Article

Metal-Organic Cuboctahedra for Synthetic Ion
Channels with Multiple Conductance States
Ryuji Kawano, Nao Horike, Yuh
Hijikata, ..., Susumu Kitagawa,
Shoji Takeuchi, Shuhei Furukawa
rjkawano@cc.tuat.ac.jp (R.K.)
takeuchi@iis.t-tokyo.ac.jp (S.T.)
shuhei.furukawa@icems.kyoto-u.ac.jp(S.F.)HIGHLIGHTS
Metal-organic cuboctahedra(MOPs) were used for creating
artificial channels in bilayers
These artificial channels have two
distinct conductance states
Changes in chemical functionalityof the MOPs switched between
the two states
A high-throughput lipid bilayer
system unveiled the ion-transportmechanism

Kawano and colleagues show two distinct ion conductance states by embedding asingle metal-organic porous molecule with Archimedean cuboctahedrongeometry into a planar lipid bilayer. The triangular and square apertures in the
cuboctahedron work independently as ion-transporting pathways. By changing
the aliphatic chain length introduced on the periphery of the cuboctahedron, theauthors identified that the rotational dynamics of the cuboctahedron regulate theopen pore time of each conductance state through distinct apertures and theswitching between them.

Kawano et al., Chem 2, 393–403
March 9, 2017 ª 2017 Elsevier Inc.
http://dx.doi.org/10.1016/j.chempr.2017.02.002

Article

Metal-Organic Cuboctahedra for Synthetic Ion
Channels with Multiple Conductance States
Ryuji Kawano,1,2,* Nao Horike,3 Yuh Hijikata,4 Mio Kondo,3,5 Arnau Carné-Sánchez,3 Patrick Larpent,3
Shuya Ikemura,3 Toshihisa Osaki,2 Koki Kamiya,2 Susumu Kitagawa,3 Shoji Takeuchi,2,6,*
and Shuhei Furukawa3,7,*

SUMMARY

The Bigger Picture

Emulation of biological ion channels by synthetic molecules is not only a challenge for chemists in designing highly complex (supra)molecules but also key
to developing a new tool for exploring subcellular electrochemical activity.
Despite efforts to create a single pore in a lipid bilayer by synthetic channels,a general synthetic strategy for realizing more complex two-pore channels
has yet to be proposed. Here, we demonstrate two distinct ion conductancestates by embedding a single metal-organic porous molecule with the geometryof an Archimedean cuboctahedron into an artificially reconstructed lipid bilayer
membrane in which triangular and square apertures in the cuboctahedron work
independently as ion-transporting pathways. By changing the aliphatic chain
length introduced on the periphery of the cuboctahedron, we found that therotational dynamics of the cuboctahedron regulate the open pore time ofeach conductance state through distinct apertures and the switching between
them.

Trans-membrane ion channels
with multiple conductance states
are studied intensively because of
their significance in biologicalevents ranging from Ebola virus
entry into host cells to neuronal
dysfunction. Emulation of thesefunctions by synthetic molecules
gives an opportunity to cure ionchannel-related diseases(channelopathies). Because of thevoids inherently present in porous
molecules, they are prime
candidates for designing
synthetic ion channels. Here, we
propose a strategy forconstructing synthetic channels
with multiple conductance statesby using metal-organic
cuboctahedra with distinct squareand triangular apertures forindependent ion pathways. These
inorganic ion channels embeddedin planar lipid bilayers
demonstrate two distinct
conductance states by switching
apertures. This work opens new
avenues for creating extensive
synthetic ion channels withcomplicated functions. Further
developments along these lines
would realize the synthesis ofstimuli-responsive synthetic
channels and thus potential drugs
for channelopathies.

INTRODUCTION
Ion channels regulate membrane potentials and cellular excitability by transporting
ions. Such subcellular electrochemical activity is crucial for intercellular communications and electrical signaling in the nervous and muscle systems.1,2 In parallel with
the investigation into understanding the relationship between structural dynamics
and the transport properties of biological ion channels, the creation of artificial
ion channels has been attempted with synthetic molecules or peptides to emulate
their structure or function.3–5 Chemists have attempted the synthesis of ion channels
on the basis of designer organic molecules and their supramolecular assemblies tocontrol cellular signaling by synthetic molecules.6,7 More recently, other functional
materials such as DNA origami8,9 and carbon nanotubes10 have been introduced
as potential candidates for artificial ion channels. Studies on synthetic channels
have focused on approaches that effectively generate channels or holes in the lipid
bilayer and implement ion selectivity for Na+, K+, Cl–, etc.3,4,11
Most ion channels consist of a single pore; however, a unique family of ion channels
with multiple ion pathways has been identified, for instance, two-pore domain channels12 or two-pore channels (TPCs)13 as a superfamily of voltage-gated ion channels.
The former are exclusively present in nerve or muscle cells, and the latter are
expressed ubiquitously in animals and plants, and both control the resting membrane potential by using dual channels. Their ubiquitous presence and potential
implications in diseases have led to increased research on these channels inrecent decades.14,15 Because of their characteristic multiple conductance states,

Chem 2, 393–403, March 9, 2017 ª 2017 Elsevier Inc.

