PubMed · 68708
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E Neher, C F Stevens. 1977. Conductance fluctuations and ionic pores in membranes.. https://doi.org/10.1146/annurev.bb.06.060177.002021
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We showed in a previous study that associations of antimicrobial peptides (AMPs), which are key components of the innate immune systems of all living species, with the fluoroquinolone enrofloxacin can successfully cure HeLa cell cultures of Mycoplasma fermentans and M. hyorhinis contamination. In the present work, the in vitro susceptibility of M. pulmonis, a murine pathogen, to enrofloxacin and four AMPs (alamethicin, globomycin, gramicidin S, and surfactin) was investigated, with special reference to synergistic associations and the effect of the mycoplasma cell concentration. Enrofloxacin and globomycin displayed the lowest MICs (0.4 microM), followed by gramicidin S (3.12 microM), alamethicin (6.25 microM), and surfactin (25 microM). When the mycoplasma cell concentration was varied from 10(4) to 10(8) CFU/ml, the MICs of enrofloxacin and globomycin increased while those of the three other molecules remained essentially constant. The minimal bactericidal concentration of enrofloxacin (0.8 microM) was also lower than those of the peptides (6.25 to 100 microM), but the latter killed the mycoplasma cells much faster than enrofloxacin (2 h versus 1 day). The use of the AMPs in association with enrofloxacin revealed synergistic effects with alamethicin and surfactin. Interestingly, the mycoplasma-killing activities of the two combinations enrofloxacin (MIC/2) plus alamethicin (MIC/4) and enrofloxacin (MIC/2) plus surfactin (MIC/16) were about 2 orders of magnitude higher than those of the three molecules used separately. These results support the interest devoted to AMPs as a novel class of antimicrobial agents and pinpoint their ability to potentiate the activities of conventional antibiotics, such as fluoroquinolones.
Alamethicin is a 19-amino-acid residue hydrophobic peptide that produces voltage-dependent ion channels in membranes. Analogues of the Glu(OMe)(7,18,19) variant of alamethicin F50/5 that are rigidly spin-labeled in the peptide backbone have been synthesized by replacing residue 1, 8, or 16 with 2,2,6,6-tetramethyl-piperidine-1-oxyl-4-amino-4-carboxyl (TOAC), a helicogenic nitroxyl amino acid. Conventional electron paramagnetic resonance spectra are used to determine the insertion and orientation of the TOAC(n) alamethicins in fluid lipid bilayer membranes of dimyristoyl phosphatidylcholine. Isotropic (14)N-hyperfine couplings indicate that TOAC(8) and TOAC(16) are situated in the hydrophobic core of the membrane, whereas the TOAC(1) label resides closer to the membrane surface. Anisotropic hyperfine splittings show that alamethicin is highly ordered in the fluid membranes. Experiments with aligned membranes demonstrate that the principal diffusion axis lies close to the membrane normal, corresponding to a transmembrane orientation. Combination of data from the three spin-labeled positions yields both the dynamic order parameter of the peptide backbone and the intramolecular orientations of the TOAC groups. The latter are compared with x-ray diffraction results from alamethicin crystals. Saturation transfer electron paramagnetic resonance, which is sensitive to microsecond rotational motion, reveals that overall rotation of alamethicin is fast in fluid membranes, with effective correlation times <30 ns. Thus, alamethicin does not form large stable aggregates in fluid membranes, and ionic conductance must arise from transient or voltage-induced associations.
As a result of the interfacial chemical heterogenity, membrane-penetrating peptides will experience a dramatic variation in environmental polarity manifested via electrical interactions with the surface and dipole potential of membranes prone to modulate the membrane insertion and folding of different peptides and proteins. Herein we present evidence demonstrating that roughly a 30 mV, phlorizin-induced lowering of the magnitude of the dipole potential of a phosphatidyilcholine membrane leads to a 4-fold increase in the electrical activity of embedded alamethicin. The effect is voltage-independent, implying that the dipole potential affects the barrier of alamethicin adsorption to the membrane rather than the translocation of it across the hydrophobic core. Our interpretation points to an enhanced interfacial accumulation of alamethicin monomers on the cis side of the membrane caused by a lower value of the cis dipole potential, which will promote an elevated activity of alamethicin oligomers across the membrane. As expected for a modestly selective ion channel, the enhancing effect of such dipole potential changes on the electrical conductivity is limited (80 +/- 3 pS before and 100 +/- 2 pS after phlorizin addition to the membrane, for the first conductive state of the channel). Our study emphasizes the possibility that, by manipulating at will the sign of change and the magnitude of the interfacial dipole field, it is possible to modulate the extent of the membrane penetration of ion-channel-forming peptides and thereby provide deeper insights into mechanisms of protein-lipid and protein-protein interactions within membranes.