Trans-bilayer ion conduction by proline containing cyclic hexapeptides and effects of amino acid substitutions on ion conducting properties

Article abstract of DOI:10.1246/bcsj.20090272

Several ion channel forming cyclic peptides have been reported over the past two decades and various ionconducting mechanisms have been proposed. In this article, we report on amino acid substitutions in cyclic hexapeptides and their effects on the ion co

Full text of DOI:10.1246/bcsj.20090272

© 2010 The Chemical Society of Japan
Bull. Chem. Soc. Jpn. Vol. 83, No. 6, 683–688 (2010)
683
Trans-Bilayer Ion Conduction by Proline Containing Cyclic
Hexapeptides and Effects of Amino Acid Substitutions
on Ion Conducting Properties
Junichi Taira,1,2 Satoshi Osada,1 Ryo Hayashi,1 Toshihisa Ueda,3 Masoud Jelokhani-Niaraki,4
Haruhiko Aoyagi,5 and Hiroaki Kodama*1
1Department of Chemistry, Faculty of Science and Engineering, Saga University, Saga 840-8502
2Department of Chemistry, Kurume University School of Medicine, Kurume 830-0011
3Department of Applied Biological Science, Faculty of Agriculture, Saga University, Saga 840-8502
4Department of Chemistry, Faculty of Science, Wilfrid Laurier University, Waterloo, Ontario N2L 3C5, Canada
5Kyushu Nutrition Welfare University, Kitakyushu 803-8511
Received October 9, 2009; E-mail: hiroaki@cc.saga-u.ac.jp
Several ion channel forming cyclic peptides have been reported over the past two decades and various ion
conducting mechanisms have been proposed. In this article, we report on amino acid substitutions in cyclic hexapeptides
and their effects on the ion conducting properties of these peptides. Cyclic hexapeptides, cyclo(Pro-Xxx-Yyy)2,
containing two Pro residues, were used as the main framework. The substitution is performed at the Xxx positions with
cationic/hydrophilic Lys or hydrophobic Leu. Yyy positions were substituted with D-Phe, D-Ala, or Gly. The peptides
which were absent Lys residues showed ion conducting profiles with clear transitions of electric currents, whereas the
peptides containing Lys residues tended to exhibit spiky or burst-like profiles. These profiles were altered single state
profiles by the protection of ¾-amino groups with aromatic protecting groups. The protected analogs exhibited significant
decrease in ion conductance. These results indicated that peptides containing Lys conduct ions without forming ring
stacked tube-like structure. Ion channel properties were also affected by conformational changes of the cyclic peptides
induced by substitution of the Yyy positions. Enhancement of intramolecular ¢-turn structures of cyclic peptides tended
to decrease their ion conductance values.
Conduction of ionic species across phospholipid bilayers is
restricted due to the hydrophobic nature of the bilayer core,
nonetheless ion conduction via the bilayer is allowed by ion
channel peptides.1 Naturally occurring ion channel peptides
have generally linear structures consisting of 10 to 20 amino
acid residues. Details of relationships between molecular
structure and ion conducting mechanisms of linear peptides
have been exhaustively investigated.2 A better understanding of
fundamental structures of ion channel peptides allows for
artificial ion channel design.3,4 Ion channel forming of cyclic
peptides has been also reported and utilizations of cyclic
peptides for the design of artificial ion channels have been
attempted.5-7 In comparison with linear peptides, the reduced
number of amino acid residues in cyclic peptides makes their
design easier. Tailor-made ion channel forming cyclic peptides
would be not only useful in biophysical studies, but also in
other applications such as development of novel anti-infective
agents, nanosize functional material.8 These channel forming
cyclic peptides have been believed to associate and form high-
ordered pore constructs through noncovalent intermolecular
interactions in lipid membranes, meaning that appropriate
intermolecular interactions should be required to construct
pores with desirable ion conducting properties.9,10 However,
there is still no consensus about the principles governing the
formation of pores that can assist their design. Additionally,
correlation of peptide conformations with ion channel function
also needs to be clarified for appropriate engineering of pore
constructions. It is thought that steady accumulation of knowl-
edge of the structure-activity relationships is the only way to
obtain cyclic peptides with desired ion channel properties.