393

Figure 1. Schematic Illustration of Channels with Multiple Conductance States(A) Biological two-pore channels possessing two P-domains and eight transmembrane segments(2P/8TM), for instance, TOK1, the first example of a two-P-domain K+ channel subunit.
(B) Synthetic two-pore channels based on Archimedean polyhedral molecules. In particular, themolecule with cuboctahedral morphology is displayed; the molecular dynamics in a lipid bilayer
would stochastically regulate the ion pathway through either a triangular or square aperture.

dual-conductance channels represent an interesting area of research for chemists;
however, a general strategy for incorporating two pores into a single synthetic molecule is still required.
Here, we show the design and synthesis of switchable two-pore channels based on
porous molecules with the shape of Archimedean polyhedra, in which metal clusters(vertices) are linked by organic ligands (edges): so-called metal-organic polyhedra(MOPs).16 This geometry allows porous molecules to possess more than two
different polygonal apertures and one internal cavity. In particular, we chose the cuboctahedron geometry composed of six square apertures and eight triangular apertures17,18 and embedded the metal-organic cuboctahedron molecule into a planar
lipid bilayer. For the generation of two distinct ion conductance states, ions needto pass through the internal cavity via either the square or triangular aperture
exposed to the aqueous phase (Figure 1). We functionalized the periphery of the cuboctahedron molecules with aliphatic chains to allow for a suitable interaction withthe lipid bilayers and changed the chain length to modulate its interaction with thelipids so that it can alter the molecular dynamics, which is the key to switching between the two conductance states. To distinguish between the two distinct ion
conductance states, we implemented ion conductance measurements at thesingle-molecule level by using planar lipid bilayers. We further integrated these394

Chem 2, 393–403, March 9, 2017

1Department of Biotechnology and Life Science,

Tokyo University of Agriculture and Technology,
2-24-16 Naka-cho Koganei-shi, Tokyo 184-8588,
Japan
2Artificial Cell Membrane Systems Group,

Kanagawa Academy of Science and Technology(KAST), 3-2-1 Sakado, Takatsu-ku, Kawasaki
213-0012, Japan
3Institute for Integrated Cell-Material Sciences(WPI-iCeMS), Kyoto University, Yoshida,
Sakyo-ku, Kyoto 606-8501, Japan
4Institute of Transformative Bio-Molecules(WPI-ITbM) and Department of Chemistry,
Graduate School of Science, Nagoya University,
Furo-cho, Chikusa-ku, Nagoya 464-8602, Japan
5Department of Life and Coordination-Complex

Molecular Science, Institute for Molecular
Science, Higashiyama 5-1, Myodaiji, Okazaki444-8787, Japan
6Institute of Industrial Science, University ofTokyo, 4-6-1 Komaba, Meguro-ku, Tokyo
153-8505, Japan
7Lead Contact*Correspondence: rjkawano@cc.tuat.ac.jp (R.K.),
takeuchi@iis.t-tokyo.ac.jp (S.T.),
shuhei.furukawa@icems.kyoto-u.ac.jp (S.F.)
http://dx.doi.org/10.1016/j.chempr.2017.02.002

into a high-throughput microarray device19–21 to analyze a massive dataset ofchannel currents to understand the relationship between the dynamics and ion
transportation.

