The presented EIS data allow us to evaluate the range of possible AMP modes of action. We have previously shown that channel-forming peptides, such as gramicidin, change only membrane resistance in a concentration-dependent fashion and do not change the homogeneity and the capacitance of a surface-supported membrane [23
]. Purrucker et al
have shown that channel-forming peptides do not change homogeneity of a surface-supported membrane [30
]. Similarly, addition of melittin, which acts according to the barrel-stave model, to the bilayer leads to two orders of magnitude drop in membrane resistance, while membrane capacitance and homogeneity remain practically the same [27
]. In contrast, we observed a large decrease in membrane homogeneity in the presence of the potent pore-former FSKRGY. Moreover, membrane resistance is not dependent on peptide concentration within the range tested for both the potent and poor pore-formers. Therefore, these AMPs do not act according to the barrel-stave model.
For a toroidal pore model it is expected that the pore density increases (lower membrane resistance) with higher peptide concentration. No such trend was observed for both peptides. In addition, the observed recovery of membrane homogeneity is not expected for the toroidal pore model. If the toroidal pore acts like a gate for the peptides to translocate to the other side of the membrane, the pores themselves should be transient and disintegrate with time as the concentration of the peptides on both sides of the membrane equilibrates. The pore disintegration should lead to an increase in membrane resistance, which was not observed.
According to the carpet model, peptides aggregate upon binding to the bilayer, thus decreasing bilayer homogeneity and possibly decreasing bilayer resistance. Therefore, the carpet model is consistent with our experimental observations. However, the observed recovery in membrane homogeneity in experiments with FSKRGY is somewhat unexpected. After an initial minimum is reached, a recovery to a more homogeneous membrane is always observed in EIS experiments. It is therefore possible that carpet “rafts” form within the first minute, and that the observed recovery occurs after membrane destabilization by the lateral redistribution of lipids/peptides. Since the carpet model does not explicitly describe how and why rafts destabilize the membrane, we cannot definitively rule out or support this model using the impedance data. Nonetheless, we can conclude that aggregation and membrane destabilization should occur within the first minute of the membrane exposure to the peptides, and that such destabilization should be localized, since the overall membrane remains intact and still exhibits a measurable resistance.
The observed decrease in homogeneity and membrane resistance in FSKRGY experiments is also expected from the detergent model. However, since the membrane remains intact and still exhibits measurable resistance, the membrane disruption/dissolution occurs locally and is consequently repaired by the lateral diffusion of lipids into the damaged area, and there is no large scale membrane disruption. Note that “healing” the disrupted regions by vesicle fusion should inevitably decrease the ion permeability (increase the membrane resistance), which was not observed. Therefore we have to rule out the fusion of free vesicles as a possible recovery mechanism. Thus the reparative behavior should be incorporated into the detergent model to explain the observed recovery of membrane homogeneity.
There is another important consideration for both the carpet and the detergent models. The constant value of the membrane capacitance Cm
at high frequency together with the observed decrease in homogeneity implies that the membrane thickness stays the same in some regions and gets thinner in the other regions after peptide interactions. Since membrane thinning is expected upon interaction with AMP (see eg. [31
]), these local regions of decreased thickness are most likely the sites of peptide interactions. The observed partial recovery towards a more homogeneous membrane implies that, with time, the thinned regions do equilibrate to some extent with the rest of the membrane. This recovery is reminiscent of the recovery in vesicle leakage assays: the rate of efflux becomes smaller, with a time constant of a few minutes [9
AGGKGF does not induce leakage in high-stringent assays (1/500 P/L) while FSKRGY does [25
] and both peptides exhibit anti-microbial activity by sterilizing bacterial cultures at concentrations of ~2 μM whereas only AGGKGF had significant cytotoxicity and hemolytic activity against human cells [25
We found that both peptides reduce membrane resistance, which may be correlated with antimicrobial activity. There are changes in membrane resistance even at high stringency, under conditions where AGGKGF should be inactive according to the vesicle leakage assay. The reason for the observed AGGKGF activity at high stringency might be the fact that EIS is sensitive to any ions that can leak through the membrane, while the leakage assay detects the leakage of particular encapsulated molecules. Thus EIS may be more sensitive than the vesicle leakage assays to the membrane effects that are important in antimicrobial activity. Note that some AMPs, like dermaseptins, cecropin B, cecropin P and cecropin A dissipate ion gradients at low peptide concentration, while much higher concentrations are required to release encapsulated fluorescent probe [32
]. It is possible that AGGKGF acts in a similar manner, which can explain the potent sterilization activity of AGGKGF (via dissipation of ion gradient) despite the poor vesicle leakage activity. AGGKGF may merely adhere/intercalate into the membrane and cause lipid local disordering leading to some ion leakage, thus decreasing membrane resistance. FSKRGY peptides, in addition to the above-mentioned lipid disordering, aggregate and disrupt the membrane, thus further decreasing the membrane resistance. FSKRGY aggregates more strongly at higher concentration (lower n
at 1/100 P/L than at 1/500 P/L), as expected. In contrast to FSKRGY, there is no change in homogeneity upon addition of AGGKGF. This difference in peptide behavior correlates with lytic activity in vesicles: AGGKGF does not cause vesicle leakage at high stringency, whereas FSKRGY does. This suggests that lytic activity is a consequence of peptide aggregation and of increase in membrane heterogeneity.
It is well known that the activity of AMPs strongly depends on the presence of negatively charged lipids in the target membrane. The bias potential effectively sets the charge density of the tethered membrane by means of ion accumulation on the membrane surface, thus modulating the peptide partitioning into the membrane. Membrane potentials affect the partitioning of peptides into membranes [34
], as well as the orientation and the degree of penetration of the peptides within the membranes [35
], consistent with our results. We observed that the aggregation of FSKRGY depends on the membrane potential: change in membrane potential from zero to a positive bias reduced aggregation. Thus, the membrane potential is an additional parameter that can be used to characterize and to modulate peptide activity. In general, EIS on a surface-supported bilayer offers the unique possibility to vary and maintain the membrane potential during peptide-membrane interactions, which is often impossible to do in other assays.
The results reported here illustrate the general requirements for studying AMP-membrane interaction using EIS. First, the surface-supported membrane should be spatially homogeneous, that is the lipids should form a continuous single phase without rafts. Second, the membrane should be in the fluid phase to provide lateral mobility of membrane-associated peptides. Third, the membrane should be decoupled from the solid support via a polymer cushion or spacer in order to better accommodate possible peptide-induced lateral stress or local membrane curvature. Finally, the ion permeability of the membrane should be low to maximize the range for detection of AMP interactions. Low ion permeability can be achieved when lipids are densely packed and well ordered; the addition of cholesterol to low-density disordered lipids can improve membrane resistance.