Synthetic Ion Channel Formed by Multiblock Amphiphile with Anisotropic Dual-Stimuli-Responsiveness

Article abstract of DOI:10.1021/jacs.0c09470

Transmembrane proteins within biological membranes exhibit varieties of important functions that are vital for many cellular activities, and the development of their synthetic mimetics allows for deep understanding in related biological events. Inspired by the structures and functions of natural ion channels that can respond to multiple stimuli in an anisotropic manner, we developed multiblock amphiphile VF in this study. When VF was incorporated into the lipid bilayer membranes, VF formed a supramolecular ion channel whose ion transport property was controllable by the polarity and amplitude of the applied voltage. Microscopic emission spectroscopy revealed that VF changed its molecular conformation in response to the applied voltage. Furthermore, the ion transport property of VF could be reversibly switched by the addition of (R)-propranolol, an aromatic amine known as an antiarrhythmic agent, followed by the addition of β-cyclodextrin for its removal. The highly regulated orientation of VF allowed for an anisotropic dual-stimuli-responsiveness for the first time as a synthetic ion channel.

Full text of DOI:10.1021/jacs.0c09470

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Synthetic Ion Channel Formed by Multiblock Amphiphile with
Anisotropic Dual-Stimuli-Responsiveness
Ryo Sasaki, Kohei Sato,* Kazuhito V. Tabata, Hiroyuki Noji, and Kazushi Kinbara*
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ABSTRACT: Transmembrane proteins within biological mem-
branes exhibit varieties of important functions that are vital for
many cellular activities, and the development of their synthetic
mimetics allows for deep understanding in related biological events.
Inspired by the structures and functions of natural ion channels that
can respond to multiple stimuli in an anisotropic manner, we
developed multiblock amphiphile VF in this study. When VF was
incorporated into the lipid bilayer membranes, VF formed a
supramolecular ion channel whose ion transport property was
controllable by the polarity and amplitude of the applied voltage.
Microscopic emission spectroscopy revealed that VF changed its
molecular conformation in response to the applied voltage.
Furthermore, the ion transport property of VF could be reversibly switched by the addition of (R)-propranolol, an aromatic
amine known as an antiarrhythmic agent, followed by the addition of β-cyclodextrin for its removal. The highly regulated orientation
of VF allowed for an anisotropic dual-stimuli-responsiveness for the first time as a synthetic ion channel.
can respond to biologically important signals.25,26 Over the
past four decades, synthetic molecules that can respond to a
are vital for a variety of cellular activities.1,2 In many cases, such
single external stimulus such as voltage, ligand molecules, light,
INTRODUCTION
Directional signal transductions across biological membranes
■
or changes in pH have been developed, and their ion transport
properties have been investigated.27−57 For example, some
molecules were designed to possess dipole moments along
their molecular axes so that their channel-forming properties
could be controlled by the changes in transmembrane
potential.27−32 Other examples incorporated binding sites for
ions and molecules within their structures to control the ion
transport properties.35−47 However, design and synthesis of
such molecules still remain challenging,58 and most impor-
tantly, no successful example of a synthetic ion channel with
anisotropic dual-stimuli-responsiveness has been reported to
date.