RESULTS AND DISCUSSION
Design and Synthesis of Metal-Organic Cuboctahedra as Two-Pore Synthetic
Channels
The design principle of synthetic ion channels has focused on the control of molecular assemblages in lipid bilayers.22 Indeed, many molecular systems have beendeveloped since the early 1980s for the construction of ion pathways in hydrophobic
environments.6 Recently, biological or synthetic materials with permanent porosity,
such as DNA-based architectures or carbon nanotubes, were directly inserted into
lipids for implementing ion transportation therein.23 The key to success is to design
stable porous structures inside materials and to hybridize with lipids to generate theentrance and exit apertures in the aqueous phase.
In this context, porous molecules based on MOPs are among the best candidates
because of their intrinsic porosity as a result of their cage-type structures andchemical versatility for functionalizing the outer periphery of the cages.24,25 Towardrealizing TPC-like behavior from a synthetic channel, we focused on cuboctahedron
topology with two distinct triangular and square apertures, where the upward triangular aperture is arranged parallel to the downward triangle on the opposite sideof the cuboctahedron and the square aperture is arranged parallel to anothersquare on the opposite side. This topology allows for only one of the two windows
to be exposed to the aqueous phase when embedded in lipid bilayers, thereby
generating two distinct ion pathways through either a triangular or square
aperture. To switch these two pathways, it is necessary to modulate the rotational
dynamics of the MOP in the lipid bilayer; otherwise, only single conductancethrough either the triangular or the square pathway would appear from the MOP
frozen in the lipid.
Among MOPs, we selected [Rh2(bdc)2]12, in which the dirhodium paddlewheel complexes (vertices) are connected by 1,3-benzenedicarbosylate (bdc) (edges) to form
the cuboctahedron geometry.26 This rhodium-based MOP has a rigid molecular
backbone (as a result of the metal-metal single bond between rhodium ions) andhigh thermal and chemical stability.27 In order to embed this molecule into lipid
bilayers, we functionalized the periphery of the cuboctahedron with alkoxy chains,
as previously reported by Jung et al. for the integration of copper-based MOPs
into lipids.28 To control the interaction of MOPs with lipids and their dynamics, we
synthesized two MOPs with different alkoxy chains, [Rh2(bdc-C12)2]12 (C12RhMOP)
and [Rh2(bdc-C14)2]12 (C14RhMOP), (bdc-C12 = 5-dodecyloxy-1,3-benzendicarboxylate, bdc-C14 = 5-tetradecyloxy-1,3-benzenedicarboxylate). Although the alkoxyside chains of a MOP ensure its solubility in lipid bilayers via the hydrophobic
interaction with lipid alkyl chains, we anticipated the influence of chain length on
the dynamics of the MOP molecule.
The syntheses were carried out by microwave heating of a solution mixture of dirhodium acetate with Na2(bdc-C12) or Na2(bdc-C14) in N,N-dimethylacetamide (DMA)
at 200 C for 5 min, followed by recrystallization from toluene/acetonitrile. We
unambiguously identified the compounds by nuclear magnetic resonance, infrared
spectroscopy, mass spectrometry, elemental analysis, and dynamic light-scattering
measurements (Figures S1–S7).

Chem 2, 393–403, March 9, 2017395

Figure 2. The C14RhMOP Embedded in a Lipid Bilayer Works as a Synthetic Ion Channel(A) Snapshot of molecular dynamics simulation of C 14 RhMOP embedded in a lipid bilayer andschematic diagram of a single-channel measurement by a voltage clamp method. The molecular
dynamics simulation provided insight into the molecular state of C 14 RhMOP; despite thehydrophilic environment created by the lipid bilayer, water molecules were found around andinside the MOP core structure. This is most likely because water molecules coordinating to the axial
positions of the dirhodium paddlewheel moiety, which is exposed to the outer sphere and innercavity, support the accumulation of other water molecules close to the paddlewheel units. Indeed,
31 water molecules were observed in the internal cavity of the MOP core in the snapshot.
(B) A typical current-time trace of C 14 RhMOP in 1 M KCl PBS buffer at +200 mV.

Measurement of Multiple Conductance States from MOP Synthetic Channels
Ion transport through the C14RhMOP channel was electrophysiologically measured
with a planar lipid bilayer,29 into which the MOP molecule was introduced by the
droplet contact method (see Supplemental Information).30 Initially, the current
signal of the open channel states (shown as g1 and g2 in Figure 2) of C14RhMOP appeared infrequently when a single-lipid-bilayer device was used. The success rate of
the channel open-close observation fell within the range of 10%–30% for
C14RhMOP. Typical current and time traces showing infrequent open channel activity from the simultaneous measurement in 4/16ch are presented in Figure S8. To
clarify this infrequent channel activity, we carried out molecular dynamics simulations of the embedded structure of C14RhMOP in a lipid bilayer, as shown in Figure 2A and the Supplemental Information. The core moiety of the cuboctahedronis smaller than the thickness of the lipid bilayer and is wrapped with aliphatic alkoxy
chains. This size incompatibility leads to the long closed state in which none of theapertures of MOP are exposed to the aqueous phase. We integrated planar lipid bilayers into a microarray device to obtain 16 channel recordings independently at thesame time. This permitted us to acquire a massive dataset of current signals bychanging several parameters such as concentration, ion types, voltage, etc.21
Typical channel currents with the open-close transition from a single C14RhMOP
molecule in 1 M KCl are presented in Figure 2B; this current was produced by thetransfer of K+ and Cl ions through the C14RhMOP cavity at the single-molecule396

Chem 2, 393–403, March 9, 2017

Figure 3. Single C14RhMOP Channel Conductance and Ion Permeability(A) Current-voltage curve with linear curve fit of a single C 14 RhMOP in symmetrical 1 M KCl PBS
buffer.
(B) KCl-concentration dependence of a single C 14 RhMOP conductance under the applied voltageof 150 mV. The curve was fit to the Michaelis-Menten equation.
Error bars represent the standard deviation.

