Foldamer-Based Potassium Channels with High Ion Selectivity and Transport Activity

DOI: 10.1021/jacs.0c12128

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Journal of the American Chemical Society p. 3284 - 3288 (2021)

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Qi, Shuaiwei

Zhang, Chenyang

Yu, Hao Yu, Hao

Zhang, Jing

Yan, Tengfei

Lin, Ze Lin, Ze

Yang, Bing

Dong, Zeyuan

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Article abstract of DOI:10.1021/jacs.0c12128

Small molecules that independently perform natural channel-like functions show greatly potential in the treatment of human diseases. Taking advantage of aromatic helical scaffolds, we develop a kind of foldamer-based ion channels with lumen size varying f

Full text of DOI:10.1021/jacs.0c12128

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Foldamer-Based Potassium Channels with High Ion Selectivity and

Transport Activity

Shuaiwei Qi, Chenyang Zhang, Hao Yu, Jing Zhang, Tengfei Yan, Ze Lin, Bing Yang, and Zeyuan Dong*

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ABSTRACT: Small molecules that independently perform natural channel-like functions show greatly potential in the treatment of

human diseases. Taking advantage of aromatic helical scaﬀolds, we develop a kind of foldamer-based ion channels with lumen size

varying from 3.8 to 2.3 Å through a sequence substitution strategy. Our results clearly elucidate the importance of channel size in ion

transport selectivity in molecular detail, eventually leading to the discoveries of the best artiﬁcial K⁺channel by far and a rare sodium-

preferential channel as well. High K⁺selectivity and transport activity together make foldamers promising in therapeutic applications.

on transmembrane movement is essential in the central

nervous system and even human diseases. Normal

helical structures is conformationally stable and easily

predictable. Importantly, they can be tuned in lumen size via

sequence substitution by using various monomers. Preliminary

studies have demonstrated that helically folded polymers

perform signiﬁcant transport functions as unimolecular trans-

membrane ion channels.²⁶This exciting result inspired us to

design speciﬁc channels, to simulate a type of highly selective

synthetic K⁺channels (K⁺/Na⁺selectivity ratio of 22) based on

helical foldamers composed of pyridine-oxadiazole repeating

units.²⁵Moreover, Zeng et al. very recently reported pyridine-

oxadiazole-based helical polymer channels with a high K⁺/Na⁺

selectivity ratio of up to 16.²⁸These signiﬁcant ﬁndings

suggested that helical foldamer channels are a promising

research direction to manufacture artiﬁcial ion channels with

potential in treating human diseases. According to the

fundamental features of drug discovery, we empirically

speculate that small-molecule foldamers that can autono-

mously fulﬁll the transport functions of K⁺channels might be

better for therapeutic utilization than polymer-based models.

Furthermore, improvements in both the ion selectivity and

transport activity of foldamer channels are prerequisites for the

discovery of potential drug precursors at present. For such a

purpose, we precisely govern the lumen size to investigate the

transport properties of foldamer channels. By a sequence

substitution strategy in which the helical sequence is changed

by using diﬀerent monomers, three pore-containing helical

foldamers, 1−3, with slightly varied lumen sizes were obtained,

and importantly, all of them present excellent ion-selective

transmembrane transport functions (Figure 1b). Very excit-

ingly, tiny but deﬁnite variations in lumen size have a

remarkable inﬂuence on the ion selectivity of foldamer

physiological processes require a selective permeation pathway

for ions to cross cellular membranes.¹⁻⁴Ion channel proteins

play a central role in such a selective ion permeation

pathway.⁵⁻⁷However, ion channel dysfunctions generally

lead to ion channelopathies. Recently, several seminal reports

have demonstrated that small-molecule-mediated transmem-

brane translocation is able to compensate for missing protein

transport functions, which gives such small molecules great

potential in the treatment of human diseases.^8,9Therefore,

small molecules that independently perform natural channel-

like functions are eagerly pursued for possible therapeutic use.