Meanwhile, in order to develop fully synthetic ion channels
that mimic structures and functions of transmembrane proteins
in nature, varieties of multiblock amphiphiles have been
designed in our group.46,47,59,60 It has been demonstrated that
these amphiphiles were incorporated into the lipid bilayer
membranes vertical to the membrane axis and self-assembled
biological events are carried out by transmembrane proteins
within the membranes that respond to various external stimuli
such as light, ligand molecules, and changes in pH.3−10 Among
them, dual-stimuli-responsive transmembrane proteins are
particularly intriguing, because they can integrate multiple
types of signals and realize their functional controls in a highly
precise manner.11−14 For example, the transient receptor
potential melastatin 8 (TRPM8) channel responds to trans-
membrane potential and ligand molecules and regulates its
directional transmembrane ion transport property to control
signal transmissions of neurons.15−18 Importantly, the
orientation of the TRPM8 channel is highly biased to one
side of the membranes so that it can distinguish the difference
between intra- and extracellular environments and respond to
external stimuli in an anisotropic manner.12,19,20 In particular,
its charged channel-forming domains respond to the changes
in transmembrane potential, which subsequently induce
conformational changes of the whole channel.21,22 Further-
more, the channel possesses binding pockets for ligand
molecules so that it can modulate its ion transport property
in response to their bindings.23,24
Received: September 2, 2020
Published: January 14, 2021
The goal of this work has been to develop a synthetic
molecule that can mimic the functions of such a natural ion
channel with anisotropic dual-stimuli-responsiveness. Such an
attempt allows for further understandings in related biological
events and realizes the development of novel biosensors that
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to form supramolecular ion channels. Furthermore, their
structural flexibility allowed for dynamic regulations of their
ion transport properties.46,47,60 Based on the molecular designs
established in our group and previously reported synthetic
channels that can respond to a single external stimulus, dimeric
multiblock amphiphile VF and its monomeric analogue IF were
newly designed (Figure 1a). An asymmetric and a flexible
groups, which are connected to aromatic units, are expected to
interact with ligand molecules such as aromatic amines.47 In
addition, VF and IF consist of oligo(phenylene-ethynylene)
moieties as hydrophobic units and oligoethylene glycol (OEG)
chains as hydrophilic units (Figure 1a). OEG chains with 8 or
12 repeating units were selected so that these multiblock
amphiphiles would effectively span across the lipid bilayer
membranes (Figure S85, see Supporting Information).47 The
hydrophobic unit is functionalized with six fluorine atoms that
generate a permanent dipole moment along the molecular axis.
Based on DFT calculation, the dipole moment within the
hydrophobic unit was evaluated to be 4.4 debyes (Figure S83).
The hydrophobic unit of VF and IF was synthesized using a
Sonogashira cross-coupling reaction, and OEG chains were
introduced to the hydrophobic unit by Williamson ether
synthesis. An octaethylene glycol chain bearing a phosphate
ester group was then coupled by a phosphoramidite method
(see Supporting Information). All the newly synthesized
1
compounds were unambiguously characterized by H, 13C,
19F, and 31P NMR spectroscopy and high-resolution ESI-TOF
mass spectrometry (Figures S1−S82), and the purity of VF and
IF was further confirmed by elemental analysis (see Supporting
Information).
To investigate whether VF and IF can be incorporated into
the lipid bilayer membranes, microscopic observations of giant
unilamellar vesicles (GUVs) that incorporated VF or IF were
carried out (Figures S86 and S87).62,63 GUVs were prepared
from 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and
VF or IF was then added to the dispersion of GUVs. The phase-
contrast microscopy visualized the formation of micrometer-
sized GUVs (Figures S86a and S87a). Subsequently,
fluorescence microscopic observation of the GUVs was
performed (λex = 330−385 nm; λobsd > 420 nm), and
emissions along the vesicular edges were clearly observed
(Figures S86b and S87b). Because the hydrophobic unit within
VF and IF is emissive upon excitation by UV light (Figure S88),
these results indicate that VF and IF were successfully
incorporated into the lipid bilayer membranes.
To further study the location of VF and IF within the lipid
bilayer membranes, fluorescence depth quenching experiments
were carried out using spin-labeled phospholipids. Because the
quenching of emission depends on the distance between spin
probes and target molecules, the location of VF and IF can be
estimated by varying the position of spin probes within the
membranes.64 For this purpose, three spin-labeled phospho-
lipids, 1-palmitoyl-2-stearoyl-(X-doxyl)-sn-glycero-3-phospho-
choline (X = 5:5-doxyl PC, X = 12:12-doxyl PC, and X =
16:16-doxyl PC), which bear spin probes at different positions
within the alkyl tail, were used. After the preparation of large
unilamellar vesicles (LUVs) formed by DOPC and these spin-
labeled phospholipids, VF or IF was added to the preformed
LUVs. As a result, LUVs that incorporated either VF or IF
showed enhanced degree of quenching for 12- and 16-doxyl
PC-containing LUVs in comparison with 5-doxyl PC-
containing LUVs (Figure S88). Therefore, the hydrophobic
unit within VF and IF is not located at the membrane surface
but rather in the middle of the lipid bilayers, vertical to the
membrane axis (Figure 1b).47,59,60,64
Figure 1. (a) Molecular structures of VF and IF. (b) Schematic
illustrations of VF and IF being incorporated into the lipid bilayer
membranes with phosphate ester groups (light green) facing an
extravesicular medium. VF adopts a folded conformation within the
membranes.
multipass transmembrane structure of VF is reminiscent of the
structural features of TRP channel family members.61 We
therefore anticipated that the development of such multiblock
amphiphiles will give rise to anisotropic dual-stimuli-
responsiveness and bring about important progress in this field.