level. Two different levels of channel current (g1 and g2) were clearly observed in themeasurement. To estimate the aperture diameters from the conductance, we carriedout the following experiments. Initially, the linearity of the conductance was verified
against the applied voltage and the KCl concentration because the precise conductance must be determined with the regions proportional to the applied voltage (Figure 3A) and salt concentration (Figure 3B). The I-V curves for both large (g1) and small(g2) conductances clearly demonstrated ohmic behavior and the absence of rectification behavior in this voltage range. The dependence of the conductance on KCl
concentration under an applied voltage of 150 mV is shown in Figure 3B. By fitting
the Michaelis-Menten equation (see Supplemental Information), we estimated thehalf-maximal effective concentrations of the ion transportation (EC50) to be 0.41 M(g1) and 0.76 M (g2) with maximal single-channel conductances of gmax = 51 pS(g1) and 40 pS (g2). Next, Hille’s equation was applied at KCl concentrations below
EC50. The calculated aperture diameters of C14RhMOP were dHille = 0.58 nm (g1) and

Chem 2, 393–403, March 9, 2017397

Table 1. Conductance, Hille Diameters, and Ion Permeability from a Single-Channel
Measurement of C14RhMOP by a Planar Lipid Bilayer Methodgmax (pS)dHille (nm)dx-ray (nm)PCl-/PK+

PCa2+/PK+

Square pore

51 G 9

0.58

0.66

0.08

0.38

Triangular pore

40 G 16

0.39

0.45

0.13

0.13

0.39 nm (g2), which are consistent with the size of the square (0.66 nm) and triangular(0.45 nm) apertures of the cuboctahedron moiety as estimated from the results ofsingle-crystal X-ray diffraction experiments for the non-alkoxylated compound[Rh2(bdc)2]12.26 This result suggests that the two conductances (g1 or g2) are
ascribed to the ion transportation via the larger square and smaller triangularapertures of the cuboctahedron, respectively. These conductances and estimatedaperture diameters are listed in Table 1.
Switching of Two Conductance States Regulated by Alkoxy Chain Lengths
When assuming the switching of two distinct conductance states (g1 or g2) generated from square and triangular apertures, the cuboctahedron should rotate inside
a lipid bilayer to expose each aperture to the aqueous phase. Such rotational
dynamics can be regulated by the molecular interaction between the cuboctahedron and the lipid. Thus, we designed another cuboctahedron with shorter aliphatic
chains (C12RhMOP), which is expected to demonstrate higher mobility than
C14RhMOP because of the smaller interaction with lipid molecules. Figures 4A
and 4B show the current-time trace and histograms for the channel opening timesof C12RhMOP and C14RhMOP. Although the open probabilities of C12RhMOP and
C14RhMOP were similar to each other (33.8% and 34.4%, respectively), the channel
opening time of C12RhMOP was apparently shorter than that of C14RhMOP. The
time constants of C12RhMOP and C14RhMOP were 0.08 s and 0.63 s, respectively,
and the mean opening times (Figure 4C) of C12RhMOP and C14RhMOP were0.31 s and 0.85 s, respectively. The shorter time constants and mean opening
time of C12RhMOP than those of C14RhMOP mean that the shorter chain length of
the MOP induces faster rotational dynamics in the lipid matrix. The ratio of theopen channel probabilities between the large and small conductances (Pg1 orPg2) of C14RhMOP is estimated to be Pg1/Pg2 = 0.63 (Pg1 = 38.6% and Pg2 =61.4%; n = 210). This value almost corresponds to the calculated ratio of square/triangle apertures (6/8 = 0.75) in the cuboctahedron geometry. If open channel eventsare dominated by another mechanism, for example, alkoxy chain movement thatcovers the apertures, the ratio of Pg1/Pg2 would be inconsistent with the geometry.
Consequently, the stochastic events of channel opening most likely rely on a random
rotation of the cuboctahedron molecule. The subsecond timescale of the opening
time observed for these synthetic channels is comparable with that of gramicidin
A, which forms a trans-membrane channel by vertically aligning two monomers
present in opposing leaflets of the bilayer; its opening time is of the order of a second.31 Note that the ion channel conductance states (g1 and g2) of C12RhMOP
almost correspond with those of C14RhMOP under the same condition (Table S1),
indicating that the conductance states only attributed to the MOP geometry andthe side alkoxy chains did not affect the ion-transport behavior.
Characterization of Two-Pore MOP Channels
In order to elucidate the mechanism of ion transport through the MOP synthetic
channel, we further studied the ion permeability of C14RhMOP—which exhibits alonger opening time than C12RhMOP—by using monovalent cations (Li+, Na+, K+,
Rb+) and a divalent cation (Ca2+). The permeability ratio (PX/PK+) was estimated by398

