Unimolecular Transmembrane Na ⁺ Channels Constructed by Pore-Forming Helical Polymers with a 2.3 Å Aperture

Abstract

Open AccessCCS ChemistryCOMMUNICATION6 Jun 2022Unimolecular Transmembrane Na+ Channels Constructed by Pore-Forming Helical Polymers with a 2.3 Å Aperture Shuaiwei Qi, Jun Tian, Jing Zhang, Lei Zhang, Chenyang Zhang, Ze Lin, Jing Min, Shizhong Mao and Zeyuan Dong Shuaiwei Qi State Key Laboratory of Supramolecular Structure and Materials, College of Chemistry, Jilin University, Changchun 130012 , Jun Tian State Key Laboratory of Supramolecular Structure and Materials, College of Chemistry, Jilin University, Changchun 130012 , Jing Zhang State Key Laboratory of Supramolecular Structure and Materials, College of Chemistry, Jilin University, Changchun 130012 , Lei Zhang State Key Laboratory of Supramolecular Structure and Materials, College of Chemistry, Jilin University, Changchun 130012 , Chenyang Zhang State Key Laboratory of Supramolecular Structure and Materials, College of Chemistry, Jilin University, Changchun 130012 , Ze Lin State Key Laboratory of Supramolecular Structure and Materials, College of Chemistry, Jilin University, Changchun 130012 , Jing Min State Key Laboratory of Supramolecular Structure and Materials, College of Chemistry, Jilin University, Changchun 130012 , Shizhong Mao State Key Laboratory of Supramolecular Structure and Materials, College of Chemistry, Jilin University, Changchun 130012 and Zeyuan Dong *Corresponding author: E-mail Address: [email protected] State Key Laboratory of Supramolecular Structure and Materials, College of Chemistry, Jilin University, Changchun 130012 https://doi.org/10.31635/ccschem.021.202101144 SectionsSupplemental MaterialAboutAbstractPDF ToolsAdd to favoritesDownload CitationsTrack Citations ShareFacebookTwitterLinked InEmail To understand the relationships between channel size and ion selectivity, we have developed a new type of artificial ion channel based on pore-forming helical polymers consisting of phenanthroline-oxadiazole units with a pore aperture 2.3 Å close to the diameter of the Na+ ion (2.04 Å). Successful preparation of high molecular weight helical polymers ( HP1) gives rise to a 4.6 nm long artificial unimolecular transmembrane channel. The transport property of artificial channel HP1 was elaborately investigated by means of vesicle-based kinetic assay and symmetry/asymmetry bilayer membrane (BLM) experiments as well. These results unambiguously demonstrate that HP1 is a Na+-selective channel with extremely high transport activity (EC50 = 0.017 mol % relative to lipid). Moreover, the Na+/K+ selectivity ratio of HP1 reaches 1.9, as determined by asymmetry BLM experiments. Owing to the narrowest 2.3 Å size constraint so far, HP1 transport naked Na+ ion across the membrane, which represents a different Na+ transport mode from that of natural Na+ channels, which transports partially hydrated Na+ ions during transmembrane conduction. This study provides crucial insights on the chemical basis of ion selectivity in the field of ion channels. Download figure Download PowerPoint Introduction Ion channels embedded in cellular membranes can perform selective ion osmosis, which is crucial in normal physiological processes to balance ion gradient, govern action potential, and more.1–3 Recently, abundant mutations of ion channels have been found to cause channel malfunctions, eventually leading to therapeutically targeted ion channelopathies.4,5 Many efforts have been devoted to uncovering the structures of ion channels so as to investigate their function and disease mechanisms and also for structure-guided drug discovery.1,6–9 Despite the attractive features discovered by the structures of ion channels, the chemical basis of ion selectivity still remains underexplored, especially concerning how naturally evolutionary channels distinguish between spherical ions, K+ (2.76 Å) and Na+ (2.04 Å).10,11 Natural potassium channels have demonstrated highly selective K+ conduction by a selectivity filter (SF) with a pore diameter of 2.8 Å but immeasurably low permeation for smaller Na+.7–9 Compared with K+ channels, natural sodium channels specifically transport smaller Na+ ions in toward the cell through their SF with a larger lumen size of approximate 4.6 Å, which shows high Na+/K+ selectivity ratios ranging in value from 12 to 50.8,12–14 The size constraint underlies that the SF of potassium channels selects naked K+ ions by direct interaction via ion coordination formed by carbonyls of amino acid residues, whereas the SF of sodium channels transports partly hydrated Na+ ions during transmembrane conduction.8 It is reasonable that the dehydration free energy of Na+ ions (98 kcal/mol) is higher than that of K+ ions (80 kcal/mol).15 This observation indicates the fact that naturally evolutionary ion channels regulate ion selectivity in two different ways: one is by matching naked ion size, and the other one is by matching partially hydrated ion size. To understand the structure–function relationships and transport mechanisms of ion channels, the same channel with structurally distinct states is required, but it is difficult to be achieved,16–20 even by protein engineering and molecular simulation. Therefore, it is rather important to know if any natural or synthetic channels can transport the naked Na+ ion across the membrane through overcoming the high barrier of dehydration free energy of Na+ ion, as potassium channels do. The purpose is to reveal the size constraint as a determinant factor of ion selectivity, because the elucidation on the relationships between channel size and ion selectivity is still scarce from the structural analyses of natural ion channels. To realize this, one of the prerequisites is that one has to make an ion channel with a pore aperture close to the size of a Na+ ion (2.04 Å). Herein, for the first time, we prepare a type of unimolecular transmembrane channel with a pore aperture as small as 2.3 Å based on pore-forming helical polymers, which provides crucial insights on the chemical basis of ion selectivity. With the exception of synthetic K+ channels with relatively high K+/Na+ selectivity,21–29 artificial Na+ channels have been rarely explored in the past. The development of artificial Na+ channels is seriously constrained, because it is very difficult to distinguish between Na+ and K+ spherical ions in chemically synthetic molecule-level channel systems. Moreover, the transport mechanisms of Na+ ions are somehow ambiguous in previously reported artificial channels. Specifically, overwhelming the high barrier of dehydration free energy of Na+ ions has been underexplored to perform naked Na+ transmembrane transport in synthetic channels. Recently, we reported a class of synthetic ion channels based on pore-forming aromatic helical scaffolds consisting of pyridine-oxadiazole units. There are two unique features in pore-forming helical polymers: one is conformationally predictable that makes precision structure and channel size,30,31 and the other one is their specific channel surfaces that fully populate O and N atoms to recognize alkali cations.28 Recently, Zeng and co-workers21 further demonstrated these features of helical polymers made up of pyridine-oxadiazole repeating units, and they found that such helical polymers are very active K+ channels with a high K+/Na+ selectivity ratio of 16. Notably, the pyridine-oxadiazole-based helical polymer contains a specific channel structure with a lumen size of ca. 3.8 Å, which selectively binds K+ ions rather than smaller Na+ ions. To develop artificial Na+ channels, except one way that synthetic channels possess a cavity size close to 4.6 Å of natural Na+ channels, we alternatively chose to make a channel with a lumen size close to that of Na+ ion (2.04 Å) but smaller than that of K+ ion (2.76 Å) according to our recent findings.32 Based on this idea, we synthesized a new type of pore-forming helical polymers ( HP1) consisting of phenanthroline-oxadiazole repeating units with a channel size of ca. 2.3 Å and an average length of 4.6 nm (Figure 1a) For comparison, a pyridine-oxadiazole-based helical polymer ( HP2) was also prepared (Figure 1b). Importantly, the phenanthroline-oxadiazole-based helical polymer HP1 can cross through the lipid membrane as a unimolecular channel showing Na+-selective transport properties (Figure 1c). Figure 1 | (a) Molecular and simulation structures (the side chains were omitted for clarity) of helical polymer channels HP1 and HP2 with pore apertures of 2.3 and 3.8 Å, respectively. (b) Schematic representation on transmembrane transport functions of channels HP1 and HP2. Download figure Download PowerPoint Results and Discussion Helical polymers HP1 and HP2 were straightforwardly synthesized in a similar approach according to previously reported synthetic procedures30,31 with minor modifications and full characterization ( Supporting Information Scheme S1, and Figures S27–S55). In particular, for the synthesis of HP1, linear polymers were initially prepared by a condensation reaction of phenanthroline diacid and phenanthroline hydrazine and then further dehydrated by adding dehydration reagents to generate oxadiazole-containing helical polymers. This synthetic strategy can efficiently prepare high molecular weight rigid helical polymers.30,31 The 1H NMR spectrum of HP1 shows several broad characteristic peaks ( Supporting Information Figure S36), implying a typical polymeric structure. The structure of HP1 was further confirmed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), in which the periodic sequence structure of HP1 agrees with the molecular weight intervals of repeating units ( Supporting Information Figure S38). By gel permeation chromatography (GPC), the number average molecular weight (Mn) and PDI = 1.14 of HP1 were determined to be 12.8 kD and 1.1, respectively ( Supporting Information Figure S39). In light of the helical pitch of 0.36 nm and 2.4 units per turn in phenanthroline-oxadiazole-based helical polymers, the average length of HP1 was calculated to be ca. 4.6 nm, which can puncture the lipid membrane as a unimolecular channel. By cryo-transmission electron microscopy (cryo-TEM), the single molecular images of HP1 in a self-assembled state can be observed, and the widths of those lines are 2.0 ± 0.2 nm ( Supporting Information Figure S25), in accordance with the diameter of HP1 as demonstrated by a single-crystal study.32 At the same time, the structure of HP2 was also demonstrated by NMR, MALDI-TOF MS, and GPC, showing that the Mn and PDI = 1.32 of HP2 are 2700 and 1.3, respectively ( Supporting Information Figures S54 and S55). Owing to the high predictability of such rigid helical polymers, the lumen size of HP1 consisting of phenanthroline-oxadiazole units is determined to be ca. 2.3 Å by density functional theory (DFT) calculations and single-crystal study,32 which is much smaller than that of pyridine-oxadiazole-based HP2 (3.8 Å). The isopropyl groups are used as the side chains of helical polymers because of their good properties in terms of solubility and permeability during transmembrane transduction.28 Since the ion selectivity of channels essentially depends on their ion-binding capacity,28 the K+ and Na+ ion recognition of HP1 and HP2 is thus studied by fluorescence titrations. As shown in Figures 2a and 2b, the fluorescence intensities of both HP1 and HP2 were distinctly increased in the presence of K+ and Na+ ions. For a comparative observation, the fluorescence intensity of both HP1 and HP2 was set to be 100% in the absence of K+ and Na+ ions. Notably, the increments in fluorescence intensity of HP1 were consistenly larger with increasing amounts of Na+ than K+, suggesting that HP1 with a lumen size of 2.3 Å binds to Na+ better than K+. However, K+-induced enhancement in fluorescence intensity of HP2 definitely surpasses the intensity changes caused by the addition of Na+, indicating that HP2 with a cavity size of 3.8 Å is more favorable to K+ than Na+. Through fluorescence titration experiments of HP1 and HP2, the opposite phenomenon in their ion-binding capacity was observed, which suggests that the size constraint is a determining factor of ion selectivity. Figure 2 | Fluorescence titrations of HP1 (a) and HP2 (b) at 20 μM in acetonitrile by adding 0–22 equiv of Na+ and K+ ions, respectively. Download figure Download PowerPoint The ion-transport property of channels HP1 and HP2 was investigated by a vesicle-based kinetic assay,28 in which changes of the fluorescence intensity of 8-hydroxypyrene-1,3,6-trisulfonic acid trisodium salt (HPTS),33 a pH sensitive dye, directly reflected ion flux across the membrane. The co-assembly structure of vesicles and HP1 was tested by cryo-TEM, and at least three images of vesicles with a tubular objective across the lipid membrane were observed ( Supporting Information Figure S26). As expected, the HPTS assay demonstrated that both HP1 and HP2 can efficiently transport Na+ and K+ ions. Importantly, ion-transport selectivity highly agrees with their corresponding ion-binding capacity. As shown in Figures 3a and 3c, HP1 can transport Na+ even better than K+ across the lipid membrane, which indicates that HP1 with a lumen size of 2.3 Å is a Na+-selective channel. In contrast, HP2 with a cavity size of 3.8 Å is a K+-selective channel (Figures 3b and 3d), according with previously reported results.21 In using the HPTS assay, the K+/Na+ selectivity ratio of HP2 was calculated to be up to 4.2 based on the pseudo first-order rate constant obtained from normalized fluorescence intensity as a function of time ( Supporting Information Figures S4–S6). As a rare Na+-selective channel, the Na+/K+ selectivity ratio of HP1 was similarly calculated to be 1.3 ( Supporting Information Figures S1–S3). Although the Na+/K+ selectivity ratio of HP1 is relatively low, the reversal of ion selectivity between HP1 and HP2 unambiguously takes place in the case. It is reasonable that the 2.3 Å aperture of HP1 is between K+ (2.76 Å) and Na+ (2.04 Å), whereas the 3.8 Å aperture of HP2 is much bigger than the size of Na+ (2.04 Å). This observation strongly implies that the lumen size is key to the ion selectivity of channels. Moreover, the 2.3 Å aperture of HP1 transports only naked Na+ across the membrane by overwhelming the high barrier of dehydration free energy of Na+ ions. Figure 3 | Normalized relative intensity change of HP1 (a) at 2.5 μM and HP2 (b) at 100 μM for K+ or Na+ transport. Normalized relative intensity change of HP1 (c) and HP2 (d) at different concentrations for K+ or Na+ transport. Download figure Download PowerPoint The transport activity of channels HP1 and HP2 was assessed by Hill analyses.23,34 Via the Hill equation, the half maximal effective concentration (EC50) can be calculated. As observed, for transporting K+, the EC50 values of HP1 and HP2 are 2.7 μM (0.021 mol %) and 193 μM (1.4 mol %) ( Supporting Information Figures S7b, S9, and S10), respectively. The low EC50 (K+) value reflects extremely high transport activity of HP1, which is much lower than that of HP2 under the identical conditions. For transporting Na+, the EC50 value of HP1 is as low as 2.3 μM (0.017 mol %) ( Supporting Information Figures S7a and S8). However, the EC50 value of HP2 cannot be obtained due to its weak ability of transporting Na+. Such a low EC50 (Na+) value indicates that HP1 is a highly active ion channel, which is at the highest level of transport activity reported previously.31,35,36 The Hill analyses reveal that the ion-transport activity of HP1 is almost two orders of magnitude higher than that of HP2. The high transport activity of HP1 mainly benefits from its specific structure as a unimolecular channel. To investigate the conduction mechanisms of HP1 and HP2, single-channel conductance measurements were performed via the planar lipid bilayer membrane (BLM).34,37 As seen in Figure 4a, both of HP1 and HP2 show typical square-like signals, which demonstrates the function of ion channels in BLM. By assessing the relationship of current–voltage (Figures 4b and 4c) in which the current values were determined by histogram analyses ( Supporting Information Figures S11–S20), both of HP1 and HP2 were proved as ohmic resistors based on the fact that conductance does not change under different membrane potentials. Accordingly, the conductance values of HP1 and HP2 are calculated to be 24.9 ± 0.5 and 35.9 ± 0.6 pS, respectively. It is known that the conductance value generally decreases with decreasing pore diameter via the Hill equation,34 a smaller conductance value indicates that the lumen size of HP1 is smaller than that of HP2. This result further underpins the relationship between the lumen size and property of channels. To evaluate the ion selectivity of HP1, asymmetric BLM experiments for ion preference were thus undertaken. According to the zero-current potential obtained by the current–voltage relationship in an asymmetric gradient of 1 M NaCl and 1 M KCl electrolytes ( Supporting Information Figures S21–S24), the Na+/K+ selectivity ratio of HP1 is unambiguously determined to be 1.9 (Figure 4d), which agrees with the results found by a vesicle-based kinetic assay. This is the first example to show Na+ selectivity in artificial channels by asymmetric BLM experiments, which clearly demonstrates that HP1 is a Na+-selective channel. Compared with HP1, the channel HP2 with a bigger lumen size (3.8 Å) selectively transports K+ over Na+, and its K+/Na+ selectivity ratio had been studied previously.21 Although the Na+/K+ selectivity ratio (1.9) of HP1 is one order of magnitude lower than those (Na+/K+ selectivity ratio = 12–50) of natural Na+ channels,12–14 two different transport mechanisms between HP1 and natural Na+ channels were proposed. Since the 2.3 Å lumen size of HP1 is close to the diameter of Na+ (2.04 Å) and only half of the SF (4.6 Å) of natural Na+ channels, artificial channel HP1 must transport naked Na+ ions across the membrane due to size constraint, similar to that natural K+ channels transport K+ ion. Based on the research findings reported till now,8 natural Na+ channels transport partly hydrated Na+ ions during transmembrane conduction. Therefore, artificial Na+ channel HP1 represents a non-natural Na+ transport mode. This result underpins the transport mechanism of natural Na+ channels in another way. Figure 4 | (a) Current recordings produced by HP2 (top) in symmetrical 1 M KCl electrolytes at −100 mV, HP1 (middle) in symmetrical 1 M KCl electrolytes at +150 mV and HP1 (bottom) in asymmetrical 1M KCl and 1 M NaCl electrolytes at −150 mV. Linear relationship of current–voltage for HP2 (b) and HP1 (c) under different membrane potentials in symmetrical 1 M KCl electrolytes. (d) Linear relationship of current–voltage for HP1 in asymmetrical 1 M KCl and 1 M NaCl electrolytes. Download figure Download PowerPoint Conclusion To understand the relationships between lumen size and ion selectivity in ion channels, we developed a new type of artificial ion channel based on pore-forming helical polymers consisting of phenanthroline-oxadiazole units with a pore aperture 2.3 Å close to the diameter of Na+ ion (2.04 Å). Successful preparation of high molecular weight helical polymer HP1 gives rise to a 4.6 nm long artificial unimolecular transmembrane channel. The transport property of artificial channel HP1 was elaborately investigated by means of a vesicle-based kinetic assay as well as symmetry/asymmetry BLM experiments. These results unambiguously demonstrate that HP1 is a Na+-selective channel with extremely high transport activity. Moreover, the Na+/K+ selectivity ratio of HP1 reaches 1.9, as determined by asymmetric BLM experiments. Owing to the 2.3 Å size constraint, HP1 transports naked Na+ ions across the membrane, which represents a different Na+ transport mode from natural Na+ channels that transport partially hydrated Na+ ions during transmembrane conduction. To the best of our knowledge, HP1 is the first unimolecular transmembrane Na+-selective channel based on a helical polymer. This study not only provides crucial insights on the chemical basis of ion selectivity in the field of ion channels but also paves the way for goal-oriented design of ion channels, for example, to perfect artificial Na+-selective channels by narrowing the lumen size close to 2.0 Å. With the discovery of more natural Na+ channels,38–43 the transport mode of naked Na+ ions by size constraint, as reported in biomimetic systems, might exist in nature. Supporting Information Supporting Information is available, including experimental materials, synthetic and experimental procedures (Scheme S1), figures of characterization (Figure S1–S26), and spectra of NMR, MS, and GPC for all new compounds (Figure S27–S55). Conflict of Interest There is no conflict of interest to report. Funding Information This work was supported by the National Science Foundation of China (nos. 22071078 and 21722403) and the Program for JLU Science and Technology Innovative Research Team (JLUSTIRT) (no. 2019TD-36). References 1. Catterall W. A.From Ionic Currents to Molecular Mechanisms: The Structure and Function of Voltage-Gated Sodium Channels.Neuron2000, 26, 13–25. Google Scholar 2. Kasimova M. A.; Granata D.; Carnevale V.Chapter Nine—Voltage-Gated Sodium Channels: Evolutionary History and Distinctive Sequence Features. In Current Topics in Membranes; French R. J., Noskov S. Y., Eds.; Academic Press: Elsevier, 2016; Vol 78, pp 261–286. Google Scholar 3. 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