Effects of HNTX-VII on Kv4.2 and Kv4.3 and Molecular Determinants of Kv4.3 Interacting with HNTX-VII

Effects of HNTX-VII on Kv4.2 and Kv4.3 and Molecular Determinants of Kv4.3 Interacting with HNTX-VII | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Effects of HNTX-VII on Kv4.2 and Kv4.3 and Molecular Determinants of Kv4.3 Interacting with HNTX-VII Bo Chen, Zhaotun Hu, Chen Renzhong, He Juan, Xiongzhi Zeng This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4866716/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract HNTX-VII is a novel peptide isolated and purified from the venom of the Chinese spider Ornithoctonus hainana , with a relative molecular mass of 3830.973 Da. Electrophysiological experiments have demonstrated that HNTX-VII exhibits minimal effects on TTX-S, TTX-R, and delayed rectifier potassium channels on dorsal root ganglia (DRG), as well as on Kv1.4 and Kv4.1. However, it significantly inhibits Kv4.2 and Kv4.3 currents, with IC50 values of 299.6 ± 6.48 nM and 114.5 ± 5.36 nM, respectively, for Kv4.2 and Kv4.3. The sequence of HNTX-VII, determined by Edman degradation, is ECRYWLGTCSKTGDCCSHLSCSPKHGWCVWDWT. Composed of 33 amino acids and containing 3 pairs of disulfide bonds, this molecule represents a typical inhibitor cystine knot (ICK) motif. Furthermore, HNTX-VII alters the kinetic properties of Kv4.2 and Kv4.3 channels by causing corresponding shifts in their steady-state activation, steady-state inactivation, and inactivation recovery curves. To further investigate the molecular mechanism underlying the interaction between HNTX-VII and Kv4.3 channels, 19 mutants in the extracellular loops of the S1-S2 and S3b-S4 segments of the Kv4.3 channel were designed and constructed using site-directed mutagenesis. Electrophysiological techniques were then employed to assess the inhibitory activity of HNTX-VII against these mutants. Notably, the V282A mutant in the S3b-S4 loop exhibited the most significant reduction in sensitivity to HNTX-VII, with an IC50 value 5.37 times that of the wild-type channel. Therefore, it is inferred that the 282nd amino acid in the extracellular loop of S3b-S4 serves as a crucial site for the interaction between HNTX-VII and Kv4.3. HNTX-VII Kv 4.2 Kv 4.3 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction Many animals in nature can utilize toxins they produce to hunt prey or defend against natural enemies, such as snakes, spiders, puffer fish, ants, cone snails, scorpions, and more[1.4]. They inject venom produced within their bodies into other animals to paralyze or kill their prey. In recent years, as scientists have conducted more research in this field, they have increasingly discovered the abundance and valuable biological resources contained in animal venoms[5-7]. Potassium ion channels are the most diverse type of ion channels in the body, existing widely and being among the most complex[8]. Research has demonstrated their involvement in multiple physiological activities. As such, the study of potassium ion channel structure and function is currently a research hotspot in ion channel and even molecular biology[9]. Potassium ion channels are composed of four a-subunits and some auxiliary subunits. Like other ion channels, each subunit consists of six transmembrane domains (s1-s6), including a central pore domain formed by the tetrameric arrangement of S5 and S6 transmembrane helices and a voltage-sensing domain composed of S1 to S4 transmembrane helices[10]. Based on these two functional domains, toxin molecules acting on potassium channels are classified into pore-blocking molecules and gating-modulating molecules. In mammals, potassium ion channels are divided into two major families: voltage-gated channels and inward rectifier channels. Voltage-gated potassium channels are further classified into delayed rectifier potassium channels, A-type transient potassium channels, and calcium-activated potassium channels, which are distributed differently in the body and participate in distinct functions[11]. The Kv4 (Shal) family of voltage-gated potassium ion channels is widely distributed in various tissues of mammals, with high expression levels in the central nervous system and heart. This family belongs to A-type transient