Chem 2, 393–403, March 9, 2017

Figure 4. Effect of the Alkoxy Chain Length on the Ion Channel Property(A and B) Comparison of the mean open time of both large and small open channel responses fromC 12 RhMOP (A) and C 14 RhMOP (B) in 1 M KCl PBS buffer at +150 mV.
(C) Significance of the mean open time was assessed by a Mann-Whitney U test. Error bars
represent the standard error of mean.

the Goldman-Hodgkin-Katz equation; the permeability of K+ was defined as thestandard (PK+/PK+ = 1).1 The PX/PK+ values can be calculated from the reversal
potential at which no net current flows in the I-V curves (Figures 5A and S9 for cation
and anion selectivity). First, we examined the anion selectivity. The permeability
ratios of Cl (PCl-/PK+) for the square and triangular apertures estimated from g1
and g2 were calculated to be 0.08 and 0.13, respectively. The low permeability of
Cl suggests strong cation selectivity and similar permeabilities for the square andtriangular apertures.
The reversal potentials also showed monovalent cation (M+) selectivity (PM+/PK+)(Figure 5B). The PM+/PK+ values increased in the following order, Li+ > Na+ > K+ >Rb+ z Cs+, which corresponds to the Eisenman XI sequence.28 This sequencesuggests that the selectivity relies on the size of the completely dehydrated ion,
which is often observed for biological channels but is quite unusual for synthetic
channels.32 Because both square and triangular apertures consisted of hydrophobic
moieties—long alkoxy chains and phenyl rings—the dehydration of cations would
be favored because the hydrophobic barrier would be compensated by thecation-p interaction with one of the phenyl rings of the apertures.
One of the characteristic features of MOPs is to replace metal ions in paddlewheel
structures. Rather than a dirhodium paddlewheel, most MOPs are synthesizedwith copper paddlewheel complexes. Indeed, the monovalent cation permeabilityof the analogous copper structure of [Cu2(bdc-C12)2]12 was previously studied; asimilar tendency following the Eisenman XI sequence was observed.28 Here, we
tested the divalent cation (Ca2+) permeability of both C14RhMOP and C14CuMOP

Chem 2, 393–403, March 9, 2017399

Figure 5. Single C14RhMOP Channel Conductance and Ion Permeability(A) Single-channel current-voltage curve and linear curve fit of a single C14 RhMOP in asymmetric 2
and 0.25 M KCl (cis and trans, respectively). The reversal potential is the potential at zero current.
Error bars represent the standard deviation.
(B) Permeability ratios for monovalent cations as a function of their ion radius.

([Cu2(bdc-C14)2]12) as shown in Figure 6A. Whereas rectangular currents wereobserved for C14RhMOP, small spike-like currents—suggesting low Ca2+ permeability—were observed for C14CuMOP. This difference is attributed to the stabilityof the MOP structure.25,26 The dirhodium paddlewheel structure is highly chemicallystable as a result of the inertness of the coordination bonds between rhodium ionsand equatorial carboxylates (the stability test of RhMOP in water is summarized in
Figure S10 and Table S2). The copper-carboxylate bonds in the copper paddlewheel
structure are more labile; for instance, the copper acetate complex seems toreact with calcium ions to form a calcium copper complex.33 Therefore, the low
Ca2+ permeability would be more likely to be related to the decomposition of the
C14CuMOP structure caused by a reaction with calcium ions. Note that similar deactivation was observed for the stable C14RhMOP compound when introduced to astrong chelate such as EDTA, which definitely destroyed the C14RhMOP structure.
Similar to the transportation behavior of C14RhMOP toward monovalent cations,two distinct levels of current signals from the square and triangular apertures

400

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Figure 6. Divalent Cation Behavior in C14RhMOP and C14CuMOP in the Lipid Bilayer(A) Typical current-time trace of both C 14 RhMOP and C 14 CuMOP at +200 mV in 1 M CaCl2 .
(B) Current-voltage curve of C 14 RhMOP in asymmetric 1 M CaCl 2 and 1 M KCl (cis and trans,
respectively).

were observed. The permeability ratios of the calcium ion over the potassiumion (PCa2+/PK+) were calculated to be 0.38 and 0.13 for the large and small pores,
respectively, from the reversal potential in the asymmetrical solution (Figure 6B
and Table 1). These values are smaller than those in the monovalent cation
system, even though the ion radius of Ca2+ is almost identical to that of the Na+ion (PNa+/PK+ = 1.02 and 0.98 for the square and triangle apertures, respectively).
This result indicates that the highly charged ion enters the MOP nanocavity createdby 24 Rh2+ ions less effectively because of the electrostatic interaction between the
cation and rhodium metal ions.
Conclusions
We achieved an artificial system generating two conductance states by embedding a
synthetic MOP into lipid bilayers. Rotational dynamics is the key to switching between distinct conductance states to expose either triangular or square apertures
to the aqueous phase. The dynamics were altered by the interaction between the
MOP and lipids and optimized by the alkoxy chain length introduced at the periphery of the MOP. Further, we discovered that changing the metal ion component of
the MOP altered the ion selectivity; whereas the rhodium-based MOP had apermeability for divalent cations (Ca2+), the copper-based MOP transported only

Chem 2, 393–403, March 9, 2017401

monovalent cations. Studies to improve the infrequent open-pore state and theswitching of two conductance states by external stimuli (to promote active control
of cell signaling or even channelopathy therapy34) by synthetic multiple conductance channels are currently underway.