In the present study, a series of Pro-containing cyclic
hexapeptides were synthesized and ion channel properties were
evaluated. It was expected that the consequence of this study
would contribute to knowledge to overcome the above issues.
Structures of cyclic peptides evaluated in this study are
illustrated in Figure 1. Pro-containing hexameric cyclic pep-
tides have been utilized as models for conformational studies of
turn structures.11-14 Comparison of ion channel properties of
these cyclic peptides would be a rational way to understand the
relationships of peptide conformation and ion channel function.
Several reports of nanotube and/or further ion channel
formation by cyclic hexapeptides have been published, though
effects of peptide side chains for ion channel formation are not
well understood.8d,15 Cationic and/or hydrophobic character-
istics have been considered as important factors for both
peptide-lipid membrane and peptide-peptide interactions.9,10
Therefore, effects of cationic and hydrophobic characteristics
of cyclic peptides on ion conducting properties should be
Published on the web May 28, 2010; doi:10.1246/bcsj.20090272

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Bull. Chem. Soc. Jpn. Vol. 83, No. 6 (2010)
Ion Channel Activities of Cyclic Hexapeptides
Figure 1. Structures of designed cyclic hexapeptides. Cyclic hexapeptides are composed of a repeat of Pro-L-Xxx-D-Yyy sequences
(left). Side chain compositions of the analogs are illustrated at right. Z groups (-COOBz) in the structures of peptides 7, 8, and 9
stands for the benzyloxycarbonyl group.
addressed. On the basis of these points, hydrophobic Leu or
cationic/hydrophilic Lys were incorporated into Pro-containing
cyclic hexapeptide constructs and ion conducting properties
were compared. The details of peptide design principles are
explained in the following sections.
Results and Discussion
Peptide Design.
Although cyclo(Pro-Xxx-Yyy)2, a
common sequence of cyclo hexapeptides, has been previously
utilized to investigate the properties of ¢-turn structures in
cyclic peptides, the ion channel forming ability of these peptide
sequences was also expected due to possibilities of intermo-
lecular interactions between peptide molecules and some
experimental results.11-15 For example, Ramesh et al. reported
briefly the ion channel properties of cyclic peptides with the
same common sequence.15 The presence of the Pro residues not
only maintained peptide conformations, but also promoted
cyclization steps during peptide synthesis.11 The Xxx positions
were substituted with hydrophobic Leu residues or cationic/
hydrophilic Lys residues. Cationic and hydrophobic character-
istics of the peptides are believed to be essential factors for
peptide-peptide and peptide-membrane interactions. In addi-
tion, the amphiphilicity of the cyclic peptides and the
possibility of intermolecular hydrogen bond formation could
result in the clustering and stacking of peptide surfaces.9
Consequently, it was expected that the amino acid substitutions
would alter the intermolecular interactions and the properties of
the resulting ion channel formation in model lipid membranes.
In fact, charged residues were found to play a critical role for
the association of helical ion channel peptides in lipid bilayers.4
The Lys ¾-amino group protection of peptides with benzyl-
oxycarbonyl (Z) groups was used to verify the roles of the
Figure 2. CD spectra of peptide 1-6. All spectra were
collected in the presence of 2 mM DPPC SUVs. The
spectra were collected in the far-UV range from 190 to
260 nm. (a) CD Spectra of model peptides containing Leu.
(b) Spectra of model peptides containing Lys. Symbols
represent peptide 1 (closed triangle), peptide 2 (closed
circle), peptide 3 (closed square), peptide 4 (open triangle),
peptide 5 (open circle), and peptide 6 (open square),
respectively.
charged amino groups. The Yyy positions were substituted
with D-Phe, D-Ala, or Gly residues to assess conformational
effects on ion channel properties. Details of the conformational
changes induced by the amino acid substitutions will be
discussed in the following section.
