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2016 © The Japan Society for Analytical Chemistry

Channel Current Analysis for Pore-forming Properties of an
Antimicrobial Peptide, Magainin 1, Using the Droplet Contact
MethodHirokazu WATANABE and Ryuji KAWANO†
Department of Life Science and Biotechnology, Tokyo University of Agriculture and Technology,
2-24-16 Naka-cho, Koganei, Tokyo 184–8588, Japan

This study describes the pore-forming properties of magainin 1 in planar lipid bilayers. These bilayers were prepared by
the droplet contact method, which was executed on a microfabricated device for a high-throughput study. We arrayed four
droplet chambers parallelly in the single device, and the current measurements were carried out simultaneously. Using
this system, we measured the channel current conductance of magainin 1. We determined the pore size and the numberof assembling monomers in magainin pores in mammalian and bacterial model membranes. This system is a powerfultool for analyzing transmembrane peptides and their antimicrobial activities.
Keywords Droplet, lipid bilayers, electrophysiology, magainin 1, antimicrobial peptide(Received August 31, 2015; Accepted October 22, 2015; Published January 10, 2016)

Introduction
Magainins are antimicrobial peptides (AMPs), which werediscovered in the skin of the African clawed frog Xenopus
laevis. This frog can remain infection-free in nature;1–4 this isbelieved to be mainly because of the presence of Magainins.
Magainins have 23 amino acid residues and demonstratebroad-spectrum antibacterial and antifungal activities.5 To apply
a clinically useful antimicrobial therapeutic agent, extensive
studies, such as NMR,6,7 Raman spectroscopy,8 fluorescence,3,9and electrophysiological studies,1,10 have been performed toanalyze the antibacterial mechanism.
Among them, anelectrophysiological method that uses lipid bilayers is an
integral tool for analyzing lipid-peptide interactions and pore
formations. However, the electrophysiological experimentrequires technical skills for preparing planar lipid bilayers, andis time consuming. Therefore, efficient electrophysiologicalmethods are required.
We recently proposed stable and reproducible planar lipid
bilayer formation using microdroplets in a “droplet contact
method” (DCM).11 A planar lipid bilayer can be spontaneously
formed at the interface between contacting microdroplets in an
oil/lipid mixture solution.12,13 In this method, membranestability is improved by reducing the contact area. The long-term
stability of the membrane is notable, and remains for more than
300 h.
Moreover, we constructed a droplet array forhigh-throughput measurements.11 Using bilayer lipid membranes(BLMs), we have been measuring various membrane protein
and biological nanopores, e.g., single molecule detection using
DNA aptamers,14 high-throughput ion channel measurements at
To whom correspondence should be addressed.
E-mail: rjkawano@cc.tuat.ac.jp†

the single-protein level,11,15 solution-exchange experiment in the
microdroplet system,16 and the development of portable
nanopore sensors.17
In this paper, we report on the pore-forming properties ofmagainin 1 (GIGKFLHSAGKFGKAFVGEIMKS) from channel
current conductance measurements using the high-throughput
DCM. Both magainin 1 and magainin 2 are isolated fromXenopus laevis, and have a similar amino-acid sequence. It has
been known that magainin 2 has higher antibacterial activity
than magainin 1. Although mgainin 2 is a popular AMP and
there are extensive studies, molecular mechanism of magainin 1
is still in challenge. Magainins and many AMPs form
transmembrane pores by assembling monomers. This study
focuses on determining the pore size and the number ofassembling monomers. Thus, we mathematically estimate thisnumber from the current conductance and a polygonal model.
Our methods have significant potential for AMP analysis and inclinical applications.

