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Electrophysiological Analysis of Membrane Disruption by Bombinin
and Its Isomer Using the Lipid Bilayer System
Yusuke Sekiya,†,∥ Keisuke Shimizu,†,∥ Yuki Kitahashi,‡ Akifumi Ohyama,§ Izuru Kawamura,‡,§and Ryuji Kawano*,††Downloaded via KYOTO UNIV on March 4, 2019 at 07:27:01 (UTC).
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Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho, Koganei,
Tokyo 184-8588, Japan‡
Graduate School of Engineering, Yokohama National University, Hodogaya-ku, Yokohama 240-8501, Japan§
Graduate School of Engineering Science, Yokohama National University, Hodogaya-ku, Yokohama 240-8501, JapanS Supporting Information*

ABSTRACT: Bombinin H2 and H4 are peptides isolated fromthe skin of the frog Bombina variegata that exhibit antimicrobialactivity against Leishmania as well as bacteria. H4 is an isomerof H2 that has D-allo-Ile at position 2 from the N-terminus.
Although H4 exhibits higher antimicrobial activity than that of
H2, the molecular mechanism has remained unclear. In thisstudy, we tried to reveal the molecular mechanism in terms oflipid membrane disruption through pore formation, using
electrophysiological measurements. Based on our experiments,
we estimated the pore-forming structure, pore size, and the
kinetics in a bacteria model membrane. Stochastic analysis of
the current data indicated that peptide isomerization enables us
to accelerate the pore formation owing to the higher aﬃnity
between the peptide and lipid membrane. Additionally, the H2/H4 mixture was studied with 31P NMR and cross-linking
experiment with mass spectrometry. It was found that heterogeneous pore formation with H2 and H4 was indicated. This
electrophysiological approach will likely be promising as a useful tool for analyzing the molecular mechanism of pore-forming
peptides.
KEYWORDS: antimicrobial peptide, bombinin, isomer, electrophysiology, lipid bilayer, solid-state NMR■

INTRODUCTION
The isomerization of peptides constitutes an intriguing event ofbiosynthesis because of its unique capability of modifying thebiochemical properties of the original peptide.1 For example,
one of the opiate peptides, dermorphin, secreted in the skin of
the South America tree frog contains D-Ala at position 2 from the
N-terminus.2 This type of isomer exhibits higher binding aﬃnity
and selectivity to μ-opiate receptors. Similarly, bombinin H,
which is an antimicrobial peptide (AMP) isolated from skinsecretions of the frog Bombina variegata, contains either L-Ile orD-allo-Ile at the position of the second amino acid from the Nterminus, as all L-peptide bombinin H2 (IIGPVLGLVGSALGGLLKKI-NH2) or its diastereomer H4 (Figure 1a).3
Several studies have reported that bombinin H4 showed higher
antimicrobial activity than that of H2.4 In particular, bombinins
are potent candidates as medicine for Leishmania, whichconstitutes an infectious disease with a global incidence of 10
to 12 million people infected.5 Bombinins can directly impact
Leishmania, whereas only a few other AMPs have been known to
exhibit leishmanicidal activity, the majority of which acts mainlyon the insect stage of the parasite (promastigotes).6 Although
the bombinins are expected to function as unique AMPs upon
© XXXX American Chemical Society

Figure 1. Schematic diagrams of L- and D-bombinins and their pore
formation in the planar lipid bilayer system. (a) Schematic structure ofbombinins H2 (left) and H4 (right). (b) Schematic diagram of thechannel current measurement using a planar bilayer lipid membrane(pBLM).

isomerization even against Leishmania, the molecular mechanism of their activity remains under debate.
In this study, we hypothesize that the main activity ofbombinins is probably due to the membrane disruption as
indicated from several prior experiments.7,8 Thus, we examined
Received: December 27, 2018
Accepted: February 20, 2019
Published: February 20, 2019
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Figure 2. Classiﬁcation of the channel current signals and possible models of the transmembrane structure of bombinins from among those previously
proposed. The assignment between the current signals and pore-forming models has been proposed in our previous study (ref 22).

