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Title: 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 has 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 ofmore » 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. Furthermore, this methodology establishes the use of parallel electrophysiology for efficiently studying membrane asymmetry in arrays of DIBs.« less

Authors:
 [1];  [1];  [1];  [2]; ORCiD logo [3];  [1]
+ Show Author Affiliations
  1. The Univ. of Tennessee, Knoxville, TN (United States)
  2. The Univ. of Georgia, Athens, GA (United States)
  3. Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States)
Publication Date:
Research Org.:
Oak Ridge National Laboratory (ORNL), Oak Ridge, TN (United States)
Sponsoring Org.:
USDOE
OSTI Identifier:
1484993
Grant/Contract Number:  
AC05-00OR22725
Resource Type:
Accepted Manuscript
Journal Name:
Biochimica et Biophysica Acta. Biomembranes
Additional Journal Information:
Journal Volume: 1861; Journal Issue: 1; Journal ID: ISSN 0005-2736
Publisher:
Elsevier
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES

Citation Formats

Taylor, Graham J., Nguyen, Mary -Anne, Koner, Subhadeep, Freeman, Eric, Collier, C. Patrick, and Sarles, Stephen A. Electrophysiological interrogation of asymmetric droplet interface bilayers reveals surface-bound alamethicin induces lipid flip-flop. United States: N. p., 2018. Web. doi:10.1016/j.bbamem.2018.07.001.
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Taylor, Graham J., Nguyen, Mary -Anne, Koner, Subhadeep, Freeman, Eric, Collier, C. Patrick, & Sarles, Stephen A. Electrophysiological interrogation of asymmetric droplet interface bilayers reveals surface-bound alamethicin induces lipid flip-flop. United States. https://doi.org/10.1016/j.bbamem.2018.07.001
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Taylor, Graham J., Nguyen, Mary -Anne, Koner, Subhadeep, Freeman, Eric, Collier, C. Patrick, and Sarles, Stephen A. Tue . "Electrophysiological interrogation of asymmetric droplet interface bilayers reveals surface-bound alamethicin induces lipid flip-flop". United States. https://doi.org/10.1016/j.bbamem.2018.07.001. https://www.osti.gov/servlets/purl/1484993.
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@article{osti_1484993,
title = {Electrophysiological interrogation of asymmetric droplet interface bilayers reveals surface-bound alamethicin induces lipid flip-flop},
author = {Taylor, Graham J. and Nguyen, Mary -Anne and Koner, Subhadeep and Freeman, Eric and Collier, C. Patrick and Sarles, Stephen A.},
abstractNote = {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 has 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. Furthermore, this methodology establishes the use of parallel electrophysiology for efficiently studying membrane asymmetry in arrays of DIBs.},
doi = {10.1016/j.bbamem.2018.07.001},
journal = {Biochimica et Biophysica Acta. Biomembranes},
number = 1,
volume = 1861,
place = {United States},
year = {Tue Jul 10 00:00:00 EDT 2018},
month = {Tue Jul 10 00:00:00 EDT 2018}
}
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Journal Article:
Free Publicly Available Full Text
Publisher's Version of Record
https://doi.org/10.1016/j.bbamem.2018.07.001
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Cited by: 25 works
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Figures / Tables:

Figure 1 Figure 1: A) 10× magnified image of the opposing T-junction droplet generators used to form droplets of alternating aqueous composition. Oil flow rate: 0.4–1.0 μL/ min. Aqueous flow rate: 0.005–0.5 μL/min. B) 10× magnified image of the microfluidic droplet routing and trapping pathway used to form asymmetric DIBs, including squaremore » electrode pads deposited on the glass surface under each droplet trap. The central electrodes (green dashed line overlays) are connected and used as a common ground connection (G), while each black electrode (E1-E8) is connected to an independent channel of the 8-channel patch clamp amplifier headstage. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)« less
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