A synthetic ion channel with anisotropic ligand response

Biological membranes play pivotal roles in the cellular activities. Transmembrane proteins are the central molecules that conduct membrane-mediated biochemical functions such as signal transduction and substance transportation. Not only the molecular functions but also the supramolecular properties of the transmembrane proteins such as self-assembly, delocalization, orientation and signal response are essential for controlling cellular activities. Here we report anisotropic ligand responses of a synthetic multipass transmembrane ion channel. An unsymmetrical molecular structure allows for oriented insertion of the synthetic amphiphile to a bilayer by addition to a pre-formed membrane. Complexation with a ligand prompts ion transportation by forming a supramolecular channel, and removal of the ligand deactivates the transportation function. Biomimetic regulation of the synthetic channel by agonistic and antagonistic ligands is also demonstrated not only in an artificial membrane but also in a biological membrane of a living cell. Transmembrane proteins are important for cellular functions and synthetic analogues are of interest. Here the authors report on the design and testing of a synthetic multipass transmembrane channel which shows anisotropic responses to agonistic and antagonistic ligands.

C ellular and organelle membranes play pivotal roles in controlling and maintaining biological activities, including environmental sensing, energy conversion, signal transduction and substance transportation. A significant part of these functions is realized by a series of proteins so-called transmembrane proteins, and their structure-function relationships have been attracting interest not only in biology and medicine, but also in chemistry and materials science for developing functional molecules and nanodevices [1][2][3] . Inspired by the proteinic channels, for example, numerous types of synthetic supramolecular ion channels have been developed [4][5][6] , and ion transportation through biological membranes have been demonstrated 7,8 . On the other hand, it has also been recognized that not only the structure of the proteins itself, but also other features such as self-assembly, delocalization and orientation of the molecules in the membrane as well as their dynamic properties like stimuli-responsiveness, are also responsible for their functions. In this context, control of these features for synthetic molecules remains important challenges, and draws increasing interest to develop sophisticated stimuli-responsive systems like ligand-, light-, voltage-and tension-gated ion channels [9][10][11] . Indeed, only a few successful examples to control the orientation of the synthetic transmembrane molecules have been demonstrated, which critically limits the applicability of the synthetic molecules to sensing and separation devices 12,13 .
In this study, we report a totally synthetic multipass transmembrane channel that can be introduced in lipid bilayers unidirectionally, and shows anisotropic responses to ligands allowing reversible regulation of ion transportation through lipid bilayers. This synthetic transmembrane molecule shows agonistic and antagonistic responses to different ligands, and functions not only in an artificial membrane but also in a plasma membrane of a living cell.

Results
Molecular design and synthesis. By mimicking a multipass transmembrane (MTM) structure seen in the proteinic ion channels constructed through folding of iterative hydrophilic and hydrophobic domains, we newly designed amphiphiles 1mer and 2mer (Fig. 1). These molecules have a multiblock structure with one and two hydrophobic (R)-1,11-dimethyl-3,9-bis[4-(phenylethynyl)phenyl]-5,7-dihydrodibenzo[c,e]oxepine (BPO) units and hydrophilic oligoethylene glycol (oligoEG) chains, where each terminus is capped with a hydrophobic triisopropylsilyl (TIPS) group, respectively (Fig. 1). While BPO unit and two oligoEG chains are connected via phosphate groups in the case of 1mer, those of 2mer are connected directly at one side and via a phosphate group at the other sides. These phosphate groups are expected to allow 1mer and 2mer to be dispersible to water and also to increase the interaction with aromatic amines as ligands. Furthermore, the BPO unit includes fluorescent and chiral components, which visualize the localization, conformational changes and assembling of these amphiphiles in the bilayer membranes through fluorescence and circular dichroism (CD) spectral measurements and microscopic observations. In addition, our previous study suggests that the TIPS groups at the termini of the oligoEG chains encourage the ion channel formation 7 .

Conformations and self-assembling properties in solution.
Conformations and self-assembling properties of 1mer and 2mer were investigated by spectroscopic and light scattering measurements. Dynamic light scattering (DLS) measurements indicated that 1mer was dispersed in tetrahydrofuran (THF) and formed aggregates in water ( Supplementary Fig. 4a). 1mer displayed blue shift of the absorption band with an emergence of a characteristic split Cotton effect upon increasing the ratio of water in THF, suggesting intermolecular H-type assembly of BPO units (λ abs = 325 nm in THF, 314 nm in water, Fig. 2a, b). The fluorescence band of 1mer showed a red-shift with a decrement of the intensity upon increase of the solvent polarity (λ em = 383 nm in THF and 444 nm in water corresponding to the monomer and excimer emissions, respectively; fluorescence lifetime τ 383 = 0.54 ns, τ 444 = 2.13 ns, Supplementary Fig. 5).