potassium channels, characterized by rapid activation and inactivation. Currently, Kv4.1, Kv4.2, Kv4.3, and Kv1.4 have been cloned and belong to this channel family. The transient outward potassium current in cardiomyocytes is primarily encoded by three genes: Kv1.4, Kv4.2, and Kv4.3. These genes, especially Kv4.3, participate in the repolarization of cardiomyocyte membrane potential, playing a crucial role in regulating the amplitude and duration of cardiac action potentials, thereby maintaining normal cardiac function. For this reason, this channel is considered a drug target for arrhythmia, and its specific inhibitors hold promise as anti-arrhythmic drugs[12,13]. We isolated HNTX-VII from the Hainan bird-eating spider. HNTX-VII significantly inhibits Kv4.2 and Kv4.3 potassium channel currents in a concentration- and time-dependent manner, with half-maximal inhibitory concentrations (IC50) of 299.6 ± 6.48 nM and 114.5 ± 5.36 nM, respectively. HNTX-VII also shifts the steady-state activation, steady-state inactivation, and recovery from inactivation curves of Kv4.2 and Kv4.3, altering the kinetic properties of the channels. Further electrophysiological experiments demonstrated that HNTX-VII had no effect on TTX-resistant (TTX-R) currents expressed in dorsal root ganglia (DRG) but slightly inhibited TTX-sensitive (TTX-S) currents, with an IC50 of 1.854 mM. To further investigate the molecular mechanism of the interaction between HNTX-VII and Kv4.3 channels, we designed and constructed 19 mutants on the extracellular loops of the S1-S2 and S3b-S4 segments of the Kv4.3 channel using site-directed mutagenesis. Electrophysiological assays to detect the inhibitory activity of HNTX-VII on these mutants revealed that the V282A mutant on the S3b-S4 segment exhibited the greatest reduction in inhibitory activity by HNTX-VII, with an IC50 5.37 times higher than that of the wild type. Therefore, we speculate that amino acid 282 on the extracellular loop of S3b-S4 is a critical site for the interaction between HNTX-VII and Kv4.3. In summary, our findings demonstrate that HNTX-VII is a novel gating modulatory toxin for Kv4.2 and Kv4.3 channels. It interacts with the voltage-sensitive domain and extracellular loop of the channel to alter the gating properties of the channel, thereby determining the opening and closing of the channel. This serves as a good tool reagent for studying Kv4.2 and Kv4.3 channels. 2. Methods and material 2.1 Experimental Materials and Animal Sources This study employed Sprague-Dawley rats from Xiangya School of Medicine, Central South University, adhering to NIH ethical guidelines. Approval was granted by Huaihua University's Animal Care Committee. Venom samples were carefully collected, freeze-dried, and experiments utilized DMEM and Sigma reagents. 2.2 Toxin Refinement Process Venom for the study originated from both sexes of Ornithoctonus hainana spiders. Venom was collected via electrical stimulation, as documented. Purification of HNTX-VII followed established protocols, utilizing reverse phase HPLC for multiple iterations[14]. All the HNTX-VII used in this study were isolated and purified from natural toxins. 2.3 Characterization of Toxin via Mass Spectrometry and N-Terminal Sequencing Molecular mass of HNTX-VII was determined using ABI's Voyager-DETM STR MALDI-TOF mass spectrometer. Its amino acid sequence was deciphered with an Applied Biosystems/Perkin-Elmer Procise 491-A protein sequencer. 2.4 cDNA cloning of HNTX-VII cDNA sequence of HNTX-VII was finished using the method of RACE. Total RNA was extracted from the venom gland which was isolated and frozen in liquid nitrogen. The whole cDNA sequence of HNTX-VII was completed by overlapping 3’ RACE and 5’ RACE fragments. 2.5 Extraction and Cultivation of DRG Neurons Dorsal root ganglion (DRG) neurons from rats were acutely dissociated and maintained in a primary culture setting, with a focus on short-term cultivation, ensuring the optimal growth and health of the neurons. 