Nature encodes a series of elegant and sophisticated

signature sequences to govern ionic ﬂow across cellular

membranes.¹⁰For instance, the selectivity ﬁlter of potassium

channels is a highly conservative amino acid sequence

containing four groups of close-spaced K⁺binding sites.^11,12

The characteristic sequences occurring inside potassium

channels give rise to extremely high selectivity for transporting

K⁺ions but not smaller Na⁺ions. Because K⁺channels are

responsible for the transmembrane conduction of K⁺ions,

their absence or dysfunctional physiological actions can cause

severe diseases, like hyperkalemia.¹³Therefore, the develop-

ment of K⁺channel mimics provides an alternative pathway to

compensate for the functional deﬁciency. Inspired by the

characteristic structure of K⁺channels with a pore aperture of

2.8 Å, chemists have endeavored to build numerous synthetic

nanopores for mimicry recently.^14,15However, the relation-

ships between channel size and ion selectivity are still

underexplored, partly because it would not only be hard to

engineer protein structures of natural ion channels but also be

extremely diﬃcult to construct a pore matching ion size in

synthetic molecule-level channel systems.¹⁶⁻²⁴

Received: November 30, 2020

Published: March 1, 2021

Recently, we reported a class of artiﬁcial ion channels based

on pore-forming aromatic helical scaﬀolds with the concept of

a pyridine-oxadiazole helical codon, in which helically folded

structures are stabilized by electrostatic repulsion of heter-

oatoms in molecular backbones.²⁵⁻²⁷This type of hollow

https://dx.doi.org/10.1021/jacs.0c12128

J. Am. Chem. Soc. 2021, 143, 3284−3288

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Communication

Figure 1. (a) Molecular structures of foldamers 1−3 and energy-

minimized conformations of 1 and 2 as well as crystal structure of 3

with pore diameters of 3.8, 2.7, and 2.3 Å, respectively. (b) Schematic

representation on ion channels formed by self-assembly of foldamers

1−3 with high K⁺or Na⁺selectivity.

Figure 2. Normalized K⁺and Na⁺transport activities of foldamer 1 at

3 mM (a), foldamer 2 at 0.01 mM (b), and foldamer 3 at 0.05 mM

(c). Hill analysis of the dose−response proﬁle of K⁺and Na⁺transport

for 1 (d), 2 (e), and 3 (f).

channels. We eventually ﬁnd a small-molecule surrogate for K⁺

channels, 2, that shows the highest K⁺selectivity and transport

activity among reported channel mimics.²⁹⁻³²

ions. As can be seen in Figure 2d−f, the concentration−activity

curves are nonlinearly ﬁtted via the Hill equation, and the half-

maximal eﬀective concentration (EC₅₀) can thus be

quantitatively evaluated (Figures S1 −S3). As listed in Table

1, EC₅₀values of foldamers 1−3 are 3.1 μM, 35 nM, and 280

Foldamers 1−3 were straightforwardly synthesized and fully

characterized (see the Supporting Information). We previously

reported a type of artiﬁcial K⁺channels with O and N atoms

fully populated inside the channel surface, such as in foldamer

1.²⁵To improve the properties of artiﬁcial channels, we thus

retain the landmark characteristic structure in the channel and

attempt to adjust the lumen size. When monomer pyridine is

substituted by o-phenanthroline, foldamer 3 can be obtained.

The substitution of helical sequence achieves the channel

aperture convergence, leading to a smaller lumen size in

foldamer 3 than in 1. At the same time, foldamer 2 was

rationally designed by combining o-phenanthroline and

pyridine monomers into helical sequences which can regulate

the lumen size between those of 1 and 3. The crystal structure

of 3 conﬁrms the helical conformation and gives a lumen size

of 2.3 Å (Figure 1a). The simulated structure of 3 is similar to

its crystal structure (Figure S21), suggesting that such aromatic

foldamers are structurally predictable. Correspondingly, the

lumen sizes from the energy-minimized structures (Figure 1a)

of foldamers 1 and 2 can reasonably be 3.8 and 2.7 Å,

respectively. These small-molecule foldamers 1−3 are thus

established with gradually constricted electron-rich pores.

Moreover, their respective stacking channel structures, formed

by self-assembly of foldamers 1−3, were conﬁrmed by atomic

force microscopy (Figure S22).²⁵

Table 1. Quantitative Analyses on the Lumen Sizes,

Transport Activities, and Ion Selectivity Ratios of

Foldamers 1−3

foldamer

lumen size

EC₅₀

S_K/Na

3.8 Å

3.1 μM

4.0−22.7

−

2.7 Å

2.3 Å

280 nM

−

35 nM

10.2−32.6

−

S_Na/K

4.2−5.2

Deﬁned by van der Waals surfaces.

nM, respectively. Notably, the transport activity of 2 is almost

2 orders of magnitude higher than that of 1. Such a low EC₅₀

value of 35 nM gives foldamer 2 great potential for therapeutic

utilization.