Analogous to 1mer, 2mer forms aggregates in water (Supplementary Fig. 4b). This aggregation formation induced a blue-shift of the absorption band and significant CD spectral change to show a strong split Cotton effect (λ abs = 329 nm and 316 nm in THF and water, respectively, Fig. 2c, d). Importantly, this CD profile in water was hardly dependent on the concentration of 2mer. Namely, similar CD patterns were observed at concentrations of 2mer in water of 7.0 μM and 2.5 mM (Supplementary Fig. 6). This similarity suggests that the CD signals in water are mostly originated from the geometry of the two aromatic units within a single molecule, thereby indicating an intramolecular assembly of BPO units of 2mer in an aqueous medium. The overall fluorescence intensity of 2mer decreased with an emergence of excimer emission upon addition of water (λ em = 384 nm, τ 384 = 0.15 ns in THF, λ em = 411 nm, τ 411 = 1.31 ns in water; Supplementary Figs. 5 and 7). Thus, it is likely that 2mer adopts a folded conformation in water, where the two BPO units are in close proximity with each other, and form H-type assembly. 1 H NMR spectroscopic measurements of 2mer at 2.5 mM also support the folding in the aqueous media, where the aromatic proton signals showed an upfield shift upon addition of D 2 O to THF-d 8  Ligand binding in solution. The folded conformation of 2mer, with intramolecularly-stacked aromatic units with neighboring phosphate groups, was considered to be advantageous to binding with amine-type ligands bearing aromatic groups by electrostatic and aromatic-aromatic interactions, as found in the β-adrenergic receptor 14 . Thus, as simple ligands, 2-phenylethylamine (PA) and (R)-1-(isopropylamino)-3-(1-naphthyloxy)-2-propanol (propranolol, PPN) were chosen in this study. Upon addition of PA, 2mer in a HEPES buffer showed continuous decrease in the intensity of CD signals (Fig. 3b). In 1 H NMR spectra, the signals corresponding to the aromatic and aliphatic protons close to the phosphate groups showed upfield shifts in response to the PA addition ( Supplementary Fig. 9). These spectral changes indicate that 2mer binds with PA in an aqueous medium presumably at the connecting points of the phosphate and aromatic units by the electrostatic and aromatic interactions. Job's plot indicates 1:1 complexation between 2mer and PA, and the association constant was evaluated to be K assoc = 1.07 × 10 2 M -1 (Supplementary Figs. 10 and 11). Since the intensities of split CD signals depend on the distance and dihedral angle between two chromophores, the observed CD spectral change suggests a conformational change of 2mer in company with the ligand accommodation. Essentially, similar spectral changes were observed in the titration of 2mer with PPN, where the evaluated association constant was K assoc = 1.04 × 10 4 M -1 (R = 0.997, Supplementary Figs. 12 and 13). The stronger interaction of 2mer with PPN than PA suggests a significant contribution of the aromatic-aromatic interaction to the formation of the complex. In contrast to 2mer, titration of 1mer with PA showed only little spectral changes (Fig. 3a, Supplementary Fig. 14). Thus, the folded conformation of 2mer likely be essential for the ligand-binding capability of this amphiphilic receptor.
Anisotropic membrane insertion and ligand binding. Having the folding and ligand-binding behaviours of 1mer and 2mer in hand, we investigated their incorporation as well as functions in liposomal membranes using giant and large unilamellar vesicles (GUVs, LUVs) prepared by the gentle hydration method. For the preparation of GUVs consisting of a mixture of 1,2-dioleoyl-snglycero-3-phosphocholine (DOPC) and 1mer, we conducted two procedures, which we called preloading and postloading methods. Interestingly, these two methods unexpectedly gave contrasting results. In the preloading method, DOPC and 1mer were dissolved in a mixture of MeOH and CHCl 3 . After evaporation of the resulting organic solution on the glass substrate, the residual film was hydrated with an aqueous solution of glucose and sucrose. Phase-contrast microscopy visualized successful formation of the GUVs (Supplementary Fig. 15a). Under fluorescence microscopic observation by irradiation with 330-385 nm light to excite the BPO units, ring-shaped images corresponding to the GUVs were visualized, indicating the localization of 1mer in the DOPC liposomal membranes ( Supplementary Fig. 15b). Detailed analysis by fluorescence depth quenching suggested a rather vertical orientation of the BPO units ( Supplementary Fig. 16) in the membranes 15 . On the other hand, in the postloading method, 1mer was added to the preformed DOPC GUVs in a buffer, which resulted in very faint fluorescent ring-shaped images of the GUVs with rather strong background fluorescence under fluorescence microscopic observation ( Supplementary Fig. 15c, d).
