel expression as well as the dynamics of channels affected by both sterol and temperature in phosphatidylcholine membranes. The results support the idea that membrane structure is a determining factor in the action of the antibiotic.

FIGURE 1

Examples of traces showing the six types of AmB channels, in this case in a lecithin membrane with 30 mol % cholesterol and a temperature of 25°C. The buffer solution used was 2 M KCl, 1 mM CaCl₂, and 10 mM Hepes at pH 8.0 and 200 mV where applied.

FIGURE 2

Differential calorimetric scanning of the IOM preparation with (30 mol %) or without cholesterol. The concentration of lipid in the solution was 4 mg/ml (2 M KCl, 1 mM CaCl₂, and 10mM Hepes, pH = 8.0).

FIGURE 3

Accumulated all-points histogram of all records analyzed in the E. coli IOM bilayer at 25°C without cholesterol and AmB concentration of 100 μM. The solution used was 2 M KCl, 1mM CaCl₂, and 10 mM Hepes at pH = 8.0. The applied membrane potential was 200 mV. The baseline at 0 current is not shown. In this case channel type V does not appear, but this histogram is shown because it corresponds to those conditions where channels are less likely to appear according to the assumption of sterols requirement.

FIGURE 4

I-V curves for AmB channels in a lecithin membrane with a 30 mol % cholesterol at 25°C. The external solution has 2 M KCl, and the micropipette solution 0.2 M KCl. The Nernst potential for the K⁺ ion is +52 mV and for the Cl⁻ ion is −52 mV.

FIGURE 5

Accumulated all-points histogIram of all records at 0 mV for the lecithin membrane with 30 mol % cholesterol at 25°C.