The ion selectivity of foldamers was investigated by vesicle-

based kinetic analysis. As can be seen in Figure 2a, foldamer 1

displays high transport selectivity for K⁺over Na⁺. Very

strikingly, foldamer 2 shows extremely high K⁺transport

selectivity. As can be observed in Figure 2a,b, the relative

ﬂuorescence intensity of 2 reaches saturation in a shorter time

and at a much lower concentration than that of 1. Based on

pseudo-ﬁrst-order initial transport rates,^35,36the K⁺/Na⁺

selectivity ratio (S_K/Na) of 2 was calculated to be 10.2−32.6

Next, the ion transport properties of foldamers 1−3 were

investigated via vesicle-based kinetic techniques.^33,34As shown

in Figure 2a−c, a signiﬁcant increase in normalized

ﬂuorescence intensity is observed, indicating that foldamers

1−3 can remarkably accelerate transmembrane movement of

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Communication

at diﬀerent concentrations, which is higher than that of 1

(S_K/Na= 4.0−22.7). It is reasonable that the S_K/Navalue will be

given as a range with the concentration change of

foldamers.^31,35Notably, the S_K/Navalue of 2 can reach up to

32. This result demonstrates that 2, with a smaller cavity (2.7

Å), shows a higher S_K/Nathan 1 (3.8 Å), indicating that the

lumen size does play a crucial role in ion selectivity. This

ﬁnding inspired us to explore what would happen to 3 with a

smaller lumen size (2.3 Å). Unexpectedly, foldamer 3

preferentially transports Na⁺over K⁺(Figure 2c), and its

selectivity ratio S_Na/Kwas calculated to be 4.2−5.2 (Figures

S10 −S14). This result indicates that 3 is a rare Na⁺-preferred

channel. This observation further supports the importance of

lumen size in ion selectivity. When the lumen size becomes

smaller, e.g., 2.3 Å in 3, the selectivity is prone to the smaller

Na⁺(2.04 Å) over K⁺(2.76 Å), eventually resulting in an

opposite Na⁺-preference phenomenon. It should be empha-

sized that the lumen size of the selectivity ﬁlter (4.6 Å) of

natural Na⁺channels is bigger than that of the selectivity ﬁlter

(2.8 Å) of natural K⁺channels.^12,37,38These results imply that

artiﬁcial sodium channel 3 performs a dehydrating Na⁺

transport, diﬀering from the natural sodium channels, of

which the ion transport mechanism involves water molecules.

Additionally, the critical transformation lumen size of K⁺and

Na⁺selectivity is in the range of 2.3−2.7 Å. We envisage that

the ion selectivity of channels originates from their ion aﬃnity.

To prove it, K⁺and Na⁺binding behaviors of foldamers 1−3

were studied by ﬂuorescence titrations. As observed (Figure

S16), the ﬂuorescence intensity of both 1 and 2 largely changes

with the addition of K⁺ions but varies slightly for Na⁺ions,

suggesting that foldamers 1 and 2 can speciﬁcally recognize K⁺

over Na⁺, and particularly that the K⁺binding capacity of 2 is

higher than that of 1 (Figures S16a,b and S17a). Surprisingly,

the ﬂuorescence intensity of foldamer 3 changes a little bit

upon addition of K⁺, but it is signiﬁcantly decreased in the

presence of Na⁺(Figures S16c and S17b). Although attempt

failed to measure the association constants of foldamers 2 and

3 due to strong self-assembly features, the results from

ﬂuorescence titrations suggest that ion binding capacities of

foldamers 1−3 are well consistent with their ion transport

selectivity. These observations demonstrate that ion selectivity

can be essentially controlled by tuning the lumen size.

Figure 3. (a) Single-channel current traces recorded for 1−3. (b)

Current−voltage relationship from asymmetric BLM experiments of

2. (c) Time-dependent ﬂuorescence intensities of safranine O in the

presence of 2, 3, and valinomycin (0.5 nM). (d) Hill analyses on

concentration-dependent K⁺transport of 1, 2, and gA.

that foldamer 2 and valinomycin have very similar capacities

for membrane polarization generated by K⁺eﬄux. In contrast,

sodium-selective channel 3 is unable to polarize the membrane

under identical conditions, which allows Na⁺inﬂux and thus

prevents safranine O from moving into the hydrophobic area

of the membrane.⁴⁴This observation provides unambiguous

evidence regarding the high K⁺selectivity of foldamer 2.