Experimental
Reagents and chemicals
The reagents used in this study were as follows: 1,2-dioleoylsn-glycero-3-phosphocholine (DOPC, Avanti Polar Lipids,
Alabaster, AL); 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine(DOPE, Avanti Polar Lipids); 1,2-dioleoyl-sn-glycero-3phospho-(1′-rac-glycerol) (DOPG, Avanti Polar Lipids);
n-decane (Sigma-Aldrich, St. Louis, MO); 3-morpholinopropane1-sulfonic acid (MOPS, Nacalai Tesque, Kyoto, Japan); andKCl (Nacalai Tesque). Buffered electrolyte solutions wereprepared from ultrapure water, which was obtained from a
Milli-Q system (Millipore, Billerica, MA). Magainin 1 (LKT
Laboratories Inc., St. Paul, MN) was obtained as a monomer

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Fig. 2 Representative current signals in the PC and PE/PG
membranes. The signals were classified into three types: step-like,
multi-level, and spike signals. The percentages of each types of signals
represent the ratio of each signals in all of signals.

Fig. 1 (a) Lipid bilayer prepared by the droplet contact method, anda photograph of the device for measuring channel currents.
(b) Magainin reconstituted in BLM forms barrel-stave and toroidal
pore by assembling monomers.

polypeptide isolated from African frogs in the form of a powder,and dissolved at a concentration of 1 mM in ultrapure water.
For use, samples were diluted to 10 μM using a buffered
electrolyte solution and stored at 4°
C.
Lipid bilayer preparation and reconstitution of magainin 1
BLMs were prepared using an arrayed device, which has
Four
four chambers fabricated by microfabrication.11,15
individual BLMs can be formed simultaneously in this device,
which allowed for a higher-throughput measurement comparedto the conventional system. Using this device, the DOPC or
DOPE/DOPG (3:1 mol/mol) mixture (lipids/n-decane, 10 mg/mL) solution (2.3 μL) was poured into each chamber. Next, thebuffer solution (4.7 μL) was poured into both chambers. The
same buffer solution (200 mM KCl, 10 mM MOPS, pH 7.0)
was used for both droplets in this study. Magainin 1 wasdissolved in an aqueous droplet at a 10 μM concentration to one
side (voltage applying side). Within a few minutes of adding
the buffer solution, two lipid monolayers connected and formed
BLMs, and magainin 1 formed nanopores by reconstitution in
the BLM. When the BLM ruptured during the process, wereformed the BLM by tracing with a hydrophobic stick at theinterface of the drops.
Channel current measurements and data analysis
Channel currents were monitored using a Jet patch clamp
amplifier (Tecella, Foothill Ranch, CA) connected to an
Ag/AgCl electrode in each chamber. These electrodes were ineach droplets when we added the solution into the chambers.
The trans side was grounded, and a constant voltage of +100 mV
was applied to the cis side. Reconstituted magainin in BLMs
allowed ions to pass through nanopores under the voltage
gradient, and we obtained the channel current signals. The
signals were detected using a 4-kHz low-pass filter at a sampling

frequency of 20 kHz. Analysis of channel signal was performed
using pCLAMP Ver. 10.5 (Molecular Devices, Sunnyvale, CA).
We classified the current signals into three types in reference
to the shape of the time–current traces. First, the step-likesignals were the signals where the current increased sharply andplateaued. We counted one step of the current increase as one
signal. Second, we regarded the current increase fluctuating
continuously as multi-level signals and counted from the start tofinish of the current increase as one signal. The end of thecurrent increase means the return to 0 pA or an increase larger
than the measurement limit. Third, the spike signal was a shortcurrent increase that momentarily returns to 0 pA, and we
counted a series of events at a similar amplitude as one signal.

Results and Discussion
The pore-forming activities of many AMPs is different between
mammalian and bacteria model membranes.3 AMPs usuallyshow a stronger activity in a negatively charged membrane (e.g.,
DOPE/DOPG) because of the greater interaction between lipid
head groups and peptides at the surface of the membrane. Thus,
we used different compositions of BLMs of DOPC (PC) or
DOPE/DOPG (PE/PG) as mammalian or bacteria model
membranes.
Figure 2 shows a representative example of current recordingsin the PC and PE/PG membrane systems. Several types ofcurrent signals appeared in both system. We classified thesesignals by reference to Fyles group’s study.18 The three types ofsignals, i.e., step-like, multi-level, and spike signals, obtainedreflect the several pore-forming modes of magainin 1. Previous
studies have proposed19 that magainin and many other AMPs
form peptide helices and associate to form a bundle with acentral lumen, similar to a barrel made of helical peptides,
which is the “barrel-stave model.” However, in the “toroidal
model,” the peptides are always associated with the lipid headgroups, even when they are perpendicularly inserted into thelipid bilayer. During the formation of the pore structure in thetoroidal model, the lipid monolayer bends continuously from
top to bottom to form a toroidal hole. The toroidal pore is linedby both peptides and lipid head groups. In channel current
recordings, the obtained current signals would reflect thesemodels, as proposed in a previous study.19 The step-like signals
correspond to a barrel-stave-model pore where the rigid-ringpore shows a stepwise transition to the upstairs direction. In
contrast, the multi-level or spike signals indicate a toroidal-

