Transient Potential Gradients and Impedance Measures of Tethered Bilayer Lipid Membranes: Pore-Forming Peptide Insertion and the Effect of Electroporation

Abstract

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.

Candidalysin is a fungal peptide toxin critical for mucosal infection

“…tBLMs with 10% tethering lipids and 90% spacer lipids (T10 slides) were formed using the solvent exchange technique46,47 according to the manufacturer’s instructions (SDx Tethered Membranes Pty Ltd, Sydney, Australia). Briefly, 8 µl of 3 mM lipid solutions in ethanol were added, incubated for 2 min and then 93.4 µl buffer (100 mM KCl, 5 mM HEPES, pH 7.0) was added.…”

Investigating Sterol and Redox Regulation of the Ion Channel Activity of CLIC1 Using Tethered Bilayer Membranes

“…Artificial membranes of ~4 nm thickness capable of incorporating proteins of up to 40 kDa were formed using methods reported in [3,22,37,38]. The monolayer tethering coating was prepared by coating freshly deposited, 100 nm pattern gold electrodes, on 25 mm × 75 mm × 1 mm polycarbonate slides, with two benzyl disulphide families, one being a spacer molecule containing a four oxygen-ethylene glycol spacer, terminated with an OH group (90%), and the second being a tethering group comprising an eleven oxygen–ethylene glycol linker group with a single C20 hydrophobic phytanyl chain (10%) as the hydrophobic tether.…”

Design, Synthesis and Biological Evaluation of N‐Sulfonylphenyl glyoxamide‐Based Antimicrobial Peptide Mimics as Novel Antimicrobial Agents

“…To test how selected potent phenylglyoxamide compounds ( 27 a, 26 b , 34 d , 35 d ) might interact with cell membranes, tethered bilayer lipid membrane (tBLM) technology was used in conjunction with electrical impedance spectroscopy ,. By anchoring a lipid membrane onto a gold substrate, it is possible to measure changes in intrinsic membrane conduction in real time upon addition of the compounds.…”