To assess the transport activity of potassium channel 2, a

native channel, gramicidin A (gA), with an approximately 4.0

Å, highly conducting pore, is chosen for comparison. As can be

seen in Figure 3d, the transport activity of gA is high (Figure

S15), and its EC₅₀can be calculated to be 5.0 nM. Very

excitingly, the small-molecule foldamer 2 also exhibits high

transport activity (EC₅₀= 35 nM), with an almost 100-fold

enhancement in transport activity compared to foldamer 1.

These results indicate that the transport activity of the self-

assembling channel from foldamer 2 is close to that of native

gA.

In conclusion, we have developed a kind of foldamer-based

ion channels with gradually constricted electron-rich pores by

means of a sequence substitution strategy. Our results clearly

elucidate the importance of channel size in ion transport

selectivity, eventually leading to the discoveries of the best

artiﬁcial K⁺channel by far as well as a rare sodium-preferential

channel. High K⁺selectivity and transport activity together

make foldamers greatly promising in therapeutic application.

Our study not only provides a clear understanding on the

relationship between channel size and ion transport selectivity

but also shows a successful proof-of-concept demonstration to

create synthetic ion channels by a size-guided approach. To

facilitate future medical applications of synthetic channels,

rational design of foldamers for better ion channels with more

speciﬁc selectivity is under investigation.

Single-channel electrophysiological experiments were carried

out to further prove the ion transport selectivity of foldamers

1−3. As shown in Figure 3a, all foldamers 1−3 exhibit clear

square signals, demonstrating the features of ion channels

rather than transporters in lipid membranes (Figures S18 and

S19).^39,40Moreover, asymmetric bilayer lipid membrane

(BLM) experiments conﬁrm that permeability ratios for K⁺/

Na⁺(P_K/Na) of 2 calculated from the reverse potential (V_rev

−75 mV, Figure 3b) by using the Goldman−Hodgkin−Katz

equation⁴⁰(Figure S20) are high, up to 18.2. The result from

asymmetric BLM experiments is in accord with that of the

LUVs assay (Table 1). To our knowledge, foldamer 2 is a K⁺

channel with the highest S_K/Naso far.^{16−19,21,41,42}

To underpin the K⁺selectivity of 2, membrane polarization

experiments were carried out by using safranine O as a

membrane-potential-sensitive probe (Figure 3c).^43,44In the

presence of the K⁺-selective transporter valinomycin, the

ﬂuorescence intensity of safranine O increases and eventually

reaches a relatively stable state. With the addition of foldamer

2, the ﬂuorescence intensity of safranine O can increase to

almost as high as that of valinomycin. This result demonstrates

ASSOCIATED CONTENT

* Supporting Information

The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/jacs.0c12128.

■

sı

Materials and methods, synthetic and experimental

details of compounds, crystal data of 3, experimental

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(5) Catterall, W. A. From Ionic Currents to Molecular Mechanisms:

The Structure and Function of Voltage-Gated Sodium Channels.

Neuron 2000, 26 (1), 13−25.

details of LUV preparation, cation transport, and planar

lipid bilayer experiments, including Figures S1−S69

(PDF)

(6) Emery, E. C.; Luiz, A. P.; Wood, J. N. Nav1.7 and other voltage-

gated sodium channels as drug targets for pain relief. Expert Opin.

Ther. Targets 2016, 20 (8), 975−983.

Accession Codes

CCDC 2046777 contains the supplementary crystallographic

data for this paper. These data can be obtained free of charge

via www.ccdc.cam.ac.uk/data_request/cif, or by emailing

data_request@ccdc.cam.ac.uk, or by contacting The Cam-

bridge Crystallographic Data Centre, 12 Union Road,

Cambridge CB2 1EZ, UK; fax: +44 1223 336033.

(7) MacKinnon, R. Potassium Channels and the Atomic Basis of

Selective Ion Condution (Nobel Lecture). Angew. Chem., Int. Ed.

2004, 43 (33), 4265−4277.