This suggests dispersion of 1mer in the extravesicular aqueous medium. In the case of 2mer, the preloading method also produced DOPC GUVs containing 2mer localized in the bilayer (Supplementary Fig. 17a, b). Interestingly, in sharp contrast to 1mer, 2mer could be localized in the DOPC GUV membranes also by the postloading method ( Supplementary Fig. 17c, d). , indicating that 2mer adopts the folded conformation in the membranes. These results suggest that the phosphate groups attached to the BPO units interfere with these molecules to pass through the DOPC membranes. Namely, while 1mer, bearing the phosphate groups at the both ends, could not be inserted in the membrane by the postloading method, the folded form of 2mer caused the unsymmetrical structure with the two phosphate groups locating at one side of the BPO units, likely allowing this molecule to be inserted into the membrane from the uncharged oligoEG chain side. This biased insertion of folded 2mer in the postloading method is expected to allow directed alignment of 2mer in the DOPC membranes. Indeed, DOPC•2mer LUVs prepared by the preloading and postloading methods (DOPC•2mer pre and DOPC•2mer post , respectively) showed significantly different ligand responses from each other. DOPC•2mer pre LUVs showed a split Cotton effect at the wavelengths corresponding to the absorption of the BPO units, suggesting the folding of 2mer in the DOPC bilayer (Fig. 3c, red line). Upon addition of PA, DOPC•2mer pre LUVs showed only slight change in the CD spectra ( Fig. 3c, Supplementary Fig. 19). In contrast, addition of PA to the extravesicular medium of DOPC•2mer post LUVs caused more prompt change in the CD profile (Fig. 3d, Supplementary Fig. 20). Importantly, addition of 2mer to the preformed DOPC LUVs, encapsulating PA in their internal aqueous phases, showed only small spectral difference from the DOPC•2mer post LUVs without PA ( Supplementary Fig. 21). These processing-dependent ligand-binding properties of 2mer strongly indicate that the orientation of 2mer in the bilayer is different in the liposomes prepared by the preloading and postloading methods, where the postloading method allows for anisotropic membrane insertion of 2mer into the DOPC bilayer that exposes the phosphate groups to the extravesicular medium. The orientation selectivity of 2mer in the DOPC bilayer prepared by the postloading method was estimated to be 95% by zeta potential measurements ( Supplementary Fig. 22).
Ligand-gated ion transportation. Our previous studies demonstrated that the multipass transmembrane structures like 2mer, consisting of alternatingly connected oligoethylene glycol chains and aromatic components are effective for ion transportation    (Fig. 4d). In contrast, addition of PA into the lower chamber of the DOPC•2mer post BLM hardly induced the intensified current signals, while the subsequent addition of PA into the upper chamber triggered the ion transportation ( Supplementary Fig. 23). As expected, BLM prepared from n-decane solution of a mixture of DOPC and 2mer (DOPC•2mer pre BLM) responded to the PA addition to both upper and lower chambers identically (Supplementary Fig. 24). Thus, the ion transportation of 2mer was controlled by the ligand complexation, where addition of 2mer to the preformed BLM allowed for the anisotropic response to the ligand. The relationship between the inner diameter d and the conductance g of an ion channel (1.4 pS) is known to be described by the Hille equation, where l and ρ are the length of the channel (3.5 nm) and resistivity of the recording solution (2.35 Ω m), respectively 17 Supplementary Fig. 25), suggesting reversible ligand-gated response of 2mer to PA. Hill analysis on the 2mer concentration dependency of the cation transportation rate in the HPTS assay indicated the Hill coefficient n = 3.06 (R = 0.998), suggesting supramolecular ion channel formation of 2mer, in which the number of 2mer molecules constructing the ion channel is likely multiple of three (Fig. 4b) 19,20 . Among the alkali metal cations, Li + was transported in the fastest rate. 2mer transported alkaline-earth metal cations such as Ca 2+ as well ( Supplementary Fig. 26). It should be noted here that, PPN, known as an antagonist for the β-adrenergic receptor, showed a contrastive effect to PA on  Fig. 27a, b). Furthermore, subsequent addition of PPN to the upper chamber of the DOPC•2mer post BLM, after addition of 2mer in the presence of PA, deactivated the current flow ( Supplementary  Fig. 27c, d). Thus, analogous to the agonistic and antagonistic effects of PA and PPN to β-adrenergic receptor, PA and PPN apparently act to 2mer like an agonist and an antagonist, respectively. NMR and modelling studies of 2mer-ligand complexes indicated that PA and PPN bind with 2mer through different interacting modes ( Supplementary Figs. 28 and 29). Namely, electrostatic interaction between the phosphate and ammonium groups is likely dominant between PA and 2mer, and PA is located at the rim of the channel. Meanwhile, hydrophobic interaction is preferred between 2mer and PPN, therefore PPN is inserted into the hydrophobic cavity of the channel to infill the pore.
Regulation of ion transportation through biological membrane. Taking advantage of the capability of 2mer to spontaneously insert into the membrane, with its ligand-gated ion transportation function, we further challenged the regulation of ion transportation through a plasma membrane of a living cell. For visualization of 2mer in the cell culture by fluorescence microscopy, cyanine3-labelled 2mer (Cy3-2mer) was prepared. The localization of Cy3-2mer in the plasma membrane was investigated by total internal reflection fluorescence (TIRF) microscopic observation with 561-nm laser excitation to visualize Cy3 fluorescence. TIRF microscopy is advantageous to observation of molecules embedded in a live-cell plasma membrane because of its high sensitivity to visualize fluorescent species near the coverslip-specimen interface.  (Fig. 6a), where addition of ionomycin, an ionophore known to transport Ca 2+ , resulted in enhancement of the fluorescence intensity of Fluo-4 (Supplementary Fig. 31). Meanwhile, since L cells do not possess βadrenergic receptors, addition of PA to L cells without 2mer did not prompt the apparent change in the fluorescence intensity of Fluo-4 ( Supplementary Fig. 32). In sharp contrast, while addition of 2mer to the cells hardly changed the fluorescence intensity ([2mer] = 10 nM, Fig. 6b and Supplementary Fig. 33), subsequent addition of PA readily prompted significant enhancement of the Fluo-4 fluorescence intensity in the cells (Fig. 6c- Supplementary Fig. 36).
In summary, we demonstrate anisotropic ligand responses of a synthetic multipass transmembrane ion channel. An unsymmetrical molecular structure enables oriented insertion of the synthetic amphiphile to a bilayer by addition to a preformed membrane. Complexation with a ligand prompts formation of a supramolecular channel for ion transportation, and removal of the ligand deactivates the transportation function. Biomimetic regulation of the supramolecular channel by agonistic and antagonistic ligands is also demonstrated not only in an artificial membrane but also in a biological membrane of a living cell.
where I 0 , I t and I lyzed represent the fluorescence intensities before addition of KOH, at t seconds after addition of KOH, and after lysis by the addition of 1.0 wt% Triton X-100, respectively.
Conductance measurements using BLM system. A CHCl 3 solution of DOPC (12.7 mM, 100 μL) was gently dried under N 2 flow, which was then dispersed in n-decane (100 μL). The n-decane suspension (   Single-molecule fluorescence tracking by TIRF. Single fluorescent-molecule observation was performed as previously described 21 . To briefly explain, an objective lens-type TIRF (Total Internal Reflection fluorescence) microscope based on an Olympus IX-81 was employed. Mouse L cells were observed at room temperature of 25°C after addition of Cy3-2mer (10 nM) in HBSS-HEPES buffer (HBSS supplemented with 10 mM HEPES, adjusted to pH 7.4 with NaOH). The bottom plasma membrane was locally illuminated with an evanescent field (an Olympus ×100, 1.49 NA objective lens; excitation at 561 nm). The fluorescent image of Cy3-2mer was projected onto a two-stage microchannel plate intensifier (C8600-03; Hamamatsu Photonics), and its output image was then lens coupled to an electron bombardment charge-coupled device camera (C7190-23; Hamamatsu Photonics). The obtained images were recorded at 30 Hz on a digital video deck for the following analysis. These instruments were also employed for fluorescence imaging of intracellular Ca 2+ mobilization (described in the next section). Modelling of 2mer and ligand complexes. A three dimensional structure of a 2mer was built by 2D Sketcher and Minimize-Selected-Atoms modules in MAESTRO (Shrödinger release 2018-2). The silicon atom at the TIPS group was replaced with a carbon atom. The folded structure of the 2mer was set to the Mshape as shown in Fig. 1. After building the one 2mer structure, other two 2mer structures were further built, and a three-2mer complex was prepared, because it was suggested that the supramolecular ion channel formation of 2mer estimated by the Hille equation was achieved by multiple of three. The initial formation of the three-2mer complex was set to a symmetric triangle form, and the directions of the three 2mer were aligned ( Supplementary Fig. 29a). The three dimensional structures of PA and PPN were built by the same protocol for 2mer. A ligand was located between the two BPO units for one 2mer, and the three ligands were arranged one-to-one with the three 2mer. The position of ligand was located at the position of benzene near the phosphorus atom in 2mer. To reduce steric hindrances among TIPS groups for each 2mer or among 2mer and PA, the structures around the phosphate part near PA and the positions of the TIPS group were modified manually. Using the modified structure of the three-2mer complex, each PA was replaced with PPN, and these positions of PA ( Supplementary Fig. 29b) or PPN ( Supplementary Fig. 29c) were matched as possible.
The three-2mer and ligand complex were embedded in DOPC membrane and water molecules using the Membrane Builder implemented in CHARMM-GUI [22][23][24][25][26][27] . The orientation of 2mer relative to a lipid bilayer was set vertically, and the buried area of 2mer was determined so that the phosphate part of 2mer and DOPC molecules were close to each other. The molecular dynamics unit cell was set to a rectangular cell. In the center of the cell, the complex was embedded in a 70 Å × 70 Å DOPC bilayer at the x-y plane, and the number of DOPC molecules at the upper and lower leaflets was 56 and 65, respectively. Along the z-axis of the unit cell, water molecules were added, and the water thickness was set to 22.5 Å. Counterions and 150 mM KCl were included. A three-2mer and PA complex embedded in DOPC membrane and water molecules is illustrated in Supplementary Fig. 29d.
The complex embedded in the membrane-water system was equilibrated by allatom molecular dynamics (MD) simulations. MD simulations were performed using the MD program package GROMACS ver. 2016.3 [28][29][30] under periodic boundary conditions. The CHARMM36m force field was used for membranes [31][32][33][34][35] and the TIP3P water model 36 , and the CHARMM General Force Field (CGenFF) 37 was used for 2mer and ligands. The CGenFF parameters were assigned by ParamChem through the Membrane Builder. For CGenFF parameters of 2mer, the parameters for each part of the oligoEG including the TIPS group, the BPO unit, the PEG were assigned piece by piece, and then these parameters were assembled. The electrostatic interaction was handled by the smooth particle mesh Ewald method 38 , and the van del Waals interaction was truncated by the switching function with the range of 10-12 Å. Bond lengths involving hydrogen atoms were constrained by the P-LINKS algorithm 39 . The temperature and pressure were 300 K and 1 atm, respectively. According to the default setup in the Membrane Builder, an energy minimization by the steepest descent method and six equilibration simulations, written as EQ1-EQ6 hereafter, were performed sequentially before the production run. The ensemble adopted in the simulations was NVT ensemble in EQ1 and EQ2 and NPT ensemble in EQ3 to EQ6 and the production run. The thermostat in the equilibration runs was the weak-coupling scheme of Berendsen 40 , and that in the production run was the Nosé-Hoover scheme 41,42 . The barostat in EQ3 to EQ6 was the semi-isotropic Berendsen algorithm 40 , and that in the production run was the semi-isotropic Parrinello-Rahman approach 43,44 . The time step was set to 1 fs in EQ1 to EQ3, and 2 fs in EQ4 to EQ6 and the production runs. The simulation length of each simulation was 125 ps for EQ1 to EQ3, 500 ps for EQ4 to EQ6, and 500 ps for a production run.
In the equilibration processes, structural restraints were imposed on 2mer, ligand, and DOPC molecules according to the default setup of the Membrane Builder. The position harmonic restraints were imposed on heavy atoms of 2mer and ligand. During the equilibration runs, the strength of the force constant was reduced gradually from 4000 to 50 kJ·mol -2 ·nm -2 . In DOPC molecules, the z-axis of the phosphorus atom was restrained by the position harmonic restraints, and its force constant was gradually reduced as 1000-0 kJ·mol -2 ·nm -2 . In addition, three dihedral angles were restrained. One was the dihedral angle among three carbon atoms at the sn-1, -2, -3 positions and an adjacent oxygen atom at the sn-2 position. The angle was restrained at 120 degree. The other was the two dihedral angles corresponding to the double bond between carbon atoms in the oleic acid part in the sn-2 and -3 positions, and the angle was restrained at the cis conformation. The force constants of these dihedral angles were reduced gradually from 1000 to 0 kJ·mol -2 ·rad -2 .
After the equilibration runs, a 500 ps production run was carried out for each the three-2mer and PA complex and the three-2mer and PPN complex. In the production runs, no restraints were imposed.