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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]
  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.
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
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.
@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 = {2018},
month = {7}
}

Journal Article:
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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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Works referenced in this record:

Regulation of transbilayer plasma membrane phospholipid asymmetry
journal, December 2002


Dynamic Transbilayer Lipid Asymmetry
journal, March 2011


Asymmetric Lipid Membranes: Towards More Realistic Model Systems
journal, May 2015


Phosphatidylserine, a death knell
journal, June 2001


Engineering asymmetric vesicles
journal, September 2003

  • Pautot, S.; Frisken, B. J.; Weitz, D. A.
  • Proceedings of the National Academy of Sciences, Vol. 100, Issue 19
  • DOI: 10.1073/pnas.1931005100

Production of Unilamellar Vesicles Using an Inverted Emulsion
journal, April 2003

  • Pautot, Sophie; Frisken, Barbara J.; Weitz, D. A.
  • Langmuir, Vol. 19, Issue 7
  • DOI: 10.1021/la026100v

Inkjet formation of unilamellar lipid vesicles for cell-like encapsulation
journal, January 2009

  • Stachowiak, Jeanne C.; Richmond, David L.; Li, Thomas H.
  • Lab on a Chip, Vol. 9, Issue 14
  • DOI: 10.1039/b904984c

Formation of Giant Lipid Vesiclelike Compartments from a Planar Lipid Membrane by a Pulsed Jet Flow
journal, October 2007

  • Funakoshi, Kei; Suzuki, Hiroaki; Takeuchi, Shoji
  • Journal of the American Chemical Society, Vol. 129, Issue 42
  • DOI: 10.1021/ja074029f

Unilamellar vesicle formation and encapsulation by microfluidic jetting
journal, March 2008

  • Stachowiak, J. C.; Richmond, D. L.; Li, T. H.
  • Proceedings of the National Academy of Sciences, Vol. 105, Issue 12
  • DOI: 10.1073/pnas.0710875105

Forming giant vesicles with controlled membrane composition, asymmetry, and contents
journal, May 2011

  • Richmond, D. L.; Schmid, E. M.; Martens, S.
  • Proceedings of the National Academy of Sciences, Vol. 108, Issue 23
  • DOI: 10.1073/pnas.1016410108

1 H NMR Shows Slow Phospholipid Flip-Flop in Gel and Fluid Bilayers
journal, February 2017


Whole-GUV patch-clamping
journal, December 2016

  • Garten, Matthias; Mosgaard, Lars D.; Bornschlögl, Thomas
  • Proceedings of the National Academy of Sciences, Vol. 114, Issue 2
  • DOI: 10.1073/pnas.1609142114

Translocation of Phospholipids Is Facilitated by a Subset of Membrane-spanning Proteins of the Bacterial Cytoplasmic Membrane
journal, July 2003

  • Kol, Matthijs A.; van Dalen, Annemieke; de Kroon, Anton I. P. M.
  • Journal of Biological Chemistry, Vol. 278, Issue 27
  • DOI: 10.1074/jbc.M301875200

Phospholipid Flop Induced by Transmembrane Peptides in Model Membranes Is Modulated by Lipid Composition
journal, January 2003

  • Kol, Matthijs A.; van Laak, Adrianus N. C.; Rijkers, Dirk T. S.
  • Biochemistry, Vol. 42, Issue 1
  • DOI: 10.1021/bi0268403

Peptide-Induced Asymmetric Distribution of Charged Lipids in a Vesicle Bilayer Revealed by Small-Angle Neutron Scattering
journal, August 2011

  • Qian, Shuo; Heller, William T.
  • The Journal of Physical Chemistry B, Vol. 115, Issue 32
  • DOI: 10.1021/jp204045t

Measuring Lipid Asymmetry in Planar Supported Bilayers by Fluorescence Interference Contrast Microscopy
journal, February 2005

  • Crane, Jonathan M.; Kiessling, Volker; Tamm, Lukas K.
  • Langmuir, Vol. 21, Issue 4
  • DOI: 10.1021/la047654w

Lipid Asymmetry in DLPC/DSPC-Supported Lipid Bilayers: A Combined AFM and Fluorescence Microscopy Study
journal, January 2006