the pore-forming behavior of H2 and H4 using a planar bilayer
lipid membrane (pBLM) as a model bacteria membrane (Figure1b). The electrophysiological method of pBLM will likely be auseful tool for analyzing the molecular mechanism of poreforming peptides in comparison to the conventional methods formembrane peptide analysis, which rely on spectroscopic or
simulation study. We have previously proposed high-throughput
pBLM formation by using a microfabricated device as shown in
Figure S-1 (see the details in Methods).9−13 Using this system,
we have reported the pore-forming behavior of the AMP,
magainin, and estimated the pore formation model using
stochastic current analysis.14 Based on our systematic method, inthe current study, we attempt to reveal the pore-forming activityof bombinins H2 and H4 and their mixture at the molecularbasis and discuss the relationship between the pore formation
behavior and antimicrobial activity.

RESULTS
Signal Classiﬁcation and Estimation of Pore-Forming
Mechanisms. Bombinin H2, H4, and the H2/H4 mixture wererepresented by the four diﬀerent current signals in thismeasurement. We classiﬁed these signals as shown in Figure
2; the signals were deﬁned as a step, multilevel, erratic, and spike.
These, along with their possible pore-forming models from theliterature,14−16 are described as follows.

The carpet model has been described such that peptides
bind parallel to the lipid bilayer surface, and after reaching
suﬃcient coverage, they unwrap lipids as a peptide−lipid
cluster from the membrane similar to a detergent. The
random current behavior of the erratic signal would becaused by the random size of the cluster.
(iv) Spike signal: a current suddenly raises up and then goesback to the baseline over a period of less than 20 ms, as wedeﬁne here. This signal indicates an instantaneous
membrane defect. We apply this signal to peptide
permeation20 through the membrane.
These pore-forming models of AMPs have been alreadyproposed; we have attempted to apply these models to thediﬀerent behaviors of the observed channel currents21 andrecently to assign the signals using magainin, alamethicin, and
LK model peptides as toroidal/carpet, barrel-stave, andpenetration model peptides.22 We counted these signals fromthe initial signal until more than a hundred signals, as an index ofpore stability of peptides. However, during this counting, thediﬀerent signals sometimes overlapped at the same time. Thesesignals reﬂect that several pores are formed in the lipid bilayer
membrane, and the diameter is about 100 μm. For example,
spike signals were occasionally observed on the plateau region of
the step signal, and we did not count the overlapped signals inthis case. The ratio of the signal appearance is shown in Figure 3.

(i) Step signal: a current jumps up orthogonally and maintains
a plateau state. This signal can apply to a “barrel-stave”
model17 wherein transmembrane peptides are tightly
assembled with each other and form a rigid circular pore.
(ii) Multilevel signal: current shows ﬂuctuation after jumping
up and goes back to the initial state. This current may
indicate a “toroidal” model18 whereby transmembrane
peptides form a pore with lipids. In this model, the size ofthe pore can change dynamically with or without theparticipation of the lipid between the monomers.
(iii) Erratic signal: a current represents random increases with
ﬂuctuation. We assigned this signal to a “carpet” model.19

Figure 3. Ratio of appearance for each current signal (n > 200 in each
peptide).

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Figure 4. (a) Deﬁnition of the charge ﬂux and the reaching time for analyzing the channel current recordings. The charge ﬂux (b) and the current
reaching time (c) for threshold current of 500 pA.

Figure 5. Pore diameter and stoichiometry of bombinins with a barrel-stave pore. (a) Pore diameter of each bombinin calculated from the Hille model.
(b) Mathematical estimation of the stoichiometry (number of peptide monomers for forming the pore) using the pore diameters and the monomer
size (n > 30).

