Mechano-Sensitive Synthetic Ion Channels

Article abstract of DOI:10.1021/jacs.7b09515

Mechanical stress is a ubiquitous stimulus sensed by membrane proteins, but rarely by synthetic molecules. Inspired by mechano-sensitive ion channels found in cell membranes, tension-responsive transmembrane multiblock amphiphiles were developed. In membranes, a single-transmembrane amphiphile responds to both expanding and contracting tensions to weaken and strengthen the stacking of membrane-spanning units, respectively, and ion transportation is triggered by expanding tension to form a supramolecular channel, while little transportation is observed under a tensionless condition. In contrast, a three-transmembrane amphiphile showed little spectroscopic response to tensions, likely due to weaker stacking of membrane-spanning units than in the single-transmembrane amphiphile. Nevertheless, the three-transmembrane amphiphile shows ion transportation by forming a unimolecular channel even under a tensionless condition, and the ion-transporting activity decreased with expanding tension. Interestingly, the estimated operating force of these synthetic systems was comparable to that of the mechano-sensitive proteins. This study opens the door toward new mechano-sensitive molecular devices.

Full text of DOI:10.1021/jacs.7b09515

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Article
Mechano-Sensitive Synthetic Ion Channels
Takahiro Muraoka, Kaori Umetsu, Kazuhito V. Tabata, Tsutomu
Hamada, Hiroyuki Noji, Takashi Yamashita, and Kazushi Kinbara
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.7b09515 • Publication Date (Web): 27 Oct 2017
Downloaded from http://pubs.acs.org on October 27, 2017
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Journal of the American Chemical Society
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Mechano-Sensitive Synthetic Ion Channels
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,
†,‡
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⊥
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Takahiro Muraoka,* Kaori Umetsu, Kazuhito V. Tabata, Tsutomu Hamada, Hiroyuki Noji, Ta-
#
,†,§
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kashi Yamashita and Kazushi Kinbara*
†
School of Life Science and Technology, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama 226-
8503, Japan.
‡
Precursory Research for Embryonic Science and Technology, Japan Science and Technology Agency, 4-1-8 Honcho, Ka-
waguchi, Saitama 332-0012, Japan.
§
Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, 2-1-1, Katahira, Aoba-ku, Sendai 980-
8
¶
577, Japan.
Department of Applied Chemistry, School of Engineering, The University of Tokyo, Bunkyo-ku, Tokyo 113-8656, Japan.
⊥
School of Materials Science, Japan Advanced Institute of Science and Technology, 1-1 Asahidai, Nomi, Ishikawa 923-1292,
Japan.
#
Department of Pure and Applied Chemistry, Tokyo University of Science, 2641 Yamazaki, Noda-shi, Chiba 278-8510, Ja-
pan.
ABSTRACT: Mechanical stress is a ubiquitous stimulus sensed by membrane proteins, but rarely by synthetic molecules. Inspired
by mechano-sensitive ion channels found in cell membranes, tension-responsive transmembrane multi-block amphiphiles were
developed. In membranes, a single-transmembrane amphiphile responded to both expanding and contracting tensions to weaken
and strengthen the stacking of membrane-spanning units, respectively, and ion transportation is triggered by expanding tension to
form a supramolecular channel, while little transportation is observed under a tensionless condition. In contrast, a three-
transmembrane amphiphile showed little spectroscopic response to tensions likely due to weaker stacking of membrane-spanning
units than the single-transmembrane amphiphile. Nevertheless, the three-transmembrane amphiphile shows ion transportation by
forming a unimolecular channel even under a tensionless condition, and the ion transporting activity decreased by expanding ten-
sion. Interestingly, the estimated operating force of these synthetic systems was comparable to that of the mechano-sensitive pro-
teins. This study opens the door toward new mechano-sensitive molecular devices.
2
4–28
INTRODUCTION
air–water interface using Langmuir monolayer.
However,
the original functions of MSCs, i.e., the control of substance-
transportation through a membrane by mechanical stimuli,
remains a significant challenge in development of smart bio-
mimetic molecular devices. Herein, we demonstrate a mecha-
no-sensitive ion transportation switch by synthetic transmem-
brane molecules, which respond to both expanding and con-
tracting tensions applied to the membrane.
Mechanochemistry gains increasing attention at the interface
between chemistry and mechanical engineering. Not only
detection of shear forces for bond cleavages but also induction
of chemical reactions, non-covalent bond formation, and phase
1
–17
transitions have actively been demonstrated.
These exam-
ples have mostly been realized by polymers, liquid crystals,
and organic/inorganic crystalline materials, where the macro-
scopic forces are applied to the bulk substances. Meanwhile,
in the biological systems, microscopic mechanical forces pro-
duced by sound, gravity or osmotic pressure are sensed by
membrane proteins in a cytoplasmic membrane such as mech-
ano-sensitive channels (MSCs). MSCs respond to the changes
in the membrane tension so as to function as safety valves
against the osmotic shock in bacteria, and also as sensors for
In recent years, we have been developing synthetic mimics
of multipass transmembrane (MTM) proteins, consisting of
repeating oligo(ethylene glycol) chains and aromatic units.
We found that the aromatic units of the MTM mimics tend to
self-assemble through intra- and intermolecular interactions in
the lipid bilayer, which leads to formation of supramolecular
ion channels. These facts, coupled with the precedent exam-
2
9–33
3
4
18
ples of luminescent tension-sensing molecules, inspired us to
realize that the intra- and intermolecular aromatic stacking of
MTM mimics would possibly be responsive to the change in
the membrane tension, thereby influencing the transportation
activity. In order to acquire the sensitivity to the membrane
tension, moderately destabilized aromatic stacking is likely
advantageous, which could be attained by substituents on the
aromatic units causing steric hindrance among the π-π assem-
bling moieties. On the basis of these backgrounds, we de-
signed synthetic MTM mimics 1 and 2 in this study, which
have a multi-block structure including alternating hydrophilic
touch and sound waves in higher organisms. The membrane
tension triggers conformational changes of MSCs to open the
channel and flow ions or small signaling molecules, which
releases osmotic stress and activates signaling cascades. In-
spired by such mechano-sensitive proteins, Matile and
coworkers developed supramolecular systems probing a transi-
tion between liquid-ordered and solid-ordered membrane
phases, where changes in the physical properties such as vis-
cosity and elastic modulus could be visualized by fluorescence
1
9–23
signals.
recognition by synthetic molecules were demonstrated at the
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A conformational change and control of guest

Journal of the American Chemical Society
Page 2 of 18
and hydrophobic units. Indeed, octaethylene glycol (OEG)
chains and 3,3’-dimethyl-5,5’-bis(phenylethynyl)-2,2’-
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bipyridine (BPBP) units were introduced as the hydrophilic
and hydrophobic units, respectively, where the BPBP units are
expected to span the hydrophobic layer of the lipid bilayer.
Since fluorescence emission provides important information
regarding the conformation and assembly of the hydrophobic
parts, bipyridyl units, having higher fluorescence quantum
3
5
yield than biphenyl, were introduced in the core. In addition,
methyl groups were introduced at the 2- and 2’-positions of
the BPBP units to generate a steric hindrance. We found that
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and 2 (Figure 1) respond to the membrane tension by chang-
ing the stacking mode of the BPBP so as to influence on the
ion transportation efficiency.
1
Figure 2. H nuclear magnetic resonance spectra of (a) 1 and (b)
2 in the mixtures of D O and THF-d at various ratios at 25 °C.
1] = 3.0 mM and [2] = 1.0 mM ([BPBP units] = 3.0 mM for both
cases).
2
8
[
Figure 1. Molecular structures of multi-block amphiphiles 1 and
2.
RESULTS AND DISCUSSION
Self-Assembly of MTM Mimics in Aqueous Media.
1 was synthesized by the coupling reaction between 5,5'-
diethynyl-3,3'-dimethyl-2,2'-bipyridine
iodophenoxy)-3,6,9,12,15,18,21-heptaoxatricosan-1-ol,
and
23-(4-
and
this scheme was also extended to the synthesis of 2 (see Sup-
porting Information). Since 1 and 2 are amphiphilic, we first
investigated the self-assembling behaviors of 1 and 2 in aque-
ous environments. Dynamic light scattering (DLS) measure-
ment of 1 in THF at 25 °C hardly showed scattering signals,
while aggregates with an average size of 834 nm were visual-
ized in water at 25 °C ([1] = 7.5 µM, Figure S1a in the Sup-
1
porting Information). H nuclear magnetic resonance (NMR)
spectrum of 1 in THF-d
8
at 25 °C displayed signals at 8.29,
7
.52, 7.21, and 6.70 ppm corresponding to the protons at the
BPBP unit ([1] = 3.0 mM, Figure 2a). Upon addition of deu-
terated water, the aromatic signals showed upfield shift likely
due to magnetic shielding effect by the self-assembled BPBP
3
6,37
units (THF-d
8
/D
2
O = 90/10 and 50/50 v/v).
The absorption
Figure 3. (a,b) Absorption and (c,d) fluorescence spectra of (a,c)
and (b,d) 2 in the mixtures of THF and water at 25 °C. The
band of 1 showed a blue-shift upon addition of water, suggest-
ing H-aggregate formation ([1] = 7.5 µM, 25 °C; 325 nm in
THF, 321 nm in water; Figure 3a). The fluorescence spectra
1
fluorescence spectra were measured upon excitation with 330 nm
light for 1 and 328 nm light for 2, respectively. Optical path
length: 1.0 cm. [1] = 7.5 µM and [2] = 2.5 µM ([BPBP units] =
7.5 µM for both cases).
showed a significant red-shift of the emission band, from λEm
=
4
10 nm in THF to 449 nm in water, which was accompanied
by an emergence of a shoulder around 520 nm (λEx = 330 nm,
Figure 3c). Here, fluorescence anisotropy r of 1 decreased
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Journal of the American Chemical Society
of 1 and 2 locate at the similar position with the spin probes of
upon increment of the solvent polarity (r = 0.39 in THF, 0.18
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in THF/water = 75/25, and 0.14 in water; [1] = 7.5 µM). In
12- and 16-Doxyl PCs inside the lipid bilayer, thereby sug-
gesting that the BPBP units of 1 and 2 span the membrane.
The lower quenching efficiency of 5-Doxyl PC than the others
is likely due to the shorter length of the BPBP unit than the
thickness of the hydrophobic layer of the DOPC bilayer.
general, a slowly tumbling object, in other words a larger ob-
3
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ject, shows a larger fluorescence anisotropy value. Since the
solvent polarity increment elicited the aggregation formation,
the observed decrease in fluorescence anisotropy indicates
intermolecular energy transfer in the aggregates of 1, namely,
excimer formation of the BPBP unit of 1. Excimer formation
is also suggested from the significantly longer fluorescence
lifetime (τ = 1.4 ns) at 550 nm in water than that at 450 nm (τ
Mechano-Responses of MTM Mimics in Mem-
branes. Mechano-responses of membrane-embedded 1 and
2 to membrane tensions were assayed by applying an osmotic
pressure ΔΠ to the vesicles as performed in the previous stud-
=
0.51 ns) in THF ([1] = 7.5 µM, Figure S2a,b). Analogous to
42–44
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ies.
The osmotic pressure ΔΠ is controllable by the con-
1
, the DLS and spectroscopic studies displayed that the as-
centration difference ΔC of the solute (glucose) between the
internal and external media of the vesicles as described below:
sembly of the BPBP units of 2 is encouraged by increase in the
solvent polarity (Figure 2b, 3b,d, also see Supporting Infor-
mation Section 4 for details).
ΔΠ = ΔCRT
ΔC = Cint –Cext
where R and T represent the gas constant and temperature,
Introduction of MTM Mimics into Bilayer Mem-
brane. Giant unilamellar vesicles (GUVs) of 1,2-dioleoyl-
sn-glycero-3-phosphocholine (DOPC) containing 1 or 2 were
prepared in 200 mM sucrose aq. by the gentle hydration meth-
od ([DOPC] = 200 µM, [1] = 20 µM, [2] = 6.7 µM, [BPBP
40
respectively. On the basis of the Young-Laplace equation,
an osmotic pressure causes a membrane tension Δσ as de-
scribed by the following equation:
ΔΠr
2
Δσ =
39
units] = 20 µM for both cases). Phase-contrast microscopy
displayed successful formation of the GUVs in each case
where r represents the radius of the vesicle. Since Δσ corre-
lates with r, different size vesicles were prepared under a con-
trolled condition in the following study. Indeed, extrusion of
DOPC liposomal suspensions containing 1 and 2 provided
LUVs, and the size of the LUVs was successfully controlled
by the pore size of the extrusion membrane (Small (S): 50 nm,
Medium (M): 100 nm, and Large (L): 200 nm). By the extru-
(
Figure S3a,c). Fluorescent microscopy with an excitation
light at 330–385 nm visualized spherical images for both
DOPC•1 and DOPC•2 GUVs, indicating that 1 and 2 are em-
bedded in the bilayer of DOPC (Figure S3b,d). Importantly,
addition of 10 mM glucose aq. to the external aqueous phase
of the vesicles resulted in membrane fluctuation of DOPC•1
and DOPC•2 GUVs (see Supporting Movies 1 and 2 for
DOPC•1 and DOPC•2 GUVs, respectively). This observation
suggests that glucose hardly passes through the DOPC bilayer
containing 1 and 2, which allows for regulation of an osmotic
pressure by controlling the concentration difference of glucose
M
sion through a 100-nm pore size membrane, DOPC•1 LUV
M
and DOPC•2 LUV with average sizes of 135 nm and 147 nm
were prepared in 200 mM glucose aq., respectively
([1]/[DOPC] = 0.050, [2]/[DOPC] = 0.017, [DOPC] = 0.20
mM, [BPBP units] = 10 µM for both LUVs; Figure 4f). The
4
0
M
across the bilayer.
obtained DOPC•1 LUV showed fluorescence at λ = 443 nm
FL
The location and orientation of 1 and 2 in the bilayer were
studied by a fluorescence depth quenching method using
phospholipids bearing a spin probe, where the efficiency of
fluorescence quenching depends on the distance between the
spin probe and a fluorescent chromophore (namely BPBP
(λEx = 325 nm, Figures 4a and S5a, blue solid line). The red-
shifted fluorescence of 1 compared to that in THF (λFL = 410
nm, Figures 4a and S5a, black dotted line) suggests that the
BPBP unit of 1 forms intermolecular self-assembly in the bi-
M
layer. On the other hand, DOPC•2 LUV showed fluores-
4
1
unit).
Three spin-labeled phospholipids, 1-palmitoyl-2-
cence at 412 nm, suggesting that the stacking of the BPBP
units of 2 in the bilayer is much weaker than those of 1 (Fig-
ures 4b and S5b, blue solid line). Upon increase in tension to
M
stearoyl-(X-doxyl)-sn-glycero-3-phosphocholine (X = 5: 5-
Doxyl PC, X = 12: 12-Doxyl PC, and X = 16: 16-Doxyl PC),
bearing a spin probe at different positions in the alkyl tail were
used. Computational molecular modeling indicates that the
spin probes of 5-, 12-, and 16-Doxyl PCs locate at 1.2, 0.6 and
–
1
Δσ = 20 mN m to expand the membrane, DOPC•1 LUV
displayed blue-shift of the fluorescence band with decrease in
–
the excimer emission intensity (λFL = 443 nm at Δσ = 0 mN m
1
–1
0
.2 nm away from the tip of the hydrophobic tail of DOPC,
, 434 nm at 20 mN m ; Figures 4a and S5a, black solid line),
respectively (Figure S4). DOPC large unilamellar vesicles
including 1 or 2 (DOPC•1 LUV, DOPC•2 LUV) with/without
a Doxyl PC were prepared by freezing-and-thawing followed
by extrusion through a 100-nm pore size membrane. Incorpo-
ration of 10-mol% 5-Doxyl PC into DOPC•1 LUVs resulted in
while the contracting tension prompted red-shift of the fluo-
rescence band with intensified excimer emission (λFL = 445
–
1
nm at Δσ = –34 mN m , Figures 4a and S5a, red dashed line).
Importantly, upon increase in tension to expand the membrane,
M
DOPC•1 LUV showed increment in the fluorescence anisot-
5
=
1
1% decrease of the fluorescence intensity of 1 at 443 nm (λEx
325 nm, Figure S4a). Meanwhile, 10-mol% incorporation of
2- and 16-Doxyl PCs comparably quenched the fluorescence
ropy r, while its size was almost constant (r = 0.18 at Δσ = –34
–
1
–1
–1
mN m , 0.19 at 0 mN m , 0.22 at 20 mN m ). These obser-
vations indicate that the degree of intermolecular energy trans-
fer decreases due to dissociation of self-assembled 1 mole-
cules. Namely, 1 responds to the expanding and contracting
tensions by dissociation and association of the BPBP units,
of 1 with higher efficiency than 5-Doxyl PC (65% and 67%
decreases, respectively). Likewise, for DOPC•2-LUVs, 12-
Doxyl and 16-Doxyl PCs comparably showed higher quench-
ing efficiency for the fluorescence at 412 nm (λEx = 325 nm;
45
M
respectively. Here, 100-fold dilution of 1 in DOPC•1 LUV
([1]/[DOPC] = 0.00050) resulted in slightly blue-shifted fluo-
6
2% and 65% decreases, respectively; Figure S4b) than 5-
–
1
Doxyl PC (43%). The nearly comparable quenching effi-
ciencies of 12- and 16-Doxyl PCs indicate that the BPBP units
rescence peaks at 441 nm at 0 mN m and 432 nm at 20 mN
–1
m
(Figure S6a), respectively. This concentration-dependent
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Page 4 of 18
cence band of 2, where emission wavelength is mostly insensi-
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tive to the membrane tensions. In addition to the fluorescence
spectral change mentioned above, the excitation spectrum of
M
DOPC•1 LUV displayed red-shift (disassembly) and blue-
shift (assembly) in response to the expanding and contracting
M
tensions, respectively (Figure 4c). In contrast, DOPC•2 LUV
hardly showed changes in the excitation spectrum in response
to the tensions (Figure 4d). Hence, the membrane tension
elicits the changes in the intermolecular stacking of the BPBP
units of 1 at the ground state, while initial intramolecular
stacking of the BPBP units in 2 is likely too weak for the de-
tection of the tension-triggered conformational changes by the
spectroscopic analyses.
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The fluorescence-spectral responses of 1 and 2 in DOPC bi-
layer to the tension hardly depend on the curvature of the LUV.
DOPC•1 LUVs with smaller and larger sizes showed fluores-
cence at 443 nm with similar spectral profile to DOPC•1
M
S
L
LUV (DOPC•1 LUV : 82 nm, DOPC•1 LUV : 175 nm;
1]/[DOPC] = 0.050, [DOPC] = 0.20 mM, Figure S5c,d).
Upon exposure to the osmotic pressures to induce membrane
[
–
1
S
tensions to Δσ = 20 and –34 mN m , DOPC•1 LUV and
L
DOPC•1 LUV showed blue- and red-shifts of the fluorescen-
ce in a similar manner to DOPC•1 LUV , respectively.
M
Meanwhile, almost unchanged fluorescence spectra were ob-
served in any sizes of DOPC•2 LUVs (Figure S5e,f).
Mechano-Sensitive Ion Transportation in Lipo-
somal and Black-Lipid Membranes. Previous studies
3
0,32
in our group
revealed that the assembly of the membrane
46–53
spanning amphiphiles allows for ion transportation
by
forming supramolecular channels. Since above-mentioned
fluorescence study indicated the tension-responsive dissocia-
tion/association of the BPBP units of 1, we expected that the
membrane tension could modulate the ion transportation func-
tion. At first, ion transportation of 1 and 2 was investigated on
the vesicles by monitoring a fluorescence intensity change
Figure 4. (a,b) Fluorescence and (c,d) excitation spectra of (a,c)
DOPC•1 and (b,d) DOPC•2 LUV s in water (solid and dashed
lines) under varying membrane tensions and in THF (black dotted
lines) at 25 °C. The fluorescence spectra were measured upon
excitation with 325 nm light. The excitation spectra were meas-
ured monitoring the fluorescence at 440 nm. [1]/[DOPC] = 0.050,
M
M
using a pH sensitive probe. DOPC•1 LUV encapsulating 8-
hydroxypyrene-1,3,6-trisulfonate (HPTS) in the internal aque-
[
[
2]/[DOPC] = 0.017 ([BPBP units] = 10 µM for both cases),
DOPC] = 0.20 mM. The whole spectra (375–600 nm) of (a) and
ous phase was prepared in 20 mM HEPES buffer containing
M
5
0
0 mM KCl at pH 7.1 (DOPC•1 LUV ⊃HPTS, [1]/[DOPC] =
(b) are displayed in the Supporting Figure S5a,b. (e) Absolute
.050). HPTS emits 510-nm fluorescence upon excitation
M
values of the fluorescence peak shift of DOPC•1 LUV from λFL
with 450-nm light at pH higher than 5, and the fluorescence
–
1
=
443 nm at Δσ = 0 mN m in water as a function of the mem-
54
intensity increases upon enhancement of pH.
Under the
brane tension. The plot was analyzed by curve-fitting with Hill
tensionless condition or exposure to the contracting tension,
equation (R = 0.9868). (f) Volume-averaged DLS profiles of
M
M
M
DOPC•1 LUV ⊃HPTS hardly showed increment of the fluo-
DOPC•1 LUV (blue) and DOPC•2 LUV (red) in water under
–1
rescence intensity upon addition of KOH (Δσ = 0.0 or –34 mN,
Figure 5a, blue thick and dotted lines, respectively). In sharp
contrast, under exposure to the expanding tension, DOPC•1
tensionless (solid lines) and tensioned (–34 mN m : dashed lines,
0 mN m : dotted lines) conditions. [1]/[DOPC] = 0.050,
–1
2
[
2]/[DOPC] = 0.017 ([BPBP units] = 10 µM for both cases),
M
[DOPC] = 0.20 mM. Mean hydrodynamic diameters: (DOPC•1
LUV ⊃HPTS displayed elevation of the fluorescence intensi-
M
–1
–1
LUV ) 129 nm at –34 mN m , 135 nm at 0 mN m , 143 nm at
ty readily upon addition of KOH (Δσ = 20 mN, Figure 5a, blue
thin line), indicating the transportation of potassium ions into
the vesicles to raise the intravesicular pH. The Hill analysis
on the concentration dependency of the cation transportation
rate afforded the Hill coefficient n = 2.98 ± 0.26 (R = 0.993),
suggesting supramolecular ion channel formation of 1, possi-
–
1
M
–1
2
0
0 mN m , (DOPC•2 LUV ) 126 nm at –34 mN m , 147 nm at
mN m , 171 nm at 20 mN m .
–
1
–1
fluorescence change is likely due to intermolecular stacking of
the BPBP unit. Fluorescence spectra of 2 in the bilayer
showed much smaller shift from the fluorescence in THF
compared to 1, and hardly showed responses to the membrane
tensions (Figures 4b and S5b). Importantly, 100-fold dilution
5
5
bly by the trimeric assembly (Figure 5b, blue thin line). On
M
the other hand, DOPC•2 LUV ⊃HPTS showed a rapid in-
crease in the fluorescence intensity upon addition of KOH
even under the absence of osmotic pressure (Figure 5a, red
thick line; [2]/[DOPC] = 0.017). However, interestingly,
while similar fluorescence enhancement was observed under
M
of 2 in DOPC•2 LUV ([2]/[DOPC] = 0.00017) also hardly
influenced the fluorescence wavelength regardless of the
strength of the applied tensions (λFL = 412 nm, Figure S6b).
These results suggest that the weak intramolecular stacking of
the BPBP units mainly dictates the slight shift of the fluores-
–1
the contracting tension (Δσ = –34 mN m , Figure 5a, red dot-
ted line), the expanding tension caused a significant decline of
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Journal of the American Chemical Society
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2
3
4
5
6
7
8
9
Figure 5. (a) Changes in fluorescence intensity of HPTS en-
capsulated in DOPC•1 and DOPC•2 LUV s in HEPES buffer
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
M
at 20 °C as a function of time after the addition of KOH at 0
sec followed by addition of 1.0 wt% triton X-100 at 100 sec
(
3
[DOPC] = 0.20 mM, [1] = 1.0 µM, [2] = 0.33 µM, [HPTS] =
0 µM, 20 mM HEPES, 50 mM KCl, pH 7.1, excitation at 460
nm, emission at 510 nm). ΔpH = 0.8 (7.1 to 7.9). Tensions
M
added to DOPC•1 LUV ⊃HPTS (blue lines) and DOPC•2
M
LUV ⊃HPTS (red lines): 0 (thick lines), 20 (thin lines) and –
–1
3
4 mN m (dotted lines). (b) Plots of relative 510-nm fluo-
rescence intensities of HPTS encapsulated in DOPC•1 and
M
DOPC•2 LUV s at 30 s after the addition of KOH in 20 mM
HEPES buffer containing 50 mM KCl at 20 °C as a function
of concentration of 1 and 2. Curve-fitting analyses were car-
M
ried out by the Hill equation (R = 0.993 (DOPC•1 LUV at 20
–
1
M
–1
mN m ), 0.996 (DOPC•2 LUV at 0.0 mN m ), 0.995
(
M
–1
DOPC•2 LUV at –34 mN m )). Colors and line types cor-
respond to those in (a).
–
1
the ion transportation rate (Δσ = 20 mN m , Figure 5a, red
thin line). The Hill coefficients of 2 under the tensionless and
contracting-tension conditions were evaluated to be 1.22 ±
0.07 (R = 0.996) and 1.27 ± 0.03 (R = 0.995), respectively,
suggesting formation of the unimolecular channel as the major
active species (Figure 5b, red thick and dotted lines, respec-
M
tively). It should be mentioned that DOPC LUV ⊃HPTS
without 1 or 2 showed no ion transportation at any tensions
(
Figure S7a). Thus, it was demonstrated that 1 responds to the
expanding tension to prompt the ion transportation by forming
a supramolecular ion channel, while 2 shows ion transporta-
tion through a unimolecular ion channel under the tensionless
condition and responds to the expanding tension to reduce the
transportation rate likely by closing or collapsing the ion
Figure 6. Current traces at the applied voltage of 120 mV of a
DOPC BLM containing (a,b) 1 or (c,d) 2 ([1]/[DOPC] = 0.010,
[2]/[DOPC] = 0.0033) in HEPES buffer (20 mM HEPES, 50 mM
56
channel.
2
KCl, 2.0 mM MgCl , pH 7.5) under (a,c) low tension (Δσ < 5 mN
For further detailed investigation on the mechanosensitive ion
transporting behaviors by direct detection of the ion transpor-
tation, 1 and 2 were embedded into a DOPC black lipid mem-
brane (BLM) containing n-decane. The BLM is formed hori-
zontally at the bottom of the upper chamber containing elec-
trolyte buffer solution. Membrane tension Δσ to expand the
BLM was applied by hydrostatic pressure produced by the
difference in the height Δh of the solution surfaces between
the upper and lower chambers. Microscopic fluorescence
spectroscopy of 1 in BLM displayed a blue-shift of the fluo-
rescence peak λFL from 443 nm to 436 nm upon increase in Δh
from 1.0 mm to 9.0 mm (λEx = 295–347 nm, Figure S9). Here,
on the basis of the relationship between membrane tension Δσ
and fluorescence band λFL of 1 in DOPC bilayer (Figure 4e),
the membrane tension Δσ in the BLM system caused by Δh
could be estimated by the microscopic fluorescence spectro-
scopic analysis.
–1
–1
m ) or (b,d) increased expanding tension (Δσ = 16 mN m ) at
0 °C.
2
Ion transportation through the BLM was directly detected in
real time as a current between the electrodes in the upper and
–
1
lower chambers. At a low-tension condition (Δσ < 5 mN m ),
1 hardly showed current flow, while 2 exhibited significant
current flows (Figure 6a,c). A rise of the expanding tension to
–
1
16 mN m triggered ion transportation of 1 (Figure 6b), which
is in line with the trend observed in the DOPC•1
M
LUV ⊃HPTS system (Figure. 5a, blue thin line). At the ex-
–
1
panding tension of 16 mN m , 2 showed a significantly re-
duced frequency of the current flow (Figure 6d; 4.3-times re-
duction in average compared to the low-tension condition),
which is also consistent with the result observed in the
M
DOPC•2 LUV ⊃HPTS system (Figure 5a, red thin line). It
should be mentioned here that DOPC bilayer in the BLM sys-
ACS Paragon Plus Environment

Journal of the American Chemical Society
tem without 1 or 2 hardly showed ion transportation at any
tensions under these experimental conditions, indicating that 1
and 2 function to transport ions (Figure S10).
Page 6 of 18
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
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3
3
3
3
3
3
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6
Under the ion-transporting conditions, 2 showed a higher
conductance than 1 (1: g = 8.2 pS, 2: 12 pS). The voltage-
clamp traces of 1 and 2 in DOPC BLM show “square-top”
shape of the current flow, where the current abruptly switches
between two modes, off- and on-states maintaining constant
current intensities. Such a profile is typically observed in the
5
5,57
case of ion transportation by ion channels,
thereby suggest-
ing the formation of ion channels by 1 and 2. It is known that
the channel diameter d correlates with conductance g on the
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
8
basis of the Hille equation,
1
g
lρ
π(d/2)
ρ
d
=
2
+
where l and ρ are the length of channel (3.5 nm) and the resis-
tivity of recording solution (2.35 Ω m), respectively. Analyses
5
9
on the Hille equation including the Sansom correction factor
revealed d of 1 and 2 to be ca. 0.74 and 0.92 nm, respective-
6
0
ly.
Here, it would be significant to discuss about the difference
in the responses of 1 and 2 to the expanding and contracting
tensions. It is known that the increase in the expanding ten-
3
4
sion enhances the membrane fluidity. Under the tensionless
and contracting-tension conditions, where the mobility of the
lipid molecules is relatively restricted, the fluorescence study
of 1 visualized the formation of the developed intermolecular
π-π stacking of the BPBP units in the lipid bilayer, which like-
ly stabilizes closely-stacked assemblies rather than the for-
mation of the pore-forming channels (Figure 4a, blue solid and
red dashed lines; Figure 7a (i) and (ii)). By rise of the mem-
brane expanding tension to increase the mobility of the lipid
molecules, the intermolecular stacking is attenuated, as was
visualized by the fluorescence study (Figure 4a, black solid
line). It is likely that the increase in the mobility of the BPBP
units allows for the formation of a supramolecular channel
with a short life-span (milli-second order) (Figure 5a, blue thin
line; Figures 6b, 7a (iii)). In contrast, as described above, the
stacking of the BPBP units of 2 in the bilayer would be much
less efficient compared to those of 1 under the tensionless
condition (Figure 4b, blue line), suggesting the restricted ge-
ometry of the BPBP units. The less efficient stacking of the
BPBP units would permit a certain degree of mobility of each
BPBP unit to allow for the tentative formation of a channel
Figure 7. Schematic illustrations displaying the responses of (a)
1 and (b) 2 to (i) membrane contraction and (iii) expansion from
ii) a tensionless condition in a DOPC bilayer. Orange, blue and
gray parts denote the hydrophobic and hydrophilic parts in 1 and 2
and DOPC, respectively. Yellow spheres represent a cation.
Yellow arrows depict the direction of membrane tension.
(
–1
erage rupture force, is 13.6 mN m . The bacterial MSC pro-
teins, MscS and MscL, are known to operate at tensions of 5.5
–1
–1
mN m and 12 mN m to open the channel, respectively,
where they play roles as safety valves to prevent the cells from
61,62
lysis.
It is of interest that 1 senses a similar amplitude of
tension to MscL, although their molecular structures and sizes
are largely different from each other. The conductance meas-
urement of 2 also suggested that 2 operates at similar tensions
to MSC proteins. Another interesting point is that while both
MSC proteins and 1 form supramolecular ion channels and
sense the expanding tension to trigger ion transportation in
common, 2 forming a unimolecular channel shows the con-
trasting response to the expanding tension to deactivate the ion
transportation.
(
Figure 5a, red thick line; Figures 6c, 7b (ii)), while the re-
striction of the geometry likely inhibits closer packing in re-
sponse to the contracting tension (Figure 5a, red dotted line;
Figure 7b (i)). On the other hand, the enhanced mobility of
the membrane due to the expanding tension likely causes fur-
ther increase in the flexibility of the BPBP units to rather de-
stabilize the channel structure (Figure 5a, red thin line; Figures
6d, 7b (iii)).
SUMMARY
We demonstrated the mechano-sensitive synthetic ion chan-
nels consisting of membrane-spanning amphiphiles 1 and 2,
where the tension triggers changes in the conformation and
self-assembly of the amphiphiles, and modulates ion transpor-
tation. The BPBP unit of 1 forms intermolecular self-
assembly in the bilayer, which dissociate and associate in re-
sponse to the expanding and contracting tensions, respectively.
While 1 hardly shows ion transportation under the tensionless
or the contracting-tension conditions, 1 responds to the ex-
panding tension to prompt the ion transportation by forming a
supramolecular ion channel. The BPBB units of 2, on the
Finally, it would be interesting to make a comparison be-
tween the present systems with the biological mechano-
sensitive ion channels. The rupture force to dissociate intermo-
lecular stacking of the BPBP unit of 1 could be estimated on
the basis of the tension-induced fluorescence shift. The de-
M
pendence of the fluorescence peak shift of DOPC•1 LUV on
the expanding tension displayed
a
sigmoidal profile
(
[1]/[DOPC] = 0.050, [DOPC] = 0.20 mM, Figure 4e). The
curve-fitting analysis with Hill equation revealed that the ten-
sion causing 50% fluorescence peak shift, regarded as the av-
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Page 7 of 18
Journal of the American Chemical Society
mL, pH 7.1) was added an aqueous solution of KOH (0.60 M,
other hand, form weak intramolecular stacking. 2 shows ion
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
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5
5
5
6
transportation through a unimolecular ion channel under the
tensionless condition and responds to the expanding tension to
reduce the transportation activity likely by closing or collaps-
ing the ion channel. The operating force of these membrane-
spanning amphiphiles indicates that the amplitude of the ten-
sion sensed by the amphiphiles is similar to those sensed by
the MSC proteins. It is known that the MSC protein opens its
pore by membrane expanding force, which works as a molecu-
lar device for tactile and auditory senses. We believe that this
study opens the door to the development of membrane-
embedded synthetic molecular devices responsive to external
physical stimuli for bio-related applications.
10 µL, ΔpH = 0.8) by a syringe in the dark at 20 °C. Fluores-
cence intensity of HPTS at 510 nm upon excitation with 460
nm-light was monitored as a function of time until the addition
of 1.0 wt% Triton X-100 (40 µL) at 100 s. Relative fluores-
cence intensity of HPTS in response to the pH enhancement
was evaluated by the equation of
t 0
I – I
lyzed – I
0
I =
I
0 t
where I , I and Ilyzed represent the fluorescence intensities be-
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
fore addition of KOH, at t seconds after addition of KOH, and
after lysis by the addition of 1.0 wt% Triton X-100, respec-
tively.
METHODS
Conductance Measurements and Tension Calcu-
Giant Unilamellar Vesicles Preparation. To a mix-
lation in BLM System. Planar lipid bilayer was prepared
6
3
ture of CHCl
tube, was added a CHCl
a MeOH solution of glucose (10 mM, 12 µL) and a CH
3
/MeOH (2/1 v/v, 10 µL) was put in a glass test
solution of DOPC (2.0 mM, 20 µL),
Cl
by the reported procedure. A mixture of DOPC (12.7 mM,
100 µL) in CHCl and an MTM mimic (1: 1.0 mM, 12.5 µL,
or 2: 0.10 mM, 42 µL) in CH Cl was gently dried under N
2
3
3
2
2
2
2
solution of an MTM mimic (1: 0.10 mM, 40.0 µL, or 2: 0.10
mM, 13.3 µL). The resulting mixture was gently dried under
Ar flow to produce thin lipid film. The film was subsequently
dried under vacuum over 3 h at 25 °C and hydrated overnight
with 200 mM sucrose aq. (200 µL) under Ar at 37 °C.
flow, which was then dispersed in n-decane (100 µL) and was
painted on an orifice (diameter 150 µm) sandwiched by two
chambers containing HEPES buffer (upper chamber: trans,
lower chamber: cis, 20 mM HEPES, 50 mM KCl, 2.0 mM
2
MgCl , pH 7.5, 0.30 mL each). Current was measured with a
Large Unilamellar Vesicles Preparation. To a mix-
Nihon Kohden CEZ2400 amplifier and stored on a computer
using an AD Instruments PowerLab at 40 kHz sampling rate.
Recordings were filtered at 1 kHz. All the current recordings
were performed at 20 °C. Membrane tension Δσ was applied
by hydrostatic pressure to expand the planar membrane, where
the hydrostatic pressure was produced by the difference in
height Δh of the water surface between the two chambers.
ture of CHCl
were added a CHCl
a CH Cl solution of an MTM mimic. The resulting mixture
3
/MeOH (2/1 v/v, 50 µL) put in a glass test tube,
3
solution of DOPC (2.0 mM, 100 µL) and
2
2
was gently dried under Ar flow to produce thin lipid film,
which was subsequently dried under vacuum over 3 h at 25 °C
and hydrated overnight with 200 mM glucose aq. (1.0 mL)
under Ar at 37 °C. The resulting mixture was stirred on a
–
1
shaker (203 min ) for 1 h at 37 °C, followed by vortex mixing
for 10 s, freezing-and-thawing for three times, and subsequent
vortex mixing for 10 s. After being left standing overnight at
37 °C, the resulting mixture was passed through a polycar-
bonate membrane of defined pore size (50 nm: LFM-50, 100
nm: LFM-100 or 200 nm: LFM-200) attached in a LiposoFast-
Basic device by pushing the sample back and forth between
the two gas-tight syringes over 11 times.
ASSOCIATED CONTENT
Supporting Information. Materials; Instrumentation; Syn-
thesis; Self-Assembly of 2; DLS and Fluorescence Lifetime
Measurements of 1 and 2; Optical Microscopic Observations of
DOPC•1 and DOPC•2 GUVs; Fluorescence Depth Quenching
Studies, Size- and Concentration-Dependencies of Fluorescence
Spectral Profiles, HPTS Assays with Different Cations, DLS
Measurements of DOPC•1 and DOPC•2 LUVs; Microscopic Flu-
orescence Spectroscopic Measurements of DOPC•1 BLM; Con-
ductance Measurements of DOPC BLM. Movies of the fluctuat-
ing DOPC•1 and DOPC•2 GUVs upon addition of glucose aq.
This material is available free of charge via the Internet at
http://pubs.acs.org.
M
LUV s for fluorescence depth quenching were prepared by
following the above procedure using a mixture of DOPC and
5
-Doxyl PC, 12-Doxyl PC, or 16-Doxyl PC ([DOPC]/[Doxyl
PC] = 90/10).
M
LUV s encapsulating HPTS in the inner aqueous phase
were prepared by following the above procedure, where 20
mM HEPES buffer containing 50 mM KCl (pH 7.1) and 30
µM HPTS was used as the hydration medium. After the extru-
sion process, the obtained suspension was dialyzed at 4 °C in
AUTHOR INFORMATION
Corresponding Author
kkinbara@bio.titech.ac.jp
2
0 mM HEPES buffer containing 50 mM KCl (pH 7.1, 1.0 L,
ACKNOWLEDGMENT
This work was partially supported by Grant-in-Aid for Scientific
Research B (16H04129 to KK), Yamada Science Foundation
three times) using Spectra/Por Dialysis Membrane (MWCO
3
500).
The osmotic pressure ΔΠ to LUVs was controlled by the
concentration difference (ΔC) of glucose between the internal
(
“
KK), Grant-in-Aid for Scientific Research on Innovative Areas
π-System Figuration (No.2601)” (26102001 to ΤΜ), JST PRES-
and external media of the LUVs. Appling tension higher than
–1
–1
TO program “Molecular Technology and Creation of New Func-
tions” (TM), and the Management Expenses Grants for National
Universities Corporations from MEXT.
2
0 mN m or lower than –34 mN m resulted in little scat-
tering or polydispersed particle size distribution profiles in
DLS, suggesting disruption or deformation of LUVs.
Fluorescence Measurement for Ion Transporta-
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change, because the tension-dependent change in the fluorescen-
(
15) Sagara, Y.; Kato, T. Angew. Chem. Int. Ed. 2011, 50, 9128–9132.
(16) Caruso, M. M.; Davis, D. A.; Shen, Q.; Odom, S. A.; Sottos, N.
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M
(
17) Davis, D. A.; Hamilton, A.; Yang, J.; Cremar, L. D.; Gough, D.
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White, S. R.; Moore, J. S.; Sottos, N. R. Nature 2009, 459, 68–
ce anisotropy of DOPC•1 LUV should be caused mainly by as-
sociation and dissociation of 1 changing the degree of energy
transfer.
7
2.
(46) Sakai, N.; Matile, S. Langmuir 2013, 29, 9031–9040.
(47) Kim, Y.; Li, W.; Shin, S.; Lee, M. Acc. Chem. Res. 2013, 46,
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988–8991.
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25) Sakakibara, K.; Joyce, L. A.; Mori, T.; Fujisawa, T.; Shabbir, S.
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+
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H.; Hill, J. P.; Anslyn, E. V.; Ariga, K. Angew. Chem. Int. Ed.
(56) Among Li , Na and K , both channels consisting of 1 and 2
show faster transportation for the smaller cations. On the basis
of the Eisenman theory on the ion permeability, this trend sug-
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that cation–π interaction at the aromatic portions of 1 and 2 is
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Eisenman, G.; Horn, R. J. Membrane Biol. 1983, 76, 197–225).
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Matsukura, M.; Tsukube, H.; Suzuki, Y.; Kanekiyo, Y.; Ariga, K.
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Tsumoto, K.; Li, R.; Kinbara, K. J. Am. Chem. Soc. 2014, 136,
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(60) In fact, we confirmed Me
+
(
31) Shima, T.; Muraoka, T.; Hoshino, N.; Akutagawa, T.; Kobayashi,
4
N
(ionic diameter: 0.566 nm) was
+
Y.; Kinbara, K. Angew. Chem. Int. Ed. 2014, 53, 7173–7178.
transported by 1 and 2, while n-Bu
4
N (0.830 nm) were trans-
ported very inefficiently by either of them probably due to the
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insufficient channel sizes (Figure S7, Ue, M. J. Electrochem.
Soc. 1994, 141, 3336–3342).
61) Sukharev, S. Biophys. J. 2002, 83, 290–298.
62) Sukharev, S. I.; Sigurdson, W. J.; Kung, C.; Sachs, F. J. Gen.
Physiol. 1999, 113, 525–539.
(63) Tabata, K. V.; Sato, K.; Ide, T.; Nishizaka, T.; Nakano, A.; Noji,
1
H. EMBO J. 2009, 28, 3279–3289.
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Figure 1. Molecular structures of multi-block amphiphiles 1 and 2.
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Figure 2. H nuclear magnetic resonance spectra of (a) 1 and (b) 2 in the mixtures of D
2
O and THF-d8 at
various ratios at 25 °C. [1] = 3.0 mM and [2] = 1.0 mM ([BPBP units] = 3.0 mM for both cases).
2
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Figure 3. (a,b) Absorption and (c,d) fluorescence spectra of (a,c) 1 and (b,d) 2 in the mixtures of THF and
water at 25 °C. The fluorescence spectra were measured upon excitation with 330 nm light for 1 and 328
nm light for 2, respectively. Optical path length: 1.0 cm. [1] = 7.5 µM and [2] = 2.5 µM ([BPBP units] =
7
.5 µM for both cases).
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Figure 4. (a,b) Fluorescence and (c,d) excitation spectra of (a,c) DOPC•1 and (b,d) DOPC•2 LUV s in water
solid and dashed lines) under varying membrane tensions and in THF (black dotted lines) at 25 °C. The
(
fluorescence spectra were measured upon excitation with 325 nm light. The excitation spectra were measꢀ
ured monitoring the fluorescence at 440 nm. [1]/[DOPC] = 0.050, [2]/[DOPC] = 0.017 ([BPBP units] = 10
M for both cases), [DOPC] = 0.20 mM. The whole spectra (375–600 nm) of (a) and (b) are displayed in
the Supporting Figure S5a,b. (e) Absolute values of the fluorescence peak shift of DOPC•1 LUV from λFL
443 nm at ∆σ = 0 mN m in water as a function of the memꢀbrane tension. The plot was analyzed by
curveꢀfitting with Hill equation (R = 0.9868). (f) Volumeꢀaveraged DLS profiles of DOPC•1 LUV (blue) and
DOPC•2 LUV (red) in water under tensionless (solid lines) and tensioned (–34 mN m : dashed lines, 20
mN m : dotted lines) conditions. [1]/[DOPC] = 0.050, [2]/[DOPC] = 0.017 ([BPBP units] = 10 ꢁM for both
ꢁ
M
=
–1
M
M
–1
–1
–1
cases), [DOPC] = 0.20 mM. Mean hydrodynamic diameters: (DOPC•1 LUVM) 129 nm at –34 mN m , 135
nm at 0 mN m , 143 nm at 20 mN m , (DOPC•2 LUVM) 126 nm at –34 mN m , 147 nm at 0 mN m , 171
–1
–1
–1
–1
–1
nm at 20 mN m
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Figure 5. (a) Changes in fluorescence intensity of HPTS en-capsulated in DOPC•1 and DOPC•2 LUVMs in
HEPES buffer at 20 °C as a function of time after the addition of KOH at 0 sec followed by addition of 1.0
wt% triton X-100 at 100 sec ([DOPC] = 0.20 mM, [1] = 1.0 µM, [2] = 0.33 µM, [HPTS] = 30 µM, 20 mM
HEPES, 50 mM KCl, pH 7.1, excitation at 460 nm, emission at 510 nm). ∆pH = 0.8 (7.1 to 7.9). Tensions
M
M
added to DOPC•1 LUV ⊃HPTS (blue lines) and DOPC•2 LUV ⊃HPTS (red lines): 0 (thick lines), 20 (thin
–1
lines) and –34 mN m (dotted lines). (b) Plots of relative 510-nm fluo-rescence intensities of HPTS
M
encapsulated in DOPC•1 and DOPC•2 LUV s at 30 s after the addition of KOH in 20 mM HEPES buffer
containing 50 mM KCl at 20 °C as a function of concentration of 1 and 2. Curve-fitting analyses were car-
M
–1
M
–
ried out by the Hill equation (R = 0.993 (DOPC•1 LUV at 20 mN m ), 0.996 (DOPC•2 LUV at 0.0 mN m
1
M
–1
)
, 0.995 (DOPC•2 LUV at –34 mN m )). Colors and line types cor-respond to those in (a).
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Figure 6. Current traces at the applied voltage of 120 mV of a DOPC BLM containing (a,b) 1 or (c,d) 2
(
7
[1]/[DOPC] = 0.010, [2]/[DOPC] = 0.0033) in HEPES buffer (20 mM HEPES, 50 mM KCl, 2.0 mM MgCl
2
, pH
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C.
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Figure 7. Schematic illustrations displaying the responses of (a) 1 and (b) 2 to (i) membrane contraction
and (iii) expansion from (ii) a tensionless condition in a DOPC bilayer. Orange, blue and gray parts denote
the hydrophobic and hydrophilic parts in 1 and 2 and DOPC, respectively. Yellow spheres represent a
cation. Yellow arrows depict the direction of membrane tension.
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