Circular Dichroism (CD) Measurements.
Structural
properties of model peptides were evaluated by CD spectra.
Figure 2 exhibits the CD spectra of peptides 1-6 collected in

J. Taira et al.
Bull. Chem. Soc. Jpn. Vol. 83, No. 6 (2010)
685
Figure 3. Representative profiles of ion conductions by peptides 1-6. The experiment was carried out by pipette dipping patch
clamp at 25 « 2 °C and data were filtered at 1 or 2 kHz frequency. The pipettes were filled with a 0.5 M KCl solution buffered with
5 mM HEPES (pH 7.4) containing 1 ¯M peptides. Symbols C and O (1, 2, 3,²) denote the zero (closed) current level and open
levels of channels, respectively.
Table 1. Parameters Obtained from Ion Channel Activity
Measurements
the presence of 2 mM dipalmitoylphosphatidylcholine (DPPC)
small unilamellar vesicles (SUVs). All spectra showed double-
minima around 200 and 220 nm, respectively, and positive
ellipticities were detected around shorter wavelengths (with
maxima below 190 nm). Although it was difficult to distinguish
the details of conformational differences from the obtained
spectra, a set of “rules” correlating amino acid sequences with
preferences for particular types of ¢-turn structures in the
cyclic hexapeptides have been proposed by Gierasch et al.13
They concluded that the sequence -L-Pro-L-Yyy-Gly- (Yyy
not Val) leads to type I ¢-turn structures, and sequence -L-Pro-
L-Yyy-D-Xxx- leads to type II¤ ¢-turn structures when they
repeated in cyclic hexapeptides. According to these rules,
the spectra of peptides 3 and 6, possessing -Pro-Xxx-Gly-
sequence can be characterized as type I ¢-turn conformations.
Correspondingly, the spectra of peptides 1, 2, 4, and 5
possessing -Pro-Xxx-D-Yyy- sequence can be characterized
as type II¤ ¢-turn structures. CD ellipticities were increased by
the substitution of D-Ala residues with D-Phe residues. Increase
in ellipticity could imply an enhancement in the type II¤ ¢-turn
conformation of the peptides.
Applied
Peptide potential (E)
/mV
Conductance
values (G)
/pSa)
Pore
radius (R)
/nmb)
Open
state
1
2
3
+100
+100
¹200
1
1
1
2
3
1
2
1
2
1
1
1
1
47
73
67
95
109
79
161
106
219
98
26
43
0.08
0.10
0.10
0.11
0.12
0.10
0.15
0.12
0.17
0.12
0.06
0.08
0.07
4
5
+100
+100
6
7
8
9
+100
+100
+100
+100
40
a) The conductance values (G) were calculated as G = I/E.
b) The pore radii (r) were estimated according to eq 1. The
membrane thickness (l) is 3.0 nm and the bulk solution
resistivity (µ) of 500 mM KCl is 0.13 ³ m.16
Ion Channel Properties of Cyclic Hexapeptides.
Ion
channel activities of model peptides were measured in
diphytanoylphosphatidylcholine (DPhPC) bilayers. Typical
traces of ion conduction by peptides 1-6 are illustrated in
Figure 3 and parameters obtained from the traces are summa-
rized in Table 1. The pore radiuses were estimated from
observed conductance values and specific interactions between
the constructed pores and ions were not considered in the
calculation (see experimental section). The exhibited traces
were reproduced at least three times under comparable
conditions. All peptides exhibited current fluctuations with
conductance values between 50-200 pS. Generally, carrier or
channel conducting mechanisms have been suggested for ion
transport by cyclic peptides or peptidomimetics. The carrier ion
transporter, such as valinomycin, accepts ions on one side of
the membrane, moves them to the opposite side and releases the
ion.17 The ion conducting velocity and efficiency of carrier
molecules are relatively less than that of channel molecules.
Observed ion conductance values corresponding to channels
(107 ions/s), but are significantly larger and distinguishable
from that of carriers (104 ions/s).18,19
The single-state conductance value of peptides 1 and 2 can
be interpreted in accordance to the paradigm of ring stacked
tube-like ion channels.18 It is reasonable to assume that due to
structural limitations, the pore diameter of the tubular structures
formed by stacking of cyclic peptides remains relatively
inflexible and these supramolecular structures could have a
single-state conductance value. Interestingly, the ion conduct-
ing profile of peptide 3, assumed to possess type I ¢-turn
conformation, was distinct from those of peptides 1 and 2. The
trace with multi-state conductance values would imply the
existence of conducting substates. Decisive interpretation for
the difference could not be done, though the conformational
difference of peptide 3 might cause the alteration of ion cannel
properties.20 It was also expected that the presence of two Gly
residues, with lower steric hindrance of their side chains, in

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Bull. Chem. Soc. Jpn. Vol. 83, No. 6 (2010)
Ion Channel Activities of Cyclic Hexapeptides
peptide 3 can contribute to the flexibility of the pore structures
formed by this peptide.
conductance value and (ii) bundle-like structures constructed
with aggregation of stacked cyclic peptide columns. These
investigators predicted that channels behaving in the latter
manner exhibit multi-state conductance values. Ghadiri’s group
reported that a cyclic peptide series composed of repeated
hydrophobic L- and D-amino acid exhibited single-state
fluctuations of electrical currents reflecting fixed pore diame-
ters.18 A self-assembled tube-like architecture composed of
stacked peptide rings with intermolecular hydrogen bonds via
peptide main chains, was proposed for the ion conducting
mechanisms of these peptides. On the other hand, it was
reported that cyclic tetrapeptides tentoxin; cyclo(N-Me-
Ala-Leu-N-Me-¦Phe-Gly) and (2S,9S)-2-amino-8-oxo-9,10-
epoxydecanoic acid (Aoe) containing HC toxin; cyclo(Ala-D-
Ala-Aoe-D-Pro) form transmembrane ionic channels without
ring-stacked tube-like structures, since these peptides exhibited
a pattern with multi-state conductance values.5,6 Cylindrical
bundle formation by cyclic peptide aggregates has been
proposed as a possible ion conducting mechanism for tentoxin
and HC toxin. These reports imply that the observed multi-state
conductance values in this study were consequence of pore
formation without ring-stacked tube-like structures. However,
peptide 3 that lacks Lys exhibited multi-state conductance
values, while peptide 6 that contains Lys exhibited single-state
values. Clearly understanding this point has not been obtained
at the present stage.
Peptides lacking the Lys residues showed ion conducting
profiles with distinct open-close transitions, whereas peptides
containing Lys residues tended to show conductance profiles
with spiky or burst-like fluctuations. Generally, positively
charged peptides, especially amphiphilic examples, accumulate
on both negatively charged, as well as zwitterionic lipid
membranes, and some of the peptides show membrane
perturbation activities.9 Nonspecific membrane disruption
could be considered as one reason for the spiky or burst-like
patterns.21 Nonetheless, well-defined independent events in
millisecond open-close time scales can indicate the existence
of ion conducting pores within lipid membranes. Additionally,
according to amplitude histogram analyses (not shown), multi-
state like conductance behavior was also detected in the
conductance traces of peptides 4 and 5 (Figure 3). Duclohier
et al. proposed a few possibilities for the ion conducting
mechanism of cyclic peptides and putative traces of those ion
conduction mechanisms as follows (Figure 4).22 These possible
mechanisms include: (i) tube-like architecture constructed with
stacking ring shaped peptides expected to show a single-state
The Effects of Side Chain Amino Groups. In order to
verify whether the characteristic ion conductance profile is
mediated by free amino groups of Lys side chains, ion channel
activities of peptides 7, 8, and 9, the respective Lys side chain
protected versions of peptide 4, 5, and 6, were evaluated.
Figure 5 illustrates typical ion conducting profiles of the amino
group protected analogs. The profiles were converted to the
patterns with single-state conductance values and the values
were significantly lower than those of the non-protected
analogs (26-43 pS: see Table 1). If these peptides formed the
Figure 4. Presumed ion channel forming by cyclic peptide.
(left) Ion conduction by membrane perturbation. (center)
Bundle-like structures constructed with aggregation of
stacked cyclic peptide column. (right) Nanotube formed by
peptide stacking. Filled circles illustrated in this figure
represent ionic species.
Figure 5. The biophysical properties of peptides 7-9. (a) CD spectra of the peptides. CD spectra were collected in the presence of
2 mM DPPC SUVs. Symbols represent peptide 7 (open triangle), peptide 8 (open circle), and peptide 9 (open square), respectively.
No significant changes of the spectra were observed by the side chain protection. (b) Representative patterns of ion conduction by
these peptides. In peptides 7-9, the characteristic conductance profiles of the corresponding peptides 4-6 were converted to
considerably lower conductance values and different open-close transitions by protecting ¾-amino group.

J. Taira et al.
Bull. Chem. Soc. Jpn. Vol. 83, No. 6 (2010)
687
conventional tubular structures by peptide stacking, the internal
structures of the pores should not be affected by the presence or
absence of the protecting groups. Therefore, this observation
provides additional evidence that relevant cyclic hexapeptides,
possessing free amino groups, form ion channels without the
tube-like structures. Feigin et al. reported that the cyclic
lipodepsipeptide, syringomycin E, formed ion channels which
show multi-state conductance values.23 Syringomycin E con-
tains three cationic residues and has a total of +2 net charges.
These researchers concluded that the pore structure was formed
by association of clustered complexes of the peptides and
charged residues gathering in the pore lumen. As aforemen-
tioned, the details of ion conducting mechanism of Lys
containing cyclic hexapeptides are uncertain, though observed
changes in the ion conductance corresponding to the amino
group protection indicates that amino groups correlated ion
channel forming and the cyclic hexapeptides have a potential
to convert their ion channel properties with the variation of
side chain groups. Nonetheless, the possibility has not been
excluded that the conductances were influenced by hydro-
phobicity or other effects of Z groups. Further investigations of
this point remain in future work.
TFA or 4 M HCl in 1,4-dioxane. After the elongations of linear
peptides up to the desired sequence, C-terminal carboxyl
groups of the linear products were converted to succinimide
ester with N-hydroxysuccinimide and DCC. After deprotection
of Boc-protected groups on the N-termini, cyclization was
performed in pyridine under highly diluted conditions. Purifi-
cation of crude products was carried out by gel-filtration
chromatography, silica gel chromatography or preparative
reverse phase high-performance liquid chromatography (RP-
HPLC) on a Wakosil 5C4-200 column (Wako Pure Chemical
Industries). Peptides 4, 5, and 6 were obtained from peptides 7,
8, and 9, respectively, by removal of Z groups on ¾-amino
groups by H2 reduction catalyzed by Pd/C.25 Purities of
synthetic peptides were confirmed by analytical RP-HPLC on a
Wakosil 5C4-200 column (Wako Pure Chemical Industries).
HPLC analyses were performed on a system comprised of an
807-IT integrator (JASCO, Tokyo, Japan), two pumps PU-980
(JASCO), a UV-970 detector (JASCO), and a Rheodyne 7125
injector (Rheodyne Inc., CA, U.S.A.). Molecular masses of
synthetic peptides were determined by matrix-assisted laser
desorption ionization time-of-flight (MALDI-TOF) mass
spectroscopy on Voyager-DERP (PerSeptive Biosystems Inc.,
Framingham, MA, U.S.A.) using ¡-CHCA as matrix reagent.
MALDI-TOF MS for cyclo(Pro-Leu-D-Phe)2 (1): C40H55N6O6
[M + H]+ 715.4, found 715.5; cyclo(Pro-Leu-D-Ala)2 (2):
C28H47N6O6 [M + H]+ 563.4, found 563.0; cyclo(Pro-Leu-
Gly)2 (3): C26H43N6O6 [M + H]+ 535.3, found 535.9; cyclo-
(Pro-Lys-D-Phe)2 (4): C40H57N8O6 [M + H]+ 745.4, found
745.9; cyclo(Pro-Lys-D-Ala)2 (5): C28H49N8O6 [M + H]+
593.4, found 592.9; cyclo(Pro-Lys-Gly)2 (6): C26H45N8O6
[M + H]+ 565.4, found 566.1; cyclo(Pro-Lys(Z)-D-Phe)2 (7):
C56H68N8O10Na [M + Na]+ 1035.5, found 1034.6; cyclo(Pro-
Lys(Z)-D-Ala)2 (8): C44H60N8O10Na [M + Na]+ 883.4, found
884.2; cyclo(Pro-Lys(Z)-Gly)2 (9): C42H57N8O10 [M + H]+
833.4, found 833.4. Amino acid compositions and peptide
concentrations of stock solutions used in CD measurements
and ion channel activity measurements were determined by
quantitative amino acid analysis as described previously.26
Amino acid analyses were performed on a Pico Tag Work-
station (Waters, Milford, MA, U.S.A.) after hydrolysis in
constant boiling HCl at 110 °C for 24 h.
Conclusion
In the present study, structures of cyclic hexapeptide
conducting pores were modified on the basis of peptide
structures. In spite of the simple amino acid substitutions, these
cyclic peptides exhibited drastic changes in their ion channel
properties, as suggested by the conversion of ion conduction
mechanisms. The ion conducting mechanism of the Lys-
containing cyclic hexapeptides could not be clearly identified,
however it is experimentally suggested that the cyclic peptides
are able to form different ion conducting structures. Conforma-
tional differences in the cyclic peptide monomers also altered
ion conductivity and/or resulted in the formation of substate
conducting levels, implying different pores correlated with
ion channel properties. Recently, computational chemistry has
been utilized to explore the ion channel formation of cyclic
peptides.19,24 Correspondence between the biophysical and
simulation techniques could further clarify the details of the ion
conducting mechanisms.
CD Measurements.
CD spectra were recorded on a
Experimental
JASCO J-720 spectropolarimeter (JASCO). A cylindrical
cuvette of 2 mm path length was used for the measurements.
The measurements were carried out in 2 mM DPPC SUVs.
Peptide concentrations were 20 ¯M. Hydrophobic peptides
were dissolved in EtOH as 1 mM stock solutions and then
diluted with the SUV containing buffer. Samples were
incubated at room temperature for 30 min before measure-
ments. Four scans were averaged for each sample and the
averaged blank spectra were subtracted respectively. DPPC
SUV was prepared according to a modified Bangham’s
method.27 DPPC lipid was dissolved in chloroform and then
dried overnight in vacuo. The lipid film was hydrated with
10 mM phosphate buffer solution (pH 7.4), vortexed at 50 °C
for 30 min, and then sonicated for 6 min using a Branson
Sonifier 250 sonicator (Branson, CT, U.S.A.). Prepared SUV
solution was centrifuged for 5 min to remove the titanium
particles from sonicator probe tip.
Materials.
tert-Butoxycarbonyl (Boc)-protected amino
acids, N,N-dicyclohexylcarbodiimide (DCC), 1-hydroxybenzo-
triazole (HOBt), and 2,2,2-trifluoroacetic acid (TFA) were
purchased from Peptide Institute (Osaka, Japan). DPhPC was
obtained from Avanti Polar Lipids (Alabaster, AL, U.S.A.) as
¹1
50 mg mL chloroform solutions. ¡-cyano-4-hydrocinnamic
acid (¡-CHCA) and DPPC were purchased from Sigma
(Tokyo, Japan). All other chemicals were obtained from
Wako Pure Chemical Industries (Osaka, Japan), and used as
received.
Peptide Synthesis.
Synthesis of peptide 3 had been
reported previously,11 and the same strategy was applied for
the syntheses of peptides 1, 2, 7, 8, and 9. Briefly, coupling
reactions of amino acids or peptide fragments were performed
with DCC and HOBt in the presence of triethylamine or N-
methylmorpholine. Removal of Boc groups was achieved with

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Bull. Chem. Soc. Jpn. Vol. 83, No. 6 (2010)
Ion Channel Activities of Cyclic Hexapeptides
6
a) F. Heitz, F. Kaddari, N. V. Mau, J. Verducchi, H. R.
Single-Channel Measurements. Single-channel recording
was carried out by pipette dipping patch-clamp at ambient
temperature (25 « 2 °C).28 The patch-clamp electrodes were
prepared by the two pulls method by a pipette puller
(Narishige, Tokyo, Japan), without fire polishing, from he-
matocrit hard glass capillaries (Narishige). Settings in the
microelectrode puller were adjusted to obtain tip diameter of
ca. 1 ¯m. Pipettes were filled with 0.5 M KCl solution, buffered
with 5 mM 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic
acid (HEPES) (pH 7.4), containing 1 ¯M cyclic peptide. The
pipette tip was immersed in a disposable Petri dish filled with
the same buffer without peptides. After immersion of the
pipette, DPhPC monolayer was spread on the surface of the
dish solution by carefully adding to the edge of the dish
Seheno, R. Lazaro, Biochimie 1989, 71, 71. b) F. Heitz, F. Kaddari,
A. Heitz, H. Raniriseheno, R. Lazaro, Int. J. Pept. Protein Res.
1989, 34, 387.
7
a) D. T. Bong, T. D. Clark, J. R. Granja, M. R. Ghadiri,
Angew. Chem., Int. Ed. 2001, 40, 988. b) J. D. Hartgerink, T. D.
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a) S. Fernandez-Lopez, H.-S. Kim, E. C. Choi, M. Delgado,
J. R. Granja, A. Khasanov, K. Kraehenbuehl, G. Long, D. A.
Weinberger, K. M. Wilcoxen, M. R. Ghadiri, Nature 2001, 412,
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¹1
0.5-1.0 ¯L of a 10 mg mL solution of the lipid dissolved
in n-hexane. Before any movement of the electrode, ca. 10 min
were allowed for the evaporation of the solvent from the
surface of the solution. Single-channel currents were amplified
using an Axpatch 1D patch-clamp amplifier (Axon Instruments,
Inc., Union City, CA, U.S.A.) controlled by pClamp 6 (Axon
Instruments, Inc.) software. Data was filtered at 2 kHz
frequency and analyzed using Axograph (Axon Instruments,
Inc.). Electric current and conductance values were evaluated
from amplitude histogram analyses of the conductance traces.
Calculation of pore diameter was performed using the
following equation.29
9
E. J. Prenner, M. Kiricsi, M. Jelokhani-Niaraki, R. N. A. H.
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G ¼ ³r2=r0ðl þ ³r=2Þ
ð1Þ
Where, G is the observed conductance value; l is the membrane
thickness (or pore length, approximately 0.3 nm); r is the radius
of pore; and r¤ is the conductance of the ionic species (0.13 ³m
for 0.5 M KCl solution).16
We are grateful to Professor Yasuyuki Shimohigashi
(Kyushu University) for the use of MALDI-TOF MS. This
work was supported in part by Grants-in-Aid for Scientific
Research (C) (No. 20550154) and for JSPS Fellows (No. 17-
6431) from the Ministry of Education, Culture, Sports, Science
and Technology of Japan.
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