Probing amphotericin B single channel activity by membrane dipole modifiers

Abstract

The effects of dipole modifiers and their structural analogs on the single channel activity of amphotericin B in sterol-containing planar phosphocholine membranes are studied. It is shown that the addition of phloretin in solutions bathing membranes containing cholesterol or ergosterol decreases the conductance of single amphotericin B channels. Quercetin decreases the channel conductance in cholesterol-containing bilayers while it does not affect the channel conductance in ergosterol-containing membranes. It is demonstrated that the insertion of styryl dyes, such as RH 421, RH 237 or RH 160, in bilayers with either cholesterol or ergosterol leads to the increase of the current amplitude of amphotericin B pores. Introduction of 5α-androstan-3β-ol into a membrane-forming solution increases the amphotericin B channel conductance in a concentration-dependent manner. All the effects are likely to be attributed to the influence of the membrane dipole potential on the conductance of single amphotericin B channels. However, specific interactions of some dipole modifiers with polyene-sterol complexes might also contribute to the activity of single amphotericin B pores. It has been shown that the channel dwell time increases with increasing sterol concentration, and it is higher for cholesterol-containing membranes than for bilayers including ergosterol, 6-ketocholestanol, 7-ketocholestanol or 5α-androstan-3β-ol. These findings suggest that the processes of association/dissociation of channel forming molecules depend on the membrane fluidity.

Figure 1. Current fluctuations corresponding opening and closing of the single AmB-channels in the planar lipid bilayers.

The membranes were made from the PC∶Chol (67∶33 mol%) and bathed in 2 M KCl 5 mM Hepes pH 7.0. V = 200 mV. Bilayer bathing solutions contain: (A) – 20 µM phloretin, (B) – no dipole modifiers, (C) – 5 µM RH 421, (D) – 20 µM quercetin, (E) – 5 µkM RH 160, (F) – 5 µM RH 237.

Figure 2. Conductance-voltage curves of the single AmB-channels.

The membranes were made from PC∶Chol (67∶33 mol%) (A) and PC∶Erg (67∶33 mol%) (B), and bathed in 2.0 M KCl 5 mM Hepes pH 7.0. Bilayer bathing solutions contain: (▪) – no dipole modifiers, (○) – 20 µM phloretin, (◂) – 20 µM quercetin, (▵) – 5 µM RH 421, (▴) – 5 µM RH 237, (•) – 5 µM RH 160.

Figure 3. The dependences of the conductance at zero transmembrane voltage, G⁰ (dash lines), and mean dwell time, τ (solid lines), of AmB-channels on the concentration of Chol (solid symbols) or Erg (open symbols) in the membrane forming PC-solution.

The membranes were bathed in 2.0 M KCl 5 mM Hepes pH 7.0.

Figure 4. The voltage dependence of the ratio of AmB-channel conductance to conductance at zero transmembrane potential (G/G⁰).

The membranes were bathed in 2.0 M KCl 5 mM Hepes pH 7.0. Bilayers were made from: (▪) – PC∶Chol (67∶33 mol%), (□) – PC∶Erg (67∶33 mol%), (•) – PC: 6-ketocholestanol (67∶33 mol%), (○) – PC: 7-ketocholestanol (67∶33 mol%), (×) – PC:5α-Androstan-3β-ol (67∶33 mol%). Solutions bathing membranes made from PC∶Chol (67∶33 mol%) contain: (▸) – 20 µM phloretin, (◂) – 20 µM quercetin, (▵) – 5 µM RH 421, (▴) – 5 µM RH 237, (◊) – 5 µM RH 160. Solutions bathing membranes made from PC∶Erg (67∶33 mol%) contain: (_*) – 20 µM phloretin, (—□) – 20 µM quercetin, (+) – 5 µM RH 421, (—○) – 5 µM RH 237, (—▵) – 5 µM RH 160.