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Fig. 3 Histograms of conductance of channel-current (a, b), pore diameter (c, d) and the number ofmonomers assembling to form a nanopore (e, f) fitted by a Gaussian distribution. Magainin formed
heterogeneous-sized nanopores in BLMs.

model pore or other instantaneous membrane defects. Thesefragile pores vary in size dynamically because lipids can diffuse
laterally from the pore to lipid membranes. Consequently, thecurrent signals in these cases were fluctuating or spike signals.
Using all current signals, we determined the rate of occurrence
for each signal, as shown in Fig. 2. The orders of occurrence
were “multi-level > step-like > spike” and “step-like > spike >multi-level” in the PC and PE/PG systems, respectively. This
result implies that magainin 1 should be predisposed to form
rigid pores in the negatively charged membrane (PE/PG). When
compared with neutral lipids, magainin 1 reconstituted innegatively charged lipid bilayers is likely to follow thebarrel-stave model because of electrostatic repulsion.
To estimate pore formation in terms of the pore size andmonomer assembling state of magainin 1, we analyzed thecurrent conductance of the “step-like” signals. Figures 3a and3b shows the histograms of the conductance. In these signals,
magainin 1 forms pores following the barrel-stave model asmentioned above. Therefore, we can ignore the lipid-mixed

pores such as those formed through the toroidal model. The
Hille equation20 was used in pore diameter estimation:

ρR = ⎛ l + πr ⎞ 2 ,⎝2 ⎠ πr(1)

where r is the pore radius, ρ is the resistivity of the buffered
solution, l is the length of the pore (7 nm from the thickness of
the BLM), and R is the resistivity of the pore. R is calculated as
V/I, where I is the current through the magainin pore and V isthe applied voltage between two chambers (100 mV). Several
pore sizes can be obtained from several levels of current
amplitudes. Histograms of the pore diameters in the PC and
PE/PG systems are presented in Figs. 3a and 3b. The mean pore
diameters fitted by a Gaussian distribution were 0.88 nm in the
PC system and 1.29 nm in the PE/PG system. Magainin 1
formed larger pores in the PE/PG system when compared with
the PC system because of the negatively charged lipids. This
result is consistent with that of the antimicrobial activity of

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magainin 2 and its analogs in human red blood and bacteria
cells.21
Using the pore diameter and the monomer size from theProtein Data Bank (PDB), the number of assembling monomers
was mathematically calculated by⎛⎞1
d = a⎜−1⎝ sin(π/n) ⎟⎠(2)

where d is the pore diameter, a is the width of a peptide
monomer from the PDB, and n is the number of assembling
monomers. Figures 3c and 3d show histograms of the numberof monomers that assembled to form pores. From thehistograms, 4 – 10 monomers formed a pore. Considering theprevious results of a mean of seven monomers of magainin 2
per channel,22 these assembling numbers are reasonable. In
addition, the assembling number in the PE/PG system was also
larger than that of the PC system. The mean assembling
numbers were 4.9 and 5.9 in the PC and PE/PG systems,
respectively. Using our DCM, the pore-forming properties ofmagainin 1 were analyzed from the channel current conductance.
These results can reflect antimicrobial activities in living cells
without in vitro lipid bilayer experiments.