Ion Leaking Activity and Kinetics of the Pore
Formation. The leaking experiment is normally performed toestimate the antimicrobial activity of membrane disruption. In
the current study, we focused on the charge ﬂux instead of theleaking experiment, which is the total amount of ion transportation through the pore.23 Figure 4a shows the typical erratic
signal and the method used to estimate the charge ﬂux. The
charge ﬂux was deﬁned as the average charge per unit time fromthe initial current increase to the threshold and never goes below
the line, as presented in the yellow area in Figure 4a. In thisestimation, the rapid current overﬂow for less than 10 s that may
indicate the membrane rupture was not counted. Figure 4b
shows the charge ﬂux of H2, H4, and H2/H4 using the thresholdof 500 pA. Although the value of H4 was slightly higher than thatof others, the diﬀerence was not clear. Therefore, we checked theother threshold values of 100 and 1000 pA. The order of values
was H4 ≥ H2/H4 ≥ H2 in all threshold conditions (Figure S-2).
The charge ﬂux should reﬂect the activity of ionic leakage thatwill correspond to the ion permeability via the lipid membrane.
In addition, the tendency of the total pore area would also be H4≥ H2/H4 ≥ H2 if the ability of ion permeability per unit porearea per time (permeability/(nm2 s)) of all bombinins isconstant.

The ratio of spike signals was the highest in all three systems of
H2, H4, and mixture, after which the order was as follows: theerratic > multilevel > step signal. This tendency is reﬂected in thepore formability of bombinins.22 In terms of pore formability of
the four models, we assume that the formality of stable pore
which means to form a more cylindrical shape is spike < erratic <multilevel < step signal. Therefore, the result of the tendency of
the ratio suggests that the capability of the stable pore formation
would be energetically unfavorable in this condition. In addition,
this estimation was conducted by counting the number ofsignals, resulting in counting a large amount of the spike anderratic signals. The appearance probability of these two signals
was relatively high, whereas the duration was shorter than othersignals. Therefore, we analyzed the duration time of the pore
formation (Table S-1). As the result, the duration time of eachsignal was: spike < erratic < multilevel < step signal as the sameorder of pore stability. This fact suggested that the stable pore
formation may strongly contribute to the membrane defect.
Compared to H2 and H4, the step and multilevel signals in H4
were more than those in H2 as opposed to the erratic signals,
suggesting that H4 may be likely to form a rigid pore. In
addition, the mixture of H2/H4 showed a higher ratio of the step
signals. This result implies the high possibility of rigid pore
formation.
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Figure 6. (a) 31P static solid-state NMR spectra of PC (black), PC-H2 (red), PC-H4 (blue), and PC-H2/H4 (green) at 30 °C. Δδ = δ∥ − δ⊥. (b)
MALDI-TOF MS spectra of cross-linked H2 (red), [1-13C]Gly3-H4 (blue), and the 1:1 mixture (green) in PE/PG using BS3 linking reagent.