Asymmetric Distribution of Phosphatidyl Serine in Supported Phospholipid Bilayers on Titanium Dioxide
journal, March 2006

  • Rossetti, Fernanda F.; Textor, Marcus; Reviakine, Ilya
  • Langmuir, Vol. 22, Issue 8
  • DOI: 10.1021/la053000r

Transbilayer Effects of Raft-Like Lipid Domains in Asymmetric Planar Bilayers Measured by Single Molecule Tracking
journal, November 2006


Coupling of Cholesterol-Rich Lipid Phases in Asymmetric Bilayers
journal, January 2008

  • Wan, Chen; Kiessling, Volker; Tamm, Lukas K.
  • Biochemistry, Vol. 47, Issue 7
  • DOI: 10.1021/bi7021552

Domain coupling in asymmetric lipid bilayers
journal, January 2009

  • Kiessling, Volker; Wan, Chen; Tamm, Lukas K.
  • Biochimica et Biophysica Acta (BBA) - Biomembranes, Vol. 1788, Issue 1
  • DOI: 10.1016/j.bbamem.2008.09.003

Stable Supported Lipid Bilayers on Zirconium Phosphonate Surfaces
journal, August 2009

  • Fabre, Roxane M.; Talham, Daniel R.
  • Langmuir, Vol. 25, Issue 21
  • DOI: 10.1021/la901920y

Composition and Asymmetry in Supported Membranes Formed by Vesicle Fusion
journal, June 2011


Asymmetric Distribution of Anionic Phospholipids in Supported Lipid Bilayers
journal, July 2012

  • Stanglmaier, S.; Hertrich, S.; Fritz, K.
  • Langmuir, Vol. 28, Issue 29
  • DOI: 10.1021/la3019887

Bilayer Asymmetry Influences Integrin Sequestering in Raft-Mimicking Lipid Mixtures
journal, May 2013


Asymmetric phospholipid: lipopolysaccharide bilayers; a Gram-negative bacterial outer membrane mimic
journal, December 2013

  • Clifton, Luke A.; Skoda, Maximilian W. A.; Daulton, Emma L.
  • Journal of The Royal Society Interface, Vol. 10, Issue 89
  • DOI: 10.1098/rsif.2013.0810

An Accurate In Vitro Model of the E. coli Envelope
journal, September 2015

  • Clifton, Luke A.; Holt, Stephen A.; Hughes, Arwel V.
  • Angewandte Chemie International Edition, Vol. 54, Issue 41
  • DOI: 10.1002/anie.201504287

Dipole potential measurements in asymmetric membranes
journal, November 1976

  • Latorre, Ramon; Hall, James E.
  • Nature, Vol. 264, Issue 5584
  • DOI: 10.1038/264361a0

Voltage-dependent capacitance in lipid bilayers made from monolayers
journal, January 1978


Capacitance and conductance as tools for the measurement of asymmetric surface potentials and energy barriers of lipid bilayer membranes
journal, December 1979

  • Schoch, Peter; Sargent, David F.; Schwyzer, Robert
  • The Journal of Membrane Biology, Vol. 46, Issue 1
  • DOI: 10.1007/BF01959975

Bilayer lipid membranes in strong electric fields
journal, January 1980

  • Chizmadzhev, Yu. A.; Abidor, I. G.
  • Journal of Electroanalytical Chemistry and Interfacial Electrochemistry, Vol. 116
  • DOI: 10.1016/S0022-0728(80)80223-3

Determination of surface charge of bilayer lipid membranes
journal, January 1980

  • Cherny, V. V.; Sokolov, V. S.; Abidor, I. G.
  • Journal of Electroanalytical Chemistry and Interfacial Electrochemistry, Vol. 116
  • DOI: 10.1016/S0022-0728(80)80266-X

Tuning lipid mixtures to induce or suppress domain formation across leaflets of unsupported asymmetric bilayers
journal, January 2008

  • Collins, M. D.; Keller, S. L.
  • Proceedings of the National Academy of Sciences, Vol. 105, Issue 1
  • DOI: 10.1073/pnas.0702970105

Formation of Bimolecular Membranes from Lipid Monolayers and a Study of Their Electrical Properties
journal, December 1972

  • Montal, M.; Mueller, P.
  • Proceedings of the National Academy of Sciences, Vol. 69, Issue 12
  • DOI: 10.1073/pnas.69.12.3561

