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© 2014 by the Biophysical Society. This article reports on the construction and predictive models for a platform comprised of an engineered tethered membrane. The platform provides a controllable and physiologically relevant environment for the study of the electroporation process. The mixed self-assembled membrane is formed via a rapid solvent exchange technique. The membrane is tethered to the gold electrode and includes an ionic reservoir separating the membrane and gold surface. Above the membrane, there is an electrolyte solution, and a gold counterelectrode. A voltage is applied between the gold electrodes and the current measured. The current is dependent on the energy required to form aqueous pores and the conductance of each pore. A two-level predictive model, consisting of a macroscopic and a continuum model, is developed to relate the pore dynamics to the measured current. The macroscopic model consists of an equivalent circuit model of the tethered membrane, and asymptotic approximations to the Smoluchowski-Einstein equation of electroporation that is dependent on the pore conductance and the energy required to form aqueous pores. The continuum model is a generalized Poisson-Nernst-Planck (GPNP) system where an activity coefficient to account for steric effects of ions is added to the standard PNP system. The GPNP is used to evaluate the conductance of aqueous pores, and the electrical energy required to form the pores. As an outcome of the setup of the device and the two-level model, biologically important variables can be estimated from experimental measurements. To validate the accuracy of the two-level model, the predicted current is compared with experimentally measured current for different tethering densities.

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.

We describe new techniques to study the insertion of pore forming antimicrobial peptides (AMPs) into tethered bilayer lipid membranes (tBLMs). A consequence of tethering a membrane to a gold surface is that electrical contact to the PBS bathing solution is intrinsically capacitive, preventing the direct application of a steady-state voltage across the bilayer. However, by using pulsed waveforms, defined potentials may be expressed across the membrane for tens to hundreds of milliseconds, and the resulting I-V plots provide valuable data about AMP insertion rates and voltage dependence (Fig 1).Using this technique in the presence of PGLa, we demonstrate how AMP insertion into zwitterionic and negatively charged lipid membranes can be rapidly measured and compared. To better understand the voltage dependence of AMP insertion into tBLMs, ramped potentials can also be applied which can determine the potential thresholds of peptide insertion and pore formation.Advantages of using tBLMs:• tBLMs are more robust and longer lasting than black lipid membranes, or micropipette patches;• Easier, quicker sample preparation than conventional voltage clamp experiments;• Physiologically relevant AMP concentrations can be used (cf. NMR).View Large Image | View Hi-Res Image | Download PowerPoint Slide

MscL channels preferentially cluster in bacterial cells in vivo [1]. Recently it was shown that MscL channels also cluster when reconstituted into liposomes suggesting that this phenomena is bilayer mediated [2]. Evidence was based on atomic force microscopy, small angle neutron scattering, confocal microscopy and patch clamp electrophysiology. However doubts remained as to whether or not proteins were introduced into the liposomes as a cluster, or whether they in fact self-assemble into clusters. In this study, we used FRET FLIM microscopy to address this problem in more detail and compared clustering of MscL with itself, MscS with itself, and MscL with MscS. Separate populations of MscL and MscS channels were labeled; one population with an Alexa Fluor 488 FRET donor and the other with an Alexa Fluor 568 FRET acceptor. Samples were reconstituted into the lipids separately and the 2 samples were allowed to mix overnight. FLIM images of donor fluorescence in the mixed lipid-protein samples were collected. Reduced donor fluorescence lifetimes, due to FRET, indicated clustering between the 2 protein populations. Fast protein liquid chromatography suggested that a portion of MscL and MscS proteins will cluster and/or form higher order oligomers in detergent buffers. These fractions were excluded from the FRET-FLIM experiments. We demonstrated that MscL will self-assemble into clusters in liposomes, and also demonstrated that MscS and MscL will co-cluster. However, there was no evidence for MscS clustering with itself. Clustering of channels is of significance as channel activity is thought to be modulated by neighboring proteins [2].We acknowledge the NH&MRC (Grant 635525). E.D. acknowledges the UWA convocation.[1] Norman, C., et al. (2005)Eur Biophys J 34: 396-402.[2] Grage, S.L., et al. (2011)Biophys J. 100: 1252-1260.

While it is known that many microbial metabolites can permeabilize phospholipid membranes, it is not easy to find quantitative comparisons across large numbers of such metabolites. Such comparisons are of use in screening likely candidates for ongoing research into the development of new and improved antibiotics. We have studied the effects of 30 microbial metabolites on the electrical conductivity of a tethered bilayer lipid membrane comprising two standard phospholipid components. The technique allows rapid quantification of the activity of these compounds. As well as the electrical conductivity, we have analyzed the changes in membrane capacitance by the insertion of metabolites which reports on the change of membrane thickness, as well as the introduction of water molecules into the membrane. Using a standard 10 µM concentration for each of the 30 metabolites, we rank them according to their impact on membrane conduction in PBS solution (Fig 1). We also demonstrate that by determining the change in conduction (ΔG) against the change in capacitance (ΔC) caused by membrane insertion it is possible to determine the complexation properties of the metabolites as they insert into the membrane.View Large Image | View Hi-Res Image | Download PowerPoint Slide