Next, the orientation of VF and IF to the membrane axis was
investigated to assess whether it would be biased to one side of
the lipid bilayer membranes. As mentioned above, such a
controlled orientation of ion channels across biological
membranes is crucial for their anisotropic dual-stimuli-
RESULTS AND DISCUSSION
■
VF and IF were designed to incorporate phosphate ester
groups, which are important for controlling the molecular
orientations across the membranes. Moreover, phosphate ester
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responsiveness.12,19 Following a previously established method
in our group using zeta potential measurements,47 approx-
imately 93% of IF was evaluated to be oriented with its
phosphate ester group facing the extravesicular medium
(Figures 1b and S89). In addition, 92% of phosphate ester
groups within VF were evaluated to be oriented to the same
direction (Figure S90), thus indicating a folded conformation
of VF within the membranes as reported in our previous
research (Figure 1b).46,47 As mentioned above, VF or IF was
added to the preformed LUVs. It seems the highly polar
phosphate ester groups within the amphiphiles prevented their
permeation across the hydrophobic layer of the membranes,
and therefore, orientation of VF and IF across the lipid bilayer
membranes was highly biased.
With the highly regulated orientation of VF and IF in hand,
their ion transport properties with transmembrane potential
were investigated using DOPC black lipid membranes (BLMs)
under applied voltages. The BLM was formed horizontally at
the orifice and sandwiched with cis (upper) and trans (lower)
chambers (Figure S84, see Supporting Information). First, the
BLM without VF or IF was confirmed not to allow any ions to
permeate across the membranes under the voltage range
(−120 to +120 mV) tested in this study (Figure S91). Then,
VF or IF was added from the cis (upper) chamber so that VF
and IF would be oriented with their phosphate ester groups
facing the cis (upper) chamber (Figure S84). When positive
voltage at +60 mV was applied, VF-containing BLM did not
show any current flows (Figure 2a). Based on the controlled
orientation of VF, the applied electric field vector under these
conditions should be parallel to the dipole moment of VF.
Quite intriguingly, upon application of negative voltage at −60
mV, with an electric field vector antiparallel to the dipole
moment of VF, the current flowed extensively with a maximum
value of −2.1 pA (Figure 2b). These results indicate that when
the applied electric field vector is antiparallel to the dipole
moment of VF, its transmembrane ion transport property
emerges. Then, the average currents over the total recording
Figure 2. (a,b) Current recordings of VF-containing BLMs ([DOPC]
= 106 μM, [VF] = 5 nM) in HEPES buffer ([HEPES] = 20 mM,
[NaCl] = 50 mM, pH 7.5) at the applied voltages of (a) +60 mV and
(b) −60 mV at 20 °C. VF was added from the cis (upper) chamber.
(c,d) Plots of average currents over the total recording time (I) as a
̅
function of applied voltage (V). VF was added from (c) the cis
(upper) chamber and (d) the trans (lower) chamber, respectively.
