Short-Chained Alcohols Make Membrane Surfaces Conducive for Melittin Action: Implication for the Physiological Role of Alcohols in Cells

Abstract

Alcohols are a part of cellular metabolism, but their physiological roles are not well understood. We investigated the effects of short-chain alcohols on Daphnia pulex and model membranes mimicking the lipid composition of eukaryotic inner mitochondrial membranes. We also studied the synergistic effects of alcohols with the bee venom membrane-active peptide, melittin, which is structurally similar to endogenous membrane-active peptides. The alcohols, from ethanol to octanol, gradually decreased the heart rate and the mitochondrial ATP synthesis of daphnia; in contrast, in combination with melittin, which exerted no sizeable effect, they gradually increased both the heart rate and the ATP synthesis. Lipid packing and the order parameter of oriented films, monitored by EPR spectroscopy of the spin-labeled probe 5-doxylstrearic acid, revealed gradual alcohol-assisted bilayer to non-bilayer transitions in the presence of melittin; further, while the alcohols decreased, in combination with melittin they increased the order parameter of the film, which is attributed to the alcohol-facilitated association of melittin with the membrane. A ¹H-NMR spectroscopy of the liposomes confirmed the enhanced induction of a non-bilayer lipid phase that formed around the melittin, without the permeabilization of the liposomal membrane. Our data suggest that short-chain alcohols, in combination with endogenous peptides, regulate protein functions via modulating the lipid polymorphism of membranes.

Keywords: 1H-NMR; ERP of spin probes; alcohols; heart rate; melittin; mitochondrial ATP production; non-bilayer structures.

Figure 1

Heart rate of Daphnia pulex as a function of alcohol chain length in the absence of melittin and in melittin-treated organisms. The heart rate was measured in beats per minute (bpm). Individual fleas were treated with either alcohols, melittin or a mixture of alcohol and melittin as described in the Materials and Methods. Concentrations of alcohols and melittin were 1.65 × 10⁻³ M and 1.65 × 10⁻⁴ M, respectively. Blue and orange bars at 0 on the horizontal axis show, respectively, the heart rates of the control sample and sample treated with melittin in the absence of alcohol. Data points represent mean values from at least three independent experiments on different fleas. The standard deviations shown in error bars were within 5.0% of the means.

Figure 2

Mitochondrial ATP synthesis of Daphnia pulex as a function of alcohol chain length in the absence of melittin and in melittin-treated organelles. ATP levels were assessed as µmol ATP synthesized per mg of mitochondrial proteins. Mitochondria were treated with either alcohols, melittin or a mixture of alcohol and melittin as described in the Materials and Methods. The bulk phospholipid concentration in the mitochondrial samples, estimated via comparing the integrated areas of the ³¹P NMR signals to that of large multilamellar liposomes, was approximately 1.65 × 10⁻² M. Concentrations of alcohols and melittin were 1.65 × 10⁻³ M and 1.65 × 10⁻⁴ M, respectively. Blue and orange bars at 0 on the horizontal axis show, respectively, the ATP levels in the control sample and in sample treated with melittin in the absence of alcohol. Each data point is the mean of three independent experiments. Standard deviations, shown as error bars, were within 5.2% of the means.

Figure 3

EPR spectra of 5-doxylstearic acid (5.5 × 10⁻⁵ M) in oriented lipid films—of 5.5 × 10⁻³ M phospholipids concentration, with a composition of 40 mol% PC, 35 mol% PE, 20 mol% CL, 3 mol% PI and 2 mol% PS—at the applied magnetic field parallel (broken line) and perpendicular (solid line) to the film’s normal in the absence of alcohols and melittin (A) and in the presence of 5.5 × 10⁻⁵ M melittin (B) and 5.5 × 10⁻⁵ M melittin and 5.5 × 10⁻⁴ M ethanol (C).

Figure 4

Effects of alcohols (5.5 × 10⁻⁴ M) in the absence and presence of melittin (5.5 × 10⁻⁵ M) on the B/C ratio of the EPR spectra of 5-DSA in oriented lipid films made of a phospholipid composition which mimics that of the IMM in eukaryotes. Concentrations of the total phospholipids and 5-DSA in the lipid films were 5.5 × 10⁻³ M and 5.5 × 10⁻⁵ M, respectively. Standard deviations, shown as error bars, were within 5.2% of the means.

Figure 5

Effects of alcohols (5.5 × 10⁻⁴ M) in the absence and presence of melittin (5.5 × 10⁻⁵ M) on parameter S of EPR spectra of 5-DSA in oriented lipid films of phospholipid composition, mimicking that of the IMM in eukaryotes. The concentrations of total phospholipids and 5-DSA in lipid films were 5.5 × 10⁻³ M and 5.5 × 10⁻⁵ M, respectively. Standard deviations shown by error bars were within 5.5% of the means.

Figure 6

The ¹H-NMR spectra derived from the N⁺(CH₃)₃ groups of PC in the inner (I_i) and the outer (I_o) leaflets of unilamellar liposomes, mimicking the lipid composition of eukaryotic IMM, in the presence of K₃[Fe(CN)₃]. (A) untreated liposomes; (B–E), respectively, liposomes treated with 1.2 × 10⁻⁴ M melittin, 1.2 × 10⁻⁴ M melittin and 1.2 × 10^–3 M ethanol, 1.2 × 10⁻⁴ M melittin and 1.2 × 10⁻³ M octanol, and 1.2 × 10⁻³ M melittin and 1.2 × 10⁻³ M ethanol. Total phospholipid concentration, 1.20 × 10⁻² M.