Inner field compensation as a tool for the characterization of asymmetric membranes and Peptide-membrane interactions

Abstract

Symmetric and asymmetric planar lipid bilayers prepared according to the Montal-Mueller method are a powerful tool to characterize peptide-membrane interactions. Several electrical properties of lipid bilayers such as membrane current, membrane capacitance, and the inner membrane potential differences and their changes can be deduced. The time-resolved determination of peptide-induced changes in membrane capacitance and inner membrane potential difference are of high importance for the characterization of peptide-membrane interactions. Intercalation and accumulation of peptides lead to changes in membrane capacitance, and membrane interaction of charged peptides induces changes in the charge distribution within the membrane and with that to changes in the membrane potential profile. In this study, we establish time-resolved measurements of the capacitance minimization potential DeltaPsi on various asymmetric planar lipid bilayers using the inner field compensation method. The results are compared to the respective ones of inner membrane potential differences DeltaPhi determined from ion carrier transport measurements. Finally, the time courses of membrane capacitances and of DeltaPsi have been used to characterize the interaction of cathelicidins with reconstituted lipid matrices of various Gram-negative bacteria.

FIGURE 1

Potential profile across an asymmetric bilayer membrane (adopted from Schoch et al. (1979)).

FIGURE 2

Chemical structure of deep rough mutant LPS from E. coli strain F515, S. Minnesota strain R595, and P. mirabilis strain R45. Negative charges are indicated by a minus within a black circle, positive charges by a plus within a white circle.

FIGURE 3

Voltage-dependence of the capacitance, amplitude, and phase of the second harmonic (A). For inner field compensation measurements, an ac excitation is applied to planar lipid membranes resulting in a current response in the first and the second harmonic (I₁ ≠ 0, I₂ ≠ 0) (B). When applying an external transmembrane voltage U = −ΔΨ, the second harmonic vanishes (I₁ ≠ 0, I₂ = 0) (C) and the inner membrane potential difference ΔΨ′ resulting from the inner membrane and the transmembrane potential profile is zero.

FIGURE 4

Comparison of the inner membrane potential differences ΔΦ (light gray bars) determined by carrier transport measurements and ΔΨ by IFC measurements (dark gray bars), respectively, for asymmetric membrane, composed of different phospholipids or lipopolysaccharides. In the figure only one leaflet is defined, the other one was from PL in all cases. The PL side consists of 81 mol % PE, 17 mol % PG, and 2 mol % DPG. Measurements were performed at 37°C in electrolyte solution containing 100 mM KCl and 5 mM MgCl₂ buffered with 5 mM HEPES and adjusted to pH 7.

FIGURE 5

Change of the capacitance (gray traces) and of the inner membrane potential difference ΔΨ (black traces) of different membranes after addition of rCAP18_106–137. Peptide was added 5 min after membrane preparation (arrow): LPS F515/PL (A), LPS R45/PL (B), and DPhyPC/PL (C). Composition of PL side and electrolyte solution as described for Fig. 4.

FIGURE 6

Change of the capacitance (gray traces) and the inner membrane potential difference ΔΨ (black traces) of F515 LPS/PL membranes after addition of rCAP18_98–117 (A) and rCAP18_106–125 (B). Peptide was added 5 min after membrane preparation (arrow). Composition of PL side and electrolyte solution as described for Fig. 4.

FIGURE 7

Schematic membrane potential profile across asymmetric PS/PL (A) and LPS/PL (B) membranes on the basis of the Schoch model. The negative charges of the lipid molecules are indicated by black circles. The arrows indicate the polarity of ΔΦ and ΔΨ. The exact potential profile in the headgroups of the LPS molecules is unknown; therefore, we indicated the possible range by a solid line and a dotted line.

FIGURE 8

Correlation between surface charge density σ of the side of peptide addition and change of the inner membrane potential difference ΔΨ of various LPS/PL and phospholipid PL membranes. Composition of PL side and electrolyte solution as described for Fig. 4.

FIGURE 9

Correlation between charge of the peptides Q and change of the inner membrane potential difference ΔΨ of LPS F515/PL membranes. Composition of PL side and electrolyte solution as described for Fig. 4.