Lipid Exchange and Flip-Flop in Solid Supported Bilayers
journal, October 2013

  • Gerelli, Yuri; Porcar, Lionel; Lombardi, Lucia
  • Langmuir, Vol. 29, Issue 41
  • DOI: 10.1021/la402708u

Lipid Bilayer Formation by Contacting Monolayers in a Microfluidic Device for Membrane Protein Analysis
journal, December 2006

  • Funakoshi, Kei; Suzuki, Hiroaki; Takeuchi, Shoji
  • Analytical Chemistry, Vol. 78, Issue 24
  • DOI: 10.1021/ac0613479

Droplet interface bilayers
journal, January 2008

  • Bayley, Hagan; Cronin, Brid; Heron, Andrew
  • Molecular BioSystems, Vol. 4, Issue 12
  • DOI: 10.1039/b808893d

Functional Bionetworks from Nanoliter Water Droplets
journal, July 2007

  • Holden, Matthew A.; Needham, David; Bayley, Hagan
  • Journal of the American Chemical Society, Vol. 129, Issue 27
  • DOI: 10.1021/ja072292a

Asymmetric Droplet Interface Bilayers
journal, May 2008

  • Hwang, William L.; Chen, Min; Cronin, Bríd
  • Journal of the American Chemical Society, Vol. 130, Issue 18
  • DOI: 10.1021/ja802089s

Novel technologies for the formation of 2-D and 3-D droplet interface bilayer networks
journal, January 2012

  • Elani, Yuval; deMello, Andrew J.; Niu, Xize
  • Lab on a Chip, Vol. 12, Issue 18
  • DOI: 10.1039/c2lc40287d

Water Permeability across Symmetric and Asymmetric Droplet Interface Bilayers: Interaction of Cholesterol Sulfate with DPhPC
journal, October 2015


Engineering plant membranes using droplet interface bilayers
journal, March 2017

  • Barlow, N. E.; Smpokou, E.; Friddin, M. S.
  • Biomicrofluidics, Vol. 11, Issue 2
  • DOI: 10.1063/1.4979045

The mechanoelectrical response of droplet interface bilayer membranes
journal, January 2016

  • Freeman, E. C.; Najem, J. S.; Sukharev, S.
  • Soft Matter, Vol. 12, Issue 12
  • DOI: 10.1039/C5SM02779A

Voltage-dependent lipid flip-flop induced by alamethicin
journal, March 1981


Measurement of voltage dependence of capacitance of planar bilayer lipid membrane with a patch clamp amplifier
journal, April 1991


Hydrodynamic trapping for rapid assembly and in situ electrical characterization of droplet interface bilayer arrays
journal, January 2016

  • Nguyen, Mary-Anne; Srijanto, Bernadeta; Collier, C. Patrick
  • Lab on a Chip, Vol. 16, Issue 18
  • DOI: 10.1039/C6LC00810K

Properties of Diphytanoyl Phospholipids at the Air–Water Interface
journal, December 2014

  • Yasmann, Anthony; Sukharev, Sergei
  • Langmuir, Vol. 31, Issue 1
  • DOI: 10.1021/la503800g

Alamethicin-induced current-voltage curve asymmetry in lipid bilayers
journal, April 1983


Heating-Enabled Formation of Droplet Interface Bilayers Using Escherichia coli Total Lipid Extract
journal, December 2014

  • Taylor, Graham J.; Sarles, Stephen A.
  • Langmuir, Vol. 31, Issue 1
  • DOI: 10.1021/la503471m

Direct in situ measurement of specific capacitance, monolayer tension, and bilayer tension in a droplet interface bilayer
journal, January 2015

  • Taylor, Graham J.; Venkatesan, Guru A.; Collier, C. Patrick
  • Soft Matter, Vol. 11, Issue 38
  • DOI: 10.1039/C5SM01005E

Capacitive Detection of Low-Enthalpy, Higher-Order Phase Transitions in Synthetic and Natural Composition Lipid Membranes
journal, August 2017


Formation of Droplets of Alternating Composition in Microfluidic Channels and Applications to Indexing of Concentrations in Droplet-Based Assays
journal, September 2004

