The RH 421 styryl dye induced, pore model-dependent modulation of antimicrobial peptides activity in reconstituted planar membranes.

BACKGROUND: Antimicrobial agents, with different pore-formation mechanisms, may be differently influenced by alteration of the dipolar electric field of a lipid membrane. METHODS: By using electrophysiological measurements on reconstituted lipid membranes, we used alamethicin, melittin and magainin to report on how controlled manipulation of the membrane dipole potential by the styrylpyridinium dye RH 421 affects the kinetic and transport features of peptides within membranes. RESULTS: Our data demonstrate that the increase of the membrane dipole potential caused by RH 421 decreases the activity and single-channel conductance of alamethicin. Surprisingly, we found that RH 421 increases the activity of melittin and magainin, suggesting that RH 421 may contribute via electrostatic repulsions, among others, to an increase in the monolayer spontaneous curvature of the membrane. We propose that RH 421-induced dipole potential and membrane elasticity changes alter the peptide-induced channel dynamics, and the prevalence of one mechanism over the other for particular classes of peptides is dictated by the electrical and mechanical interactions which rule the pore-formation mechanism of such peptides. GENERAL SIGNIFICANCE: These results point to a novel paradigm in which electrical and mechanical effects promoted by chemicals which preferentially alter the electrostatics of the membrane, may be employed to help distinguish among various pore-formation mechanisms of membrane-permeabilizing peptides.

Influence of membrane potentials upon reversible protonation of acidic residues from the OmpF eyelet.

In this research we employed single-molecule electric recording techniques to investigate effects of the transmembrane and dipole potential on the reversible protonation of acidic residues from the constriction zone of the OmpF porin. Our results support the paradigm according to which the protonation state of aspartate 113 and glutamate 117 residues from the constriction region of OmpF is influenced by the electric potential profile, via an augmentation of the local concentration of protons near these residues mediated by increasing negative transmembrane potentials. We propose that at constant bulk pH, pK(a) values for proton bindings at these residues increase as the applied transmembrane potential increases in its negative values. Our data demonstrate that the apparent pK(a) for proton binding of the acidic aminoacids from the constriction region of OmpF is ionic strength-dependent, in the sense that a low ionic strength in the aqueous phase promotes the increase of the protonation reaction rate of such residues, at any given holding potential. Supplementary, we present evidence suggesting that lower values of the membrane dipole potential lead to an increase in the values of the 'on' rate of the eyelet acidic residues protonation, caused by an elevation of the local concentration of hydrogen ions. Altogether, these results come to support the paradigm according to which transmembrane and dipole potentials are critical parameters for the titration behavior of protein sites embedded lipid membranes.

Selective transfer of energy through an alamethicin-doped artificial lipid membrane studied at discrete molecular level.

In this study we present novel evidence that strengthens the paradigm of selective transfer of energy mediated by a random gating of ion channels. Specifically, we investigated the spectral response of a noisy artificial biomembrane whose electrical properties were largely dictated by embedded alamethicin oligomers. In this respect, we first evaluated experimentally the linear transfer function of the system via the white-noise analysis method. We prove that such a system displays specific ranges of frequency over which input signals pass preferentially, depending on their spectral content and the holding potential across the artificial bilayer which contains alamethicin. By employing voltage-driven periodic stimulation of alamethicin oligomers, we demonstrate that overall response of the system obeys qualitatively the predictions inferred from the transfer function analysis of it. These results emphasize the exquisite ability of excitable membranes to behave as band-limited filters and allow for maximal transfer of energy from an external stimulus over well-defined frequency ranges.

Placement of oppositely charged aminoacids at a polypeptide termini determines the voltage-controlled braking of polymer transport through nanometer-scale pores.

Protein and solid-state nanometer-scale pores are being developed for the detection, analysis, and manipulation of single molecules. In the simplest embodiment, the entry of a molecule into a nanopore causes a reduction in the latter's ionic conductance. The ionic current blockade depth and residence time have been shown to provide detailed information on the size, adsorbed charge, and other properties of molecules. Here we describe the use of the nanopore formed by Staphylococcus aureus α-hemolysin and polypeptides with oppositely charged segments at the N- and C-termini to increase both the polypeptide capture rate and mean residence time of them in the pore, regardless of the polarity of the applied electrostatic potential. The technique provides the means to improve the signal to noise of single molecule nanopore-based measurements.

Meet me on the other side: trans-bilayer modulation of a model voltage-gated ion channel activity by membrane electrostatics asymmetry.

While it is accepted that biomembrane asymmetry is generated by proteins and phospholipids distribution, little is known about how electric changes manifested in a monolayer influence functional properties of proteins localized on the opposite leaflet. Herein we used single-molecule electrophysiology and investigated how asymmetric changes in the electrostatics of an artificial lipid membrane monolayer, generated oppositely from where alamethicin--a model voltage-gated ion channel--was added, altered peptide activity. We found that phlorizin, a membrane dipole potential lowering amphiphile, augmented alamethicin activity and transport features, whereas the opposite occurred with RH-421, which enhances the monolayer dipole potential. Further, the monolayer surface potential was decreased via adsorption of sodium dodecyl sulfate, and demonstrated that vectorial modification of it also affected the alamethicin activity in a predictive manner. A new paradigm is suggested according to which asymmetric changes in the monolayer dipole and surface potential extend their effects spatially by altering the intramembrane potential, whose gradient is sensed by distantly located peptides.

Phlorizin- and 6-ketocholestanol-mediated antagonistic modulation of alamethicin activity in phospholipid planar membranes.

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.