iy1d = ajjjj− 1zzzzk sin(π /n){

We initially considered that the charge ﬂux may be the suitableparameter for reﬂecting the membrane dysfunction of
antimicrobial peptides. However, if the pore formation is very
slow even if the pore size is large, this type of mechanism may
not show the higher microbial activity. Thus, we examined both
the dynamics and kinetics of membrane disruption. The kineticsof the pore formation was analyzed by the time to over thethreshold from the initial current appearance, termed “reachingtime”. This time indicates the kinetics for generating membrane
defects by the pore formation. Notably, the time in the H2/H4
mixture system was much slower than that in H2 or H4 systems.
The time in H2 was slightly slower than that in the H4 system, as
shown in Figure 4c.
Pore Size and Stoichiometry of Bombinin H2, H4, andthe H2/H4 Mixture. The step signals will reﬂect the stable pore
formation, as peptide monomers assemble to form ﬁxed pores ina lipid membrane. We calculated the pore diameter using theamplitude of step currents and the Hille model.24
πr y ρ
i
R = jjjl + zzz 22 { πrk(2)where d is the pore diameter; a is the width of a peptide
monomer from the PDB (a = 1.1 nm); and n is the number ofassembling monomers. Figure 5b shows the number ofmonomers: H2, H4, and H2/H4 were 11.5 ± 1.3, 9.1 ± 1.0,
and 9.5 ± 0.8 molecules, and the order was H2 > H2/H4 ≈ H4
in this estimation.
Estimation of Heterogeneity of Forming Pores Using
31
P NMR and Cross-Linking Experiment. To estimate theheterogeneity of the formed pore in the H2/H4 system, we
conducted a 31P solid-state NMR and cross-linking experiment.26 The chemical shift anisotropy (Δδ/ppm), which reﬂects
the dynamics and orientation of phospholipids, showed H2/H4< H2 ≈ H4 (Figure 6a), suggesting the heterogeneous pore
formation. If the homogeneous pore formation occurs in the
H2/H4 system, that value would show the similar value of H2and H4. In addition, we attempted to reveal the heterogeneity
based on more direct evidence using cross-linking experiment.
In this method, we synthesized and used a 13C-labeled H4monomer. The two monomers in the pore reconstituted inliposomes were cross-linked using N-hydroxysulfo-succinimide(NHS) ester in short reaction time, and then we carefully
checked the weight of the dimer using matrix-assisted laser
desorption ionization-time-of-ﬂight mass spectrometry(MALDI-TOF MS). As the results, the peak from the H2/H4
system was in between H2 and H4 systems (Figure 6b),
suggesting the heterogeneous pore formation in the mixed
system.

(1)

where r is the pore radius; ρ is the resistivity of the buﬀered
solution; l is the length of the pore (5 nm);25 and R is theresistivity of the pore. R is calculated as V/I, where I is thecurrent through the pore and V is the applied voltage between
two chambers (100 mV). The pore diameters on each bombinin
are presented in Figure 5a. The diameters of H2, H4, and H2/
H4 pores were 3.2 ± 0.5, 2.2 ± 0.3, and 2.3 ± 0.3 nm,
respectively; the order of pore diameter was H2 > H2/H4 ≈ H4
in this calculation.
Next, we estimated the number of assembling monomers
from the step signals. This signal would reﬂect a barrel-stave
pore formation as mentioned above. Because a barrel-stave poreis a cylindrical pore that consists of assembling monomers at the
periphery, we mathematically estimated the stoichiometry using
a multimeric model.14■

DISCUSSION
In the previous study of the antimicrobial activities ofbombinins, the activity estimated from the minimum inhibitory
concentration (MIC) of H4 was higher than that of H2 to Gramnegative bacteria.4 Several mechanisms for these activities are
generally considered to exist such as chemical enzyme inhibition
D

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Figure 7. Proposed models for the pore formation of bombinin H2 and H4 in this study and summary of all analyzed data of the pore formation
activities.

