Search

In this work, we present experimental data, supported by a quantitative model, on the generation and effect of potential gradients across a tethered bilayer lipid membrane (tBLM) with, to the best of our knowledge, novel architecture. A challenge to generating potential gradients across tBLMs arises from the tethering coordination chemistry requiring an inert metal such as gold, resulting in any externally applied voltage source being capacitively coupled to the tBLM. This in turn causes any potential across the tBLM assembly to decay to zero in milliseconds to seconds, depending on the level of membrane conductance. Transient voltages applied to tBLMs by pulsed or ramped direct-current amperometry can, however, provide current-voltage (I/V) data that may be used to measure the voltage dependency of the membrane conductance. We show that potential gradients >∼150 mV induce membrane defects that permit the insertion of pore-forming peptides. Further, we report here the novel (to our knowledge) use of real-time modeling of conventional low-voltage alternating-current impedance spectroscopy to identify whether the conduction arising from the insertion of a polypeptide is uniform or heterogeneous on scales of nanometers to micrometers across the membrane. The utility of this tBLM architecture and these techniques is demonstrated by characterizing the resulting conduction properties of the antimicrobial peptide PGLa. © 2014 by the Biophysical Society.

Ion channels, such as gramicidin A, selectively facilitate the transport of ions across biological and synthetic membranes. The conductance properties of ion channels are frequently characterized in synthetic bilayer lipid membranes (BLMs). The instability of BLMs has seriously limited the range of applications for these structures, and tethered bilayer lipid membranes (tBLMs) have addressed the problem through tethering many of the membrane components to a solid surface. In the present study, thin gold substrates have been used to tether thiol- and disulfide-terminated membrane components to form a tBLM electrode to provide a reservoir for ions. This study reports on the ion selectivity and apparent permeability of gramicidin channels in such tethered bilayer membranes. The investigations using electrical impedance spectroscopy indicated that the magnitude of ionic conductance varies substantially in reservoirs with different chemical structures. This study addressed the effect of changing ionic concentration, the effect of changing the species in the bulk solution above the membrane, and the influence of the chemical structure of the reservoir tethers. The effect of two-dimensional packing on membrane conductance was also investigated. The present observations suggested that (a) the reservoir region resistivity has a major influence on the overall conductivity of the membrane and in some instances can dominate conduction, (b) the conduction behavior is nonlinear and exhibits saturation with increasing electrolyte concentration, and (c) that ion pairing in the reduced dielectric (ε ∼50) reservoir region is the likely basis for the latter effect. The inferred limiting ionic mobilities of alkali chloride species in the membrane reservoir regions were 3-4 orders of magnitude less than in aqueous solution, indicating that the reservoirs resembled hydrated polymer gels.

The use of polar linkers to tether lipid bilayer membranes to a gold substrate results in a hydrophilic layer between the membrane and the gold surface. The tethering of lipid bilayer membranes to gold substrates using tetraethylene glycol chains results in a polar layer between the membrane and the gold surface. This region may sequester ions and can act as a reservoir for ions transported across the tethered lipid membrane. In the present article, we report on the electrical properties of this ionic reservoir. In particular, the Stern model of ionic distribution is used to describe the interfacial capacitance. The model combines a surface adsorption layer (Helmholtz model) and a dynamic diffuse layer of ions (Gouy-Chapman model) to describe the interfacial capacitance. This model is used to interpret data from measurements of the interfacial capacitance obtained over a range of ionic species and concentrations. Four analogues of the sulfur-tetraethylene glycol tethers have been investigated. These studies show the effects of varying the structure of the linker group and of introducing a passivation layer adjacent to the gold. Studies were also made of the influence of spacer molecules included to vary the "in-plane" two-dimensional packing. The effect of applying a dc bias potential between an external reference electrode and the gold surface was also studied. These measurements were carried out using ac impedance spectroscopy on bilayers assembled using the method of Cornell et al. Most data are successfully modeled as a constant Helmholtz capacitance in series with a diffuse region capacitance that depends on ionic concentration. The dependence on ionic concentration has been modeled by the Gouy- Chapman formalism. At low ionic concentrations (