time under different voltages (I−V plot) were plotted (see
̅
caused by supramolecular ion channels formed by VF (Figure
S96).67,68 Then, based on the current recording profile of VF-
containing BLM (Figure 2b), a histogram of the current
intensity was created, and the most frequent current was
observed at −0.49 pA (Figure S97). Using the Hille
equation,65,69 the pore diameter of the supramolecular ion
channel was thus evaluated to be 0.26 nm at the applied
voltage of −60 mV (see Supporting Information). Considering
the Hill coefficient and the pore diameter, a single supra-
molecular ion channel is likely formed by the assembly of three
molecules of VF.47,51,59,60,70 The observed transient current in
Figure 2b also implies the highly dynamic nature of the
supramolecular ion channel.28 Then, microscopic emission
spectroscopy of BLM that incorporated VF or IF was
performed under applied voltages (Figure 3a−c). Our previous
research demonstrated that such oligo(phenylene-ethynylene)
chromophores show red-shifted emissions when they are
stacked in a face-to-face manner within the lipid bilayer
membranes.62,63 Without voltage (0 mV), VF-containing BLM
showed an emission maximum at 441 nm (Figure 3a, blue
curve). Then, negative voltages with an electric field vector
antiparallel to the dipole moment of VF were applied, and an
abrupt red shift at −40 mV with an emission maximum at 452
nm was observed (Figure 3a,c). In a clear contrast, when
positive voltages with an electric field vector parallel to the
dipole moment of VF were applied, the emission maximum did
Supporting Information), and a nonlinear correlation was
observed (Figure 2c). This nonlinear profile clearly demon-
strated that VF transports ions in response to the polarity and
amplitude of the applied voltage.65 As expected, when VF was
introduced from the opposite side of the BLM, the profile
showed the similar correlation but with an opposite sign
(Figure 2d). It is noteworthy that the observed threshold
voltage of ion transport by VF (ca. 40 mV) is similar to that of
voltage-gated potassium channels found in mammalian
neurons (30 mV).66 Interestingly, the BLM that incorporated
IF showed only slight current flows under positive and negative
voltages at even 120 mV (Figure S92). The ion transport
assay using 8-hydroxypyrene-1,3,6-trisulfonate (HPTS), a pH-
sensitive fluorescent dye, also revealed that IF does not
transport ions across the membranes (Figure S93). These
results indicate that the dimeric structure of VF is essential for
the voltage-responsive ion transport.
To assess the mechanism of voltage-responsive ion transport
by VF, HPTS assays using variable concentrations of VF were
conducted (Figures S94 and S95). Hill analysis on VF verified
the Hill coefficient of n = 2.7 (R2 = 0.99), indicating that VF
self-assembles to form supramolecular ion channels (Figure
S94). The molecular diffusion assay using 5-carboxy-
fluorescein (5-CF) ensured that the observed ion transport
was not due to the nonspecific disruption of the membranes
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Figure 3. (a,b) Microscopic emission spectra of VF-containing BLMs ([DOPC] = 106 μM, [VF] = 1 μM) in HEPES buffer ([HEPES] = 20 mM,
[NaCl] = 50 mM, pH 7.5) at applied (a) negative and (b) positive voltages (0 (blue), 20 (pale blue), 40 (orange), 60 mV (red)) at 20 °C (λex
= 320−353 nm). VF was added from the cis (upper) chamber. (c) Plot of corresponding emission peak tops as a function of applied voltage. The
peak top values were obtained from moving average curves of emission spectra. (d,e) Schematic illustrations of supramolecular ion channels formed
by VF (d) without and (e) with an applied negative voltage. Black arrows represent vectors of dipole moments of VF.
Figure 4. (a−c) Current recordings of VF-containing BLMs ([DOPC] = 106 μM, [VF] = 5 nM) in HEPES buffer ([HEPES] = 20 mM, [NaCl] =
50 mM, pH 7.5) at the applied voltage of −60 mV (a) before and (b) after the addition of (R)-propranolol ([(R)-propranolol] = 200 nM) from the
cis (upper) chamber, (c) followed by the addition of β-cyclodextrin ([β-cyclodextrin] = 1 mM) from the cis (upper) chamber at 20 °C. VF was
added from the cis (upper) chamber. Schematic illustrations of the ligand response of VF are shown on the right side of the corresponding current
recordings.
not show any obvious shifts (Figure 3b,c). These spectral
changes in response to the change in transmembrane potential
showed a good agreement with the threshold voltage observed
in a I−V plot (Figure 2c). These results demonstrated that the
̅
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stacking manner of the chromophores of VF and its ion
transport property are strongly correlated with the polarity and
amplitude of the applied voltage. In particular, when an applied
electric field vector is antiparallel to the dipole moment of VF,
face-to-face stacking of its chromophore is enhanced, and the
ion transport property emerges. Interestingly, IF-containing
BLMs hardly showed clear changes in emissions under the
same voltage range (Figure S98). This result indicates that an
intramolecular face-to-face stacking of the chromophores of VF
is crucial for the observed ion transport. Because an
intermolecular aromatic interaction is generally known to be
weaker than that of an intramolecular aromatic interaction,71,72
IF did not form supramolecular ion channels.
the BLM from the cis (upper) chamber, where the phosphate
ester groups of VF and (R)-propranolol are located at the same
side of the BLM, a current flow was completely inhibited
(Figure 4b). In sharp contrast, when (R)-propranolol was
added to the BLM from the trans (lower) chamber, the current
flow was not affected to any significant extent (Figure S102).