in the metabolic pathway or physical membrane disruption.
According to previous experiments on bombinins, the measurements of ﬂuorescein leakage through the lipid membrane bybombinins using living cells or liposomes show H4 > H2.7 These
leak experiments indicate that the pore formation of bombinins
into the lipid membrane constitute the important factor of
antimicrobial activity and that the activity of H4 is much higher
than that of H2.7
Several experiments using circular dichroism (CD), Fouriertransform infrared spectroscopy (FTIR), electron microscopy,
and surface plasmon resonance (SPR) measurements haveattempted to reveal the molecular mechanism of membrane
disruption.4,6 Through these experiments, two important facts
were exhibited: (1) the α-helicity which is strongly related withthe membrane disruption of bombinins was H2 ≈ H4 based on
CD and FTIR measurements and (2) the binding aﬃnity to thelipid membrane was H4 > H2 according to SPR measurements
using a Leishmania model membrane (PE/PC/PI/PS/erg). The
α-helicity did not aﬀect the antimicrobial activity in this case,whereas the α-helicity of AMPs is generally related to theantimicrobial activity. In comparison, the lower binding aﬃnityof H2 than H4 was probably due to its reduced hydrophobicity.
Molecular dynamics simulation of the hydrated Leishmania
membrane−peptide systems showed that the D-allo-Ile inbombinin H4 plays the role of a hydrophobic anchor in themembrane.26 Other structural variances between H2 and H4have been reported using nuclear magnetic resonance,27
electron microscopy,6 and molecular dynamic simulation ofdissolved peptides;8 however, the critical explanation regarding
the molecular basis of membrane disruption was still required,even in the H2/H4 mixtures. H2/H4 mixtures would play animportant role in nature because H4 is isomerized and coexists
with H2 as the mixture.28
In our electrophysiological measurements, three notable
results were obtained: (1) ion permeability from the charge
ﬂux analysis, (2) pore-forming kinetics from the reaching timemeasurements, and (3) pore size estimation from the stepcurrent.
(1) Ion permeability, H4 > H2/H4 ≥ H2: This order isconsistent with the results of the leak experiment using
cells or liposomes in the previous reports. It is reasonablebecause the permeability and leak experiments should
reﬂect similar phenomena. The values of H2 and H2/H4
were close in this estimation.
(2) Pore-forming kinetics, H4 ≥ H2 > H2/H4: In thisestimation, the kinetics of the H2/H4 mixture waslower than that of H2 or H4, whereas the values of H2 andH4 were close to each other.
(3) Pore size, H2 ≥ H2/H4 ≈ H4: This order is consistent
with the α-helicity. Reasonably, the α-helicity is strongly

related to the transmembrane structure and pore
formation.
These values are listed in Figure 7. Summarizing these data,
we consider the pore-forming model of each bombinin as also
shown in Figure 7. Bombinin H2 forms relatively large pores,
and bombinin H4 rapidly forms smaller pores. In the case of
their mixture, the kinetics of pore formation is the lowest withmedium-sized pores. Although the insertion of a D-amino acid in
the second position of the bombinin sequence should render itdiﬃcult to form the large pore with a barrel-stave shape, it would
enhance the formation of the membrane defect owing to thehigh binding aﬃnity to the membrane.
Another question is that the formed pore of the H2/H4
system is homogeneous or heterogeneous, whereas the electrophysiological results imply the heterogeneity. The binding
between H2 and H4 may be stronger than that between thehomogeneous states of H2−H2 or H4−H4 because the kineticsof the mixture decreased compared to the homogeneous state.
Other experimental results using the solid-state 31P NMR andthe cross-linking experiments indicated the heterogeneous pore
formation. Considering these facts, although the H4 has thehighest membrane disruption capability, the pore-forming
kinetics is regulated without reducing the pore-forming ability
in the case of an H2/H4 mixture. The living system may regulate
the antimicrobial activity using the isomerization of bombinin.

■

CONCLUSIONSIn summary, we estimated the pore-forming activity of bombinin
H2, H4, and their mixtures with the channel current
measurement using the pBLM system with the model bacterial
membrane. The model membrane allows us to use the pure lipid
contents. The four diﬀerent current signals were observed andwere applied to pore formation models of AMPs that have been
already proposed. Then, the probability of the appearance of thefour current types was analyzed, estimating the pore-forming
activity of each peptide. Next, we examined the following three
behaviors: (1) ion permeability from the charge ﬂux analysis, (2)pore-forming kinetics from the reaching time measurements,and (3) pore size estimation from the step current. From theresults of the analysis, we concluded and proposed the poreforming models for bombinin H2, H4, and H2/H4 mixtures tothe bacterial model membrane. These models well explain theantimicrobial activity on a molecular basis. Our systematicelectrophysiological measurements and the signal classiﬁcation
analysis will contribute useful insights into the molecularmechanism of antimicrobial activities for transmembrane
peptides and/or other transmembrane molecules such as asynthetic channel.29,30 In addition, the D-amino acids can beused for regulating the properties of L-peptides at the molecularE