Conclusions
In summary, we measured the channel current conductance ofmagainin 1 using BLMs prepared by DCM. Three types ofcurrent signals were observed: step-like, multi-level, and spikesignals. The appearance of these signals depended on the lipid
content. The step-like signal was mainly observed in the PE/PG
system as the bacterial model membrane. We estimated thepore diameter and the number of assembling monomers, andshowed that magainin 1 uses 4 – 10 monomers to assemble apore of 0.5 – 3 nm in diameter.
Our DCM can readily prepare lipid bilayers and analyze
pore-forming properties in a high-throughput study. Thismethod will be applied to the extensive analysis and estimationof pore-forming properties of AMPs.

Acknowledgements
This work was partly supported by a Grant-in-Aid for Scientific
Research on Innovative Areas (No. 24104002) and Scientific
Research Nos. 26540160 and 25708024 of The Ministry of
Education, Culture, Sports, Science and Technology, Japan.

References
1. R. A. Cruciani, J. L. Barker, S. R. Durell, G. Raghunathan,
H. R. Guy, M. Zasloff, and E. F. Stanley, Eur. J. Pharmcol.,Mol. Pharmacol. Sect., 1992, 226, 287.
2. H. W. Huang, Biochim. Biophys. Acta, Biomembr., 2006,
1758, 1292.
3. K. Matsuzaki, Biochim. Biophys. Acta, Biomembr., 1999,
1462, 1.
4. Y. Tamba and M. Yamazaki, J. Phys. Chem. B, 2009, 113,4846.
5. M. N. Melo, R. Ferre, and M. Castanho, Nat. Rev.
Microbiol., 2009, 7, 245.
6. A. Naito and I. Kawamura, Biochim. Biophys. Acta,
Biomembr., 2007, 1768, 1900.
7. B. Bechinger, Biochim. Biophys. Acta, Biomembr., 1999,
1462, 157.
8. R. W. Williams, R. Starman, K. M. P. Taylor, K. Gable, T.
Beeler, M. Zasloff, and D. Covell, Biochemistry, 1990, 29,4490.
9. M. Z. Islam, J. M. Alam, Y. Tamba, M. A. S. Karal, and M.
Yamazaki, Phys. Chem. Chem. Phys., 2014, 16, 15752.
10. E. Gallucci, D. Meleleo, S. Micelli, and V. Picciarelli, Eur.
Biophys. J. Biophys. Lett., 2003, 32, 22.
11. R. Kawano, Y. Tsuji, K. Sato, T. Osaki, K. Kamiya, M.
Hirano, T. Ide, N. Miki, and S. Takeuchi, Sci. Rep., 2013, 3.
12. K. Funakoshi, H. Suzuki, and S. Takeuchi, Anal. Chem.,
2006, 78, 8169.
13. A. J. Heron, J. R. Thompson, A. E. Mason, and M. I.
Wallace, J. Am. Chem. Soc., 2007, 129, 16042.
14. R. Kawano, T. Osaki, H. Sasaki, M. Takinoue, S. Yoshizawa,and S. Takeuchi, J. Am. Chem. Soc., 2011, 133, 8474.
15. Y. Tsuji, R. Kawano, T. Osaki, K. Kamiya, N. Miki, and S.
Takeuchi, Anal. Chem., 2013, 85, 10913.
16. Y. Tsuji, R. Kawano, T. Osaki, K. Kamiya, N. Miki, and S.
Takeuchi, Lab Chip, 2013, 13, 1476.
17. R. Kawano, Y. Tsuji, K. Kamiya, T. Kodama, T. Osaki, N.
Miki, and S. Takeuchi, PLoS One, 2014, 9, e102427.
18. J. K. W. Chui and T. M. Fyles, Chem. Soc. Rev., 2012, 41,
148.
19. L. Yang, T. A. Harroun, T. M. Weiss, L. Ding, and H. W.
Huang, Biophys. J., 2001, 81, 1475.
20. B. Hille, “Ion Channels of Excitable Membranes”, 3rd ed.,2001, Sinauer, Sunderland, 814.
21. A. Iwahori, Y. Hirota, R. Sampe, S. Miyano, and N. Numao,
Biol. Pharm. Bull., 1997, 20, 267.
22. H. W. Huang, Biochemistry, 2000, 39, 8347.