EXPERIMENTAL PROCEDURES
Full experimental procedures and data analysis are provided in the Supplemental
Information.

SUPPLEMENTAL INFORMATION
Supplemental Information includes Supplemental Experimental Procedures, ten figures, and two tables and can be found with this article online at http://dx.doi.org/10.
1016/j.chempr.2017.02.002.

AUTHOR CONTRIBUTIONS
R.K. and S.F. conceived and designed the project. N.H., A.C., P.L., S.I., and M.K.
performed all synthetic and characterization experiments. R.K., K.K., and T.O. conducted and analyzed channel current measurements. Y.H. performed molecular dynamics simulations. S.T. and S.K. supervised the project. R.K and S.F. analyzed thedata and wrote the manuscript. All authors discussed the results and commented
on the manuscript.

ACKNOWLEDGMENTS
We thank Yoshimi Komaki and Yoshimi Nozaki for technical assistance on thechannel current experiments and Dr. Nicolas Louvain for his preliminary synthetic experiments. This work was partially supported by Japanese Grants-in-Aid for Scientific
Research (KAKENHI 24655072, 16H06043, and 15K13771). The Institute for Integrated
Cell-Material Sciences (iCeMS) and Institute of Transformative Bio-Molecules (ITbM) aresupported by the World Premier International Research Center Initiative (WPI) of the Japanese Ministry of Education, Culture, Sports, Science, and Technology (MEXT).
Received: November 1, 2016
Revised: November 22, 2016
Accepted: February 2, 2017
Published: March 9, 2017

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Chem 2, 393–403, March 9, 2017403

Chem, Volume 2

Supplemental Information

Metal-Organic Cuboctahedra for Synthetic Ion
Channels with Multiple Conductance States
Ryuji Kawano, Nao Horike, Yuh Hijikata, Mio Kondo, Arnau Carné-Sánchez, Patrick
Larpent, Shuya Ikemura, Toshihisa Osaki, Koki Kamiya, Susumu Kitagawa, Shoji
Takeuchi, and Shuhei Furukawa

SUPPLEMENTAL EXPERIMENTAL PROCEDURES
Chemicals

The reagents were obtained as follows: LiCl, KCl, NaCl, RbCl, CaCl 2, K2HPO4, KH2PO4, KOH,

MOPS, and EDTA (Wako, Japan); 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC) (Avanti

Polar Lipids); n-decane (Sigma). Buffered electrolyte solutions were prepared from Milli-Q water.

Prior to use, the regents and the buffer were stored at 4 °C.

Synthesis of C14RhMOP and C12RhMOP

A 2 mL microwave reaction vial (Biotage) was charged with Rh2(OAc)4.(MeOH)2 (10 mg, 19.8 μmol,

1 eq), Na2(bdc-C12) (16 mg, 40.5 μmol, 2 eq) or Na2(bdc-C14) (17 mg, 40.5 μmol, 2 eq) and DMA (2mL). The mixture was sonicated for 10 min at 25°C and then subjected to microwave irradiation(Biotage Initiator) for 5 min at 200 °C. This procedure was repeated 5 times for each reaction and theresulting mixtures combined prior to purification. DMA was then removed by rotary evaporation

followed by heating at 120°C under vacuum for 1h under Schlenk line. To the dark resulting residue,

CH2Cl2 (3 mL) was added. The mixture was gravity filtered through paper and the latter was furtherwashed with additional CH2Cl2 (5-10 mL). The green filtrate was collected and evaporated to dryness

by rotary evaporation. The green residue was then washed with EtOH (3 x 5 mL) and dried undervacuum at 25°C overnight affording pure C12RhMOP (18 mg) and C14RhMOP (16 mg) as green

powders. Elemental analysis calcd. for C240H348O66Rh24 C12RhMOP·6(H2O): C, 52.70; H, 6.26,

Found: C, 53.37; H, 6.82. Elemental analysis calcd. for C264H428O82Rh24 C14RhMOP·22(H2O): C,

54.61; H, 6.72, Found: C, 55.04; H, 7.14. Both compounds can be recrystallized from hot acetone orfrom a toluene/acetonitrile mixture. Both solids are soluble in CH 2Cl2, CHCl3, THF, DMF, DMA andDMSO but insoluble in MeOH, EtOH, BuOH, H2O, Et2O, CH3CN, acetone.