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temperature (ca. 23 °C) and analyzed as mean ± SE (n > 30). The
signals were detected using a 4 kHz low-pass ﬁlter at a sampling
frequency of 20 kHz. Analysis of channel current signals was performed
using pCLAMP ver. 10.5 (Molecular Devices) and Excel (Microsoft)
software.
31
P Solid-State NMR. A total of 25 mg of bombinin peptide anddimyristoylphosphatidylcholine (DMPC) lipids with a peptide:lipid
molar ratio = 1:10 was dissolved in the chloroform solvent and wasevaporated. This was hydrated with 1 mL of Tris-NaCl buﬀer (20 mM,
100 mM NaCl, pH 7.5). A freeze-and-thaw cycle was repeated 10 times
to form a multilamellar vesicle. 31P solid-state NMR experiments were
performed on a Chemagnetics CMX-400 inﬁnity spectrometerequipped with a double resonance probe at the 1H and 31P resonancefrequencies of 399.4 and 161.1 MHz, respectively. 31P chemical shift
values were referred to 85% H3PO4 as 0.00 ppm (ppm). 31P direct
detection with high-power proton-decoupling static measurement was
performed using 5.0 mm outer diameter at 30 °C for the fully hydrated
samples.
Cross-Linking Experiment. Cross-linking reactions among the
peptide−peptide in POPE/POPG lipid environments were performed
by a BS3 reagent which contains N-hydroxysulfosuccinimide (NHS)
ester. The H2, [1-13C]Gly3-labeled H4, and the mixture of H2/theisotope-labeled H4 = 1:1 in POPE/POPG = 3:1 were mixed with BS3
during short incubation for 30 min at 15 °C. After that, quenching
buﬀer was added. In order to detect the cross-linked peptide as a dimer,
above three kinds of the cross-linking product were recorded on
MALDI-TOF MS (Bruker Daltonics, Autoﬂex speed TOF/TOF)
equipped with a N2 laser, respectively. All MS results were carefully
obtained in the linear positive (LP) mode using α-cyano-4hydroxycinnamic acid as a matrix.

level, suggesting that it would be a strong tool for extensive
biomaterials in nanoscience and medical applications.