(8) Grillo, A. S.; SantaMaria, A. M.; Kafina, M. D.; Cioffi, A. G.;

Huston, N. C.; Han, M.; Seo, Y. A.; Yien, Y. Y.; Nardone, C.; Menon,

A. V.; Fan, J.; Svoboda, D. C.; Anderson, J. B.; Hong, J. D.; Nicolau,

B. G.; Subedi, K.; Gewirth, A. A.; Wessling-Resnick, M.; Kim, J.; Paw,

B. H.; Burke, M. D. Restored iron transport by a small molecule

promotes absorption and hemoglobinization in animals. Science 2017,

356 (6338), 608−616.

(9) Muraglia, K. A.; Chorghade, R. S.; Kim, B. R.; Tang, X. X.; Shah,

V. S.; Grillo, A. S.; Daniels, P. N.; Cioffi, A. G.; Karp, P. H.; Zhu, L.;

Welsh, M. J.; Burke, M. D. Small-molecule ion channels increase host

defences in cystic fibrosis airway epithelia. Nature 2019, 567 (7748),

405−408.

(10) Zakon, H. H. Adaptive evolution of voltage-gated sodium

channels: the first 800 million years. Proc. Natl. Acad. Sci. U. S. A.

2012, 109, 10619−10625.

(11) Zhou, Y.; Morais-Cabral, J. H.; Kaufman, A.; MacKinnon, R.

Chemistry of ion coordination and hydration revealed by a K⁺

channel −Fab complex at 2.0 Å resolution. Nature 2001, 414

(6859), 43−48.

(12) Doyle, D. A.; Cabral, J. M.; Pfuetzner, R. A.; Kuo, A.; Gulbis, J.

M.; Cohen, S. L.; Chait, B. T.; MacKinnon, R. The structure of the

potassium channel: molecular basis of K⁺conduction and selectivity.

Science 1998, 280 (5360), 69−77.

(13) Cummings, C. C.; McIvor, M. E. Fluoride-induced hyper-

kalemia: The role of Ca²⁺-dependent K⁺channels. AM. J. EMERG.

MED 1988, 6 (1), 1−3.

(14) Howorka, S. Building membrane nanopores. Nat. Nanotechnol.

2017, 12 (7), 619−630.

(15) Zheng, S. P.; Huang, L. B.; Sun, Z. H.; Barboiu, M. Self-

Assembled Artificial Ion-Channels Toward Natural Selection of

Functions. Angew. Chem., Int. Ed. 2021, 60 (2), 566−597.

(16) Tanaka, Y.; Kobuke, Y.; Sokabe, M. A Non-Peptidic Ion

Channel with K⁺Selectivity. Angew. Chem., Int. Ed. Engl. 1995, 34 (6),

693−694.

(17) Sun, Z. H.; Barboiu, M.; Legrand, Y.-M.; Petit, E.; Rotaru, A.

Highly Selective Artificial Cholesteryl Crown Ether K⁺-Channels.

Angew. Chem., Int. Ed. 2015, 54 (48), 14473−14477.

(18) Otis, F.; Auger, M.; Voyer, N. Exploiting Peptide Nanostruc-

tures to Construct Functional Artificial Ion Channels. Acc. Chem. Res.

2013, 46 (12), 2934−2943.

(19) Gokel, G. W.; Negin, S. Synthetic Ion Channels: From Pores to

Biological Applications. Acc. Chem. Res. 2013, 46 (12), 2824−2833.

(20) Benke, B. P.; Aich, P.; Kim, Y.; Kim, K. L.; Rohman, M. R.;

Hong, S.; Hwang, I.-C.; Lee, E. H.; Roh, J. H.; Kim, K. Iodide-

Selective Synthetic Ion Channels Based on Shape-Persistent Organic

Cages. J. Am. Chem. Soc. 2017, 139 (22), 7432−7435.

(21) Xin, P.; Kong, H.; Sun, Y.; Zhao, L.; Fang, H.; Zhu, H.; Jiang,

T.; Guo, J.; Zhang, Q.; Dong, W.; Chen, C.-P. Artificial K⁺Channels

Formed by Pillararene-Cyclodextrin Hybrid Molecules: Tuning

Cation Selectivity and Generating Membrane Potential. Angew.

Chem., Int. Ed. 2019, 58 (9), 2779−2784.

AUTHOR INFORMATION

Corresponding Author

Zeyuan Dong − State Key Laboratory of Supramolecular