I−V plots under different voltages and HPTS assays further
̅
confirmed this trend (Figures S103 and S104). These results
clearly demonstrated that the highly regulated orientation of
VF also allowed for an anisotropic response to the ligand
molecule. To the best of our knowledge, VF is the first
synthetic ion channel that enabled an anisotropic dual-stimuli-
responsiveness. Our previous research demonstrated that (R)-
propranolol interacts with phosphate ester groups and
aromatic units via electrostatic and π−π interactions and
localizes inside the channel pore to block the ion transport.47
The same mechanism is likely responsible for the observed
inhibition of ion transport in this study (Figure 4b).
Considering the fact that a flip-flop of phosphate-containing
amphiphilic molecules hardly occurs within the lipid bilayer
membranes,73 it should be reasonable to hypothesize that the
reason for the voltage-responsive ion transport by VF is mainly
due to its electronic polarization rather than its orientation
polarization. As mentioned above, VF adopts a folded
conformation within the lipid bilayer membranes, whose
dipole moments of two hydrophobic units are positioned in
parallel to each other. Therefore, without an application of
voltage, hydrophobic units of VF repel each other so that VF
would not form functional transmembrane ion channels
(Figure 3d). However, when a voltage with an electric field
vector antiparallel to the permanent dipole moment of VF is
applied, displacement of electron distribution within VF should
occur.74 It then creates an induced dipole moment within VF,
whose vector is opposite to the permanent dipole moment of
VF, thereby weakening the net dipole moment of VF.75,76 As a
result, the dipole−dipole repulsion between two hydrophobic
units of VF is weakened so that their face-to-face stacking is
reinforced, thereby enabling the formation of supramolecular
ion channels that can efficiently transport ions across the
membranes (Figures 3e). On the other hand, a voltage with an
applied electric field vector parallel to the permanent dipole
moment of VF does not weaken the net dipole moment of VF,
and therefore, the ion transport property would not emerge.
Furthermore, the ligand-binding property of VF was
investigated in order to assess the dual-stimuli-responsiveness
of the ion channel. For this purpose, (R)-propranolol, an
antiarrhythmic agent that can block voltage-gated sodium
channels, was selected.77−80 It is also noteworthy that the use
of (R)-propranolol is advantageous for current recordings,
because it does not deteriorate the membrane stability, while
its enantiomer does (Figure S99). 1H NMR spectroscopy
revealed upfield shifts of aromatic and aliphatic protons near
the phosphate ester groups of VF upon the addition of (R)-
propranolol (Figure S100). These spectral changes indicate
that (R)-propranolol binds to VF at its connection part of
phosphate esters and hydrophobic units.47 In addition, Job’s
plot revealed that (R)-propranolol and VF form a 1:1 complex,
with an association constant of Kassoc = 9.0 × 103 M−1 (R2 =
0.99) (Figure S101).81 For current recording measurements,
VF was first added to the DOPC BLM from the cis (upper)
chamber, so that VF would be oriented and its phosphate ester
groups face the cis (upper) chamber (Figure S84). Sub-
sequently, (R)-propranolol was added to the VF-containing
BLM from either the cis (upper) or trans (lower) chamber, and
current flows were recorded under negative voltage at −60 mV.
As expected, the VF-containing BLM showed the evident
current flows before the addition of (R)-propranolol (Figure
4a). Quite intriguingly, after the addition of (R)-propranolol to
Finally, with an expectation on the channel reopening, β-
cyclodextrin was used as a host molecule for (R)-propranolol.
Prior to the experiment, the association constant of (R)-
propranolol and β-cyclodextrin was evaluated to be Kassoc = 3.3
× 102 M−1 (R2 = 0.99) (Figure S105), based on the 1:1
complexation model reported previously.82,83 Then, β-cyclo-
dextrin was added from the cis (upper) chamber so that the
phosphate ester groups of VF, (R)-propranolol, and β-
cyclodextrin would be located at the same side of the BLM.
Strikingly, the inhibited current flow was again enhanced to the
original level upon the addition of β-cyclodextrin (Figure 4c).
Because the BLM with β-cyclodextrin alone does not allow any
ions to permeate across the membranes (Figure S106), the
observed recovery of the current flow clearly indicates that β-
cyclodextrin removed (R)-propranolol from the binding site of
VF and reactivated the ion transport property. Such a reversible
activation−inactivation cycle is known to be one of the
important and ubiquitous features of ion channels in nature,
because it prevents the excessive ion flux across the biological
membranes to maintain homeostasis.84−86
CONCLUSION
■
Inspired by natural transmembrane proteins with anisotropic
dual-stimuli-responsiveness, we developed a multiblock
amphiphile VF in this study. Ion transport assays using
fluorescent dyes in combination with current recordings
revealed the formation of supramolecular ion channels by VF
within the lipid bilayer membranes. The permanent dipole
moments within the fluorinated hydrophobic units of VF
allowed for controls over its ion transport properties by the
polarity and amplitude of the applied voltage. Microscopic
emission spectroscopy further revealed that the voltage-
triggered conformational change of VF was responsible for
the ion transport. Moreover, the ion transport property of VF
could be inhibited by the addition of (R)-propranolol, an
antiarrhythmic agent that blocks voltage-gated sodium
channels in nature. The highly regulated orientation of VF
allowed for anisotropic responses to the transmembrane
potential and a ligand molecule for the first time as a synthetic
ion channel. Furthermore, the blocked channel could be
reopened by an addition of β-cyclodextrin as a host molecule
for (R)-propranolol. Because heterogeneous environments
across biological membranes are universal features seen in
biology, we believe our newly developed synthetic ion channel
with anisotropic dual-stimuli-responsiveness possesses a great
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ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
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■
sı
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AUTHOR INFORMATION
Corresponding Authors
■
Kazushi Kinbara − School of Life Science and Technology,
Tokyo Institute of Technology, Yokohama 226-8501, Japan;
orcid.org/0000-0002-5945-9612; Email: kkinbara@
bio.titech.ac.jp
Kohei Sato − School of Life Science and Technology, Tokyo
Institute of Technology, Yokohama 226-8501, Japan;
orcid.org/0000-0002-8948-8537; Email: koheisato@
bio.titech.ac.jp
Authors
Ryo Sasaki − School of Life Science and Technology, Tokyo
Institute of Technology, Yokohama 226-8501, Japan;
orcid.org/0000-0002-5560-881X
Kazuhito V. Tabata − Department of Applied Chemistry,
School of Engineering, The University of Tokyo, Tokyo 113-
8656, Japan; orcid.org/0000-0002-0463-1374
Hiroyuki Noji − Department of Applied Chemistry, School of
Engineering, The University of Tokyo, Tokyo 113-8656,
Japan; orcid.org/0000-0002-8842-6836
Complete contact information is available at:
https://pubs.acs.org/10.1021/jacs.0c09470
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
■
The authors thank Suzukakedai Materials Analysis Division,
Open Facility, Tokyo Institute of Technology, for NMR
spectroscopy, EI mass spectrometry, ESI-TOF mass spectrom-
etry, MALDI-TOF mass spectrometry, and elemental analysis.
This work was supported by a Grant-in-Aid for Scientific
Research on Innovative Areas “Molecular Engine (No. 8006)”
(18H05418 and 18H05419 to K.K.), a Grant-in-Aid for
Scientific Research B (19H02831 to K.K.), a Grant-in-Aid for
Research Activity start-up (18H05971 to K.S.), and a Grant-in-
Aid for early-Career Scientists (19H15534 to K.S.). K.S. thanks
the Tokyo Institute of Technology for the Yoshinori Ohsumi
Fund for Fundamental Research and the ESPEC Foundation
for Global Environment Research and Technology (Charitable
Trust). We also thank Prof. Hirofumi Shimizu for helpful
discussions and Prof. Akira Kato for generous assistance in
electrophysiological studies.
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