■

EXPERIMENTAL SECTION

Chemicals. In this study, the reagents were obtained as follows: 1,2dioleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (DOPG; Avanti Polar
Lipids); 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE;
Avanti, Polar Lipids); n-decane (Wako Pure Chemical Industries,Ltd.); 3-morpholinopropane-1-sulfonic acid (MOPS, Nacalai Tesque);and KCl (Nacalai Tesque). Buﬀered electrolyte solutions wereprepared from ultrapure water, which was obtained from a Milli-Q
system (Millipore). Bombinin H2 and H4 were synthesized by
microwave-assisted solid-phase peptide chemistry using an Initiator+
Alstra peptide synthesizer (Biotage) and puriﬁed by reverse-phase
HPLC on a C-18 ODS column (Purity >90%, TOF-Mass data areshown in Figure S-3) and dissolved at a concentration of 1 mg mL−1 inultrapure water and stored at −20 °C.31 For use, samples were diluted
to 100 μM using a buﬀered electrolyte solution and stored at 4 °C.
Preparation of a High-Throughput pBLM Device. The
multichannel device was fabricated by machining a 6.0 mm thick, 10
× 10 mm poly(methyl methacrylate) (PMMA) plate (Mitsubishi
Rayon) using a CAD/CAM 3-D modeling machine (MM-100, Modia
Systems) (Figures S-1a and S-1b). A polymeric ﬁlm made of parylene C(polychloro-p-xylylene) with a thickness of 5 μm was patterned as asingle pore (100 μm in diameter) by a conventional photolithographymethod and then placed between PMMA ﬁlm sheets (0.2 mm thick)using an adhesive bond (Super X, Cemedine Co., Ltd.). The ﬁlms,
including the parylene ﬁlm, were inserted into the device to separate thewells (Figure S-1c and S-1d). Ag/AgCl electrodes of each device set
into a solderless breadboard (E-CALL Enterprise Co., Ltd.) wereconnected to a Jet patch clamp ampliﬁer (Tecella) using a jumper
wire.32 Two lipid monolayers were contacted together to prepare astable and reproducible lipid bilayer in the parylene ﬁlm (Figure S-1e).
This method is advantageous for preparing model cell membranes; e.g.,bacteria or mammalian cell membranes.
pBLM Preparation and Reconstitution of Bombinins. The
pBLMs were prepared using an arrayed device, which had six chamberson the breadboard. Six individual lipid bilayers could be formed
simultaneously in this device, which allowed for a higher-throughput
measurement compared to the conventional system. First, the
DOPE:DOPG = 3:1 mol mol−1 (lipids/n-decane, 10 mg mL−1)
solution (2.4 μL) was poured into all chambers. Next, the buﬀer
solution (4.7 μL) with bombinin H2 or bombinin H4 or H2/H4 mix(1:1 mol mol−1) in 100 μM (ﬁnal concentration) was poured into allchambers. In this study, the buﬀer solution (1 M KCl, 10 mM MOPS,
pH 7.0) was used for all droplets. After a few minutes of adding the
buﬀer solution, the two lipid monolayers connected and formed a lipid
bilayer, and bombinin formed nanopores by reconstitution in the lipid
bilayer. When the lipid bilayer ruptured, we reformed the lipid bilayer
by tracing with a hydrophobic stick between two droplets.
Channel Current Measurements and Data Analysis. Channel
current was monitored by using a JET patch clamp ampliﬁer (Tecella)connected to each chamber. Ag/AgCl electrodes were in each droplet
when we added the solution into the chambers. A constant voltage of+100 mV was applied to the one side, with the other side being
grounded. The reconstituted bombinin pore in the lipid bilayers
allowed ions to pass through the nanopore under the voltage gradient,so that we obtained the channel current signals. In our current
measurement, we can ﬁnd the single pore formation in the case of thestep, multilevel, and spike signals: each step current corresponds to eachpore formation the same as the case of pore-forming toxins (e.g., alphahemolysin), and the current rises up and returns to the baseline level,
suggesting the single pore formation. These single pore formations aremostly observed in the initial stage of the current recordings. Because
the lipid bilayer can be readily formed by using our droplet contact
method,33 we obtain a lot of single pore signals using our system. In theerratic signals, we cannot recognize the single pore formation because
this signal can be assigned to the “carpet model” that is detergent likelipid dissociation. All measurements were conducted at room■

ASSOCIATED CONTENT* Supporting InformationS

The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acsabm.8b00835.
The device fabrications and detailed experimental data
(PDF)

■

AUTHOR INFORMATION

Corresponding Author*E-mail: rjkawano@cc.tuat.ac.jp.
ORCID

Izuru Kawamura: 0000-0002-8163-9695
Ryuji Kawano: 0000-0001-6523-0649
Author Contributions

Y.S. and R.K. conceived the original idea and wrote the paper.
Y.S., K.S., Y.K., and A.O. conducted the experiments andanalyzed the data. R.K., Y.S., K.S., and I. K. wrote the paper. All
authors have given approval to the ﬁnal version of themanuscript.
Author Contributions∥

Equal contribution.

Notes

The authors declare no competing ﬁnancial interest.

■

ACKNOWLEDGMENTS
This work was partly supported by KAKENHI (Grant No.
25708024, 15H00803, 16H06043, and 16H00828) from
MEXT, Japan.

■

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DOI: 10.1021/acsabm.8b00835
ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX