Electrophysiological interrogation of asymmetric droplet interface bilayers reveals surface-bound alamethicin induces lipid flip-flop

Abstract

The droplet interface bilayer (DIB) method offers simple control over initial leaflet compositions in model membranes, enabling an experimental path to filling gaps in our knowledge about the interplay between compositional lipid asymmetry, membrane properties, and the behaviors of membrane-active species. Yet, the stability of lipid leaflet asymmetry in DIBs has received very little attention, particularly in the presence of peptides and ion channels that are often studied in DIBs. Herein, we demonstrate for the first time parallel, capacitance-based measurements of intramembrane potential with arrays of asymmetric DIBs assembled in a microfluidic device to characterize the stability of leaflet asymmetry over many hours in the presence and absence of membrane-active peptides. DIBs assembled from opposing monolayers of the ester (DPhPC) and ether (DOPhPC) forms of diphytanoyl-phosphatidylcholine yielded asymmetric bilayers with leaflet compositions that were stable for at least 18 h as indicated by a stable |137 mV| intramembrane potential. In contrast, the addition of surface-bound alamethicin peptides caused a gradual, concentration-dependent decrease in the magnitude of the dipole potential difference. Intermittent current-voltage measurements revealed that alamethicin in asymmetric DIBs also shifts the threshold voltage required to drive peptide insertion and ion channel formation. These outcomes take place over the course of 1 to 5 h after membrane formation, and suggest that alamethicin peptides promote lipid flip-flop, even in the un-inserted, surface-bound state, by disordering lipids in the monolayer to which they bind. Moreover, this methodology establishes the use of parallel electrophysiology for efficiently studying membrane asymmetry in arrays of DIBs.

Accelerated Molecular Dynamics Applied to the Peptaibol Folding Problem

“…A very recent and interesting work by Abbasi et al [28] reported the dampening effect of Alm ion channel on conductivity by an order of magnitude upon introducing an amiloride molecule, which does not inhibit the ion channel formation itself. Taylor et al [29] showed that Alm peptides can induce lipid flip-flop even in a surface-bound state by disordering lipids in the membrane.…”

Measuring bilayer surface energy and curvature in asymmetric droplet interface bilayers

“…Here the challenge lies in distinguishing the rate of flip-flop from the rate of lateral lipid diffusion [58] between the monolayer and bilayer, as well as lipid uptake into the bilayer [5,50,59]. However, a recent publication has demonstrated a promising technique for determining bilayer flip-flop on DIB membranes via parallel capacitance-based measurements on an integrated microfluidic device; in this study, it was successfully shown that surface-bound peptides (alamethicin) facilitate the movement of lipids between leaflets [9].…”

Methanol Accelerates DMPC Flip-Flop and Transfer: A SANS Study on Lipid Dynamics

“…In essence, both dynamical actions are intrinsically linked to bilayers and their compositional stability. Previous studies have externally induced reorganization of lipids by outside factors as seen in model phospholipid membranes upon the addition of cations (4), detergents (5), and peptides (6)(7)(8)(9). Here, we focus on how common organic solvents also impact these dynamics.…”