  • Zheng, Bo; Tice, Joshua D.; Ismagilov, Rustem F.
  • Analytical Chemistry, Vol. 76, Issue 17
  • DOI: 10.1021/ac0495743

Capacitance-voltage relationship in phospholipid bilayers containing gangliosides
journal, March 1983


Voltage-dependent energetics of alamethicin monomers in the membrane
journal, June 2006


Lipid-alamethicin interactions influence alamethicin orientation
journal, November 1991


Mechanism of alamethicin insertion into lipid bilayers
journal, November 1996


Voltage-dependent pore activity of the peptide alamethicin correlated with incorporation in the membrane: salt and cholesterol effects
journal, June 1988

  • Stankowski, Stefan; Schwarz, Udo D.; Schwarz, Gerhard
  • Biochimica et Biophysica Acta (BBA) - Biomembranes, Vol. 941, Issue 1
  • DOI: 10.1016/0005-2736(88)90208-8

The Ins and Outs of Lipid Flip-Flop
journal, December 2016


The influence of cholesterol on phospholipid membrane curvature and bending elasticity
journal, July 1997


Comparative Study of the Effects of Several n-Alkanes on Phospholipid Hexagonal Phases
journal, February 1998


Evaporation-induced monolayer compression improves droplet interface bilayer formation using unsaturated lipids
journal, March 2018

  • Venkatesan, Guru A.; Taylor, Graham J.; Basham, Colin M.
  • Biomicrofluidics, Vol. 12, Issue 2
  • DOI: 10.1063/1.5016523

Determining the Membrane Topology of Peptides by Fluorescence Quenching
journal, December 1999

  • Wimley, William C.; White, Stephen H.
  • Biochemistry, Vol. 39, Issue 1
  • DOI: 10.1021/bi991836l

A helix dipole model for alamethicin and related transmembrane channels
journal, June 1983


Incorporation kinetics in a membrane, studied with the pore-forming peptide alamethicin
journal, November 1987


Alamethicin incorporation in lipid bilayers: a thermodynamic study
journal, May 1987

  • Rizzo, Vincenzo; Stankowski, Stefan; Schwarz, Gerhard
  • Biochemistry, Vol. 26, Issue 10
  • DOI: 10.1021/bi00384a015

Thermodynamic analysis of incorporation and aggregation in a membrane: Application to the pore-forming peptide alamethicin
journal, January 1986

  • Schwarz, Gerhard; Stankowski, Stefan; Rizzo, Vincenzo
  • Biochimica et Biophysica Acta (BBA) - Biomembranes, Vol. 861
  • DOI: 10.1016/0005-2736(86)90412-8

Works referencing / citing this record:

Photopolymerized microdomains in both lipid leaflets establish diffusive transport pathways across biomimetic membranes
journal, January 2019

  • Makhoul-Mansour, Michelle M.; El-Beyrouthy, Joyce B.; Mumme, Hope L.
  • Soft Matter, Vol. 15, Issue 43
  • DOI: 10.1039/c9sm01658a

Probing membrane protein properties using droplet interface bilayers
journal, May 2019

  • Allen-Benton, Maxwell; Findlay, Heather E.; Booth, Paula J.
  • Experimental Biology and Medicine, Vol. 244, Issue 8
  • DOI: 10.1177/1535370219847939

Accelerated Molecular Dynamics Applied to the Peptaibol Folding Problem
journal, August 2019

  • Tyagi, Chetna; Marik, Tamás; Vágvölgyi, Csaba
  • International Journal of Molecular Sciences, Vol. 20, Issue 17
  • DOI: 10.3390/ijms20174268

Measuring bilayer surface energy and curvature in asymmetric droplet interface bilayers
journal, November 2018

  • Barlow, Nathan E.; Kusumaatmaja, Halim; Salehi-Reyhani, Ali
  • Journal of The Royal Society Interface, Vol. 15, Issue 148
  • DOI: 10.1098/rsif.2018.0610

Accelerated Molecular Dynamics Applied to the Peptaibol Folding Problem
journal, August 2019

  • Tyagi, Chetna; Marik, Tamás; Vágvölgyi, Csaba
  • International Journal of Molecular Sciences, Vol. 20, Issue 17
  • DOI: 10.3390/ijms20174268

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