Stability test in aqueous solutions

The small amount (ca. a few μg) of compound (C12RhMOP or C14RhMOP) was added into purewater and was stirred for 24 h. After the filtration, the residue was analyzed by IR spectroscopy andthe supernatant was analyzed by was ICP-AES (inductively coupled plasma atomic emission

spectroscopy).

Channel current recordings and data analysis

MOP channel currents were measured using planar bilayer lipid membranes using the droplet

contact method.

S1-S4

MOP dissolved in chloroform were mixed with DPhPC/n-decane (10 mg/mL) as

1 mol% to lipid molecules. Normally, 1 M KCl with, 10 mM PBS, or MOPS buffer was used for theaqueous drops (unless otherwise noted). The currents were monitored using an Axopatch 200B

(Axon Instruments) or a JET-Bilayer (Tecella, CA). The signal was detected through a 1 kHz

low-pass filter at a sampling frequency of 5 kHz (unless otherwise noted) at 23 ± 1 °C. The numberof all experimental trial was at least n>3. The typical channel recording for each experiment took 2hours. The multiple leveled signals were rarely observed, which indicates that two MOP molecules ina same lipid bilayer became simultaneously active. In that case, we ignored this type of data. The

current analysis was performed using pCLAMP ver. 10.6 (Molecular Devices, Sunnyvale) and Igor

Pro 6.2 (Wavemetrics, Oregon). The single-channel analysis is described below. The durations of

the open and closed periods were measured with a single-channel tracing analysis using pCLAMP(average data number: n = 122). Occasional long closed periods (>60 s) were excluded due toextremely low activity. In case of a high amount of noise in the data, a digitized low-pass filter (<500Hz) was used. The results are given as mean ± SD or SEM.

Calculation of channel diameter from conductanceThe channel conductance was estimated from the slope of the I–V curve (n>3 at each voltage). The

dependence of conductance on the salt concentration (n>3 at each concentration) was fitted to theMichaelis–Menten equation.

g = gMAX / (1 + EC50 /[KCl])

where gMAX is the maximum conductance. To apply Hille’s model to calculate the diameter, theconductance at a KCl concentration below EC50 (0.45 M) was required. Therefore, the conductanceat 0.25 M KCl was estimated from the curve fit, multiplied by the “correction factor,” and applied toHille’s equation.

-1

S5

2

g = 4lρ / πd + ρ / dwhere l is the length of the channel (7.0 nm) and ρ is the resistivity of the recording solution (0.348 Ω-1m ) to give diameter (d).

Verification of ion permeability.

+-

The ion permeability ratio between K and Cl (PK+/PCl-) was determined by measuring the reversal

S6

potential (Vr) in the salt gradient solution (2/0.25 M KCl, cis/trans).

PK+/PCl- was obtained using theequation derived from the Goldman–Hodgin–Katz voltage equation as follows.

PK /PCl = [aClcexp(–VrF/RT)–aClt] / [aKc–aKtexp(–VrF/RT)]+–

where aKc and aKt are the activities of K in cis and trans chambers, aClc and aClt are the same for Cl ,F is the Faraday constant, R is the gas constant, and T is the absolute temperature. Monovalent

alkali cation selectivity was similarly determined from Vr obtained under the salt gradient (1M KCl/1M

MCl, cis/trans) using the following equation.

PM /PK = (aK / aM)[exp(–VrF/RT)]+

where aM is the activity of M .

The permeability ratio with a divalent ion, PCa2+/PK+ in this case, is more complicated and can besolved as follows.

S5

PCa2+/PK+ = (aK / 4aCa)[exp(VrF/RT)][exp(VrF/RT)+1]

2+

where aCa is the activity of Ca .

Molecular dynamic simulation of MOP channel in the lipid bilayer

All calculations were performed with Universal Force Field

S7

under periodic boundary condition(PBC) using Forcite module implemented in Material studio 7.0 Package (Accelrys Inc. San Diego,

CA). The lipid bilayer model was constructed from C14RhMOP prepared from RhMOP

S8

and 98DPhPC. The ratio of C14RhMOP and DPhPC corresponds to the experimental condition. The bilayer

model was optimized by molecular mechanics (MM) calculation under PBC with lattice relaxation.

The cell parameters of a unit cell changed from a = 70.0 Å, b = 70.0 Å, c = 140.0 Å, α = 90.0˚, β =90.0˚, and γ= 90.0˚ to a = 65.4 Å, b = 66.1 Å, c = 140.0 Å, α =90.0˚, β = 90.0˚, and γ = 90.0˚ during

the optimization. Two layers of 6912 water molecules, whose density is 1.0 g/ml, were set on the top

and bottom of the bilayer. The final employed model consists of C14RhMOP, 98 DPhPC, and 13824

water molecules in a unit cell, of which the cell parameters are a = 65.4 Å, b = 66.1 Å, c = 140.0 Å, α=90.0˚, β = 90.0˚, and γ = 90.0˚. To observe the surrounding environment of C14RhMOP, weemployed NVT ensemble, where N, V, and T were the number of particles, volume of the systems,

and temperature of the system respectively, at 298K with Nosé-Hoover thermostat for the MDsimulation. We simulated for 1 ns with 2 fs time step after relaxation of the initial geometries

including water molecules by 50 ps MD simulation.

1

Figure S1: H-NMR spectra of C12RhMOP in CD2Cl2 (500 MHz). EtOH and H2O signals may be
attributed to both coordinated (S) and guest solvents.

1

Figure S2: H-NMR spectra of C14RhMOP in CD2Cl2 (500 MHz). EtOH and H2O signals may be
attributed to both coordinated (S) and guest solvents.

Figure S3: DLS measurements showing particle sizes and distribution of C12RhMOP (black) andC14RhMOP (red) in CH2Cl2. Mean sizes: C12RhMOP = 3.42 nm (error = 0.11 nm), C14RhMOP = 3.82
nm (error = 0.75 nm).

Figure S4: FT-IR spectra of C12RhMOP (black) and C14RhMOP (red).

Figure S5: TGA traces of C12RhMOP (black) and C14RhMOP (red).

Figure S6: MALDI-TOF spectrometry of C12RhMOP usingtrans-2-[3-(4-tert-Butylphenyl)-2-methyl-2-propenylidene]malononitrile (DCTB) as a matrix.

Figure S7: MALDI-TOF spectrometry of C14RhMOP usingtrans-2-[3-(4-tert-Butylphenyl)-2-methyl-2-propenylidene]malononitrile (DCTB) as a matrix.

Figure S8: Typical current and time traces of 4/16ch data for C14RhMOP. The open and closetransitions appeared only the enlargement area, suggesting the infrequent open channel events.

+Figure S9: Current and voltage curves of monovalent cation (M ) selectivity (PM/PK+) experiments for
C14RhMOP.

Figure S10: IR spectra of (a) C12RhMOP and (b) C14RhMOP before and after the 24 h treatment inaqueous solutions. No significant change was observed for all the compounds, which indicates theirstability in the solid state.

Table S1: The channel conductance of C12RhMOP and C14RhMOP molecules in the 1.5 M KCl
solution.
C12RhMOP

C14RhMOP

Square pore (g1)

42 pS

42 pS

Triangle pore (g2)

33 pS

31 pS

Table S2: The leaching of metal ion from C12RhMOP and C14RhMOP after the 24 h treatment inwater, detected by ICP-AES.

In water

C12RhMOP

C14RhMOP

0.00±0.16%

0.07±0.12%

Supplemental References

S1.

Kawano R., Tsuji Y., Sato K., Osaki T., Kamiya K., Hirano M., Ide T., Miki N., & Takeuchi S.
(2013) Automated parallel recordings of topologically identified single ion channels. Sci.
Rep. 3:1995.

S2.

Kawano R., Tsuji Y., Kamiya K., Kodama T., Osaki T., Miki N., & Takeuchi S. (2014) A
portable lpid bilayer system for environmental sensing with a transmembrane protein. PLoS
One 9(7):e102427.

S3.

Tsuji Y., Kawano R., Osaki T., Kamiya K., Miki N., & Takeuchi S. (2013) Droplet
split-and-contact method for high-throughput transmembrane electrical recording. Anal.
Chem. 85(22):10913-10919.

S4.

Tsuji Y., Kawano R., Osaki T., Kamiya K., Miki N., & Takeuchi S. (2013) Droplet-based lipid
bilayer system integrated with microfluidic channels for solution exchange. Lab Chip
13(8):1476-1481.

S5.

Hille B. (2001) Ion channels of excitable membranes (Sinauer, Sunderland, Mass.) 3rd Ed
pp xviii, 814 p.

S6.

Sakai N., Kamikawa Y., Nishii M., Matsuoka T., Kato T., & Matile S. (2006) Dendritic folate
rosettes as ion channels in lipid bilayers. J. Am. Chem. Soc. 128(7):2218-2219.

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Rappe A. K., Casewit C. J., Colwell K. S., Goddard W. A. & Skiff W. M. (1992) UFF, a fullperiodic-table force-field for molecular mechanics and molecular-dynamics simulations. J.
Am. Chem. Soc. 114(25):10024-10035.

S8.

Furukawa S., Horike N., Kondo M., Hijikata Y., Carné-Sánchez A., Larpent P., Louvain N.,
Diring S., Sato H., Matsuda R., Kawano R. & Kitagawa S. (2016) Rhodium–organic
cuboctahedra as porous solids with strong binding sites. Inorg. Chem. 55(21):10843-10846.