2.6 Electrophysiological Examinations Using an EPC-10 patch clamp amplifier (HEKA Electronics, Germany), voltage-clamp recordings were conducted with whole-cell patch-clamp techniques. Borosilicate glass pipettes, pulled with a PC-10 puller, facilitated the recordings. For sodium current analysis, the internal solution contained CsF, NaCl, Hepes, and EGTA, while the external bath solution comprised NaCl, CsCl, D-glucose, MgCl 2 , CaCl 2 , HEPES, and tetramethylammonium chloride. Neurons of varying diameters were studied to differentiate between TTX-S and TTX-R sodium currents, with 0.2 mM TTX employed to isolate TTX-R currents. Additionally, K + currents in DRG neurons and HEK293 cells were recorded using standard pipette and bath solutions. 2.7 Construction of Kv4.3 channel mutants A primer pair tailored for the mutation site's target genes was designed using Primer Premier 5.0, with both primers around 25-30 nucleotides long. PCR amplification was performed and validated by agarose gel electrophoresis. After template removal, the plasmid was transformed into DH5a (TSC-C14, Tsingke Biotechnology, China) for expression. The mutant plasmid was extracted using PureLink™HiPure Kit (K210004, Thermo Fisher Scientific, USA), following strict protocols. A 3-5 mL purified plasmid sample was sent to Changsha Tsingke Biotechnology for comprehensive sequencing to confirm successful mutation. 2.8 Data analysis Experimental data were acquired and analyzed by the program pulse + pulsefit8.0 (HEKA,Germany). Data analysises were performed using Sigmaplot (Sigma, USA). All data are showed as mean ± standard error and n is the number of independent experiments. The fitted curves of both concentration-dependent inhibition (inhibition%) and steady-state Na+ channels inactivation (I/Imax) were obtained by using the following form of the Boltzmann equation: inhibition%=100/[1+exp(C-IC 50 )/K] (1) I/Imax=1/[1+exp(V-V1/2)/K] (2) In Eq.(1) where IC 50 is the concentration of toxin at half-maximal inhibition and K is the slope factor, C is the toxin concentration. In Eq.(2) where V1/2 is the voltage of half inactivation and K is the slope factor, V is the test voltage. 3. Results 3.1 Purification, Identification and analysis of amino-acid sequences of HNTX-VII The preliminary separation and purification of pretreated crude venom from Hainan were performed using reversed-phase HPLC, resulting in the chromatogram shown in Figure 1A. The target peak, marked with an asterisk, was named HNTX-VII. Subsequent mass spectrometry identified its relative molecular mass as 3830.97 Da (Figure 1B). The sequence of HNTX-VII, determined by Edman degradation, is ECRYWLGTCSKTGDCCSHLSCSPKHGWCVWDWT, consisting of 33 amino acid residues with six cysteines forming three disulfide bonds, suggesting that it is likely a typical inhibitor cystine knot (ICK) motif molecule(Figure 1C). Concurrently, a sequence homology alignment was conducted, with conserved cysteines highlighted in black, revealing a 70% sequence similarity between Protx-1 and HNTX-VII (Figure 1D). 3.2 Inhibitory Effects of HNTX-VII on Sodium and Potassium Channels The inhibitory effects of HNTX-VII on TTX-resistant (TTX-R) and TTX-sensitive (TTX-S) sodium currents expressed in dorsal root ganglion (DRG) cells of Sprague-Dawley (SD) rats are shown in Figures 2A and 2B: In Figure 2A, 1 mM HNTX-VII can only inhibit approximately 20% of the TTX-R sodium current. In Figure 2B, 1 mM HNTX-VII inhibits approximately 30% of the TTX-S sodium current, while 5 mM HNTX-VII significantly inhibits the TTX-S current. Figure 2C illustrates that the inhibition of TTX-S sodium current by HNTX-VII is concentration-dependent, with an IC50 value of 1.854 mM for HNTX-VII against TTX-S sodium current. This suggests that HNTX-VII does not exhibit strong inhibitory activity against sodium currents.The inhibitory effect of HNTX-VII on delayed rectifier potassium currents expressed in SD rat DRG is depicted in Figure 2D: It is evident from the figure that 1 mM HNTX-VII has no effect on delayed rectifier potassium currents in rat DRG. Further experiments confirm that HNTX-VII exerts only weak inhibitory effects on Kv1.4 and Kv4.1 transiently expressed in HEK293T cells (Figures 2E and 2F). However, Inhibitory Effect of HNTX-VII on Kv4.2 (Figure 3A): Cells were clamped at -80mV, and a transient outward potassium current was elicited by applying a 300ms, +10mV test pulse. 300nM HNTX-VII significantly inhibited Kv4.2, while 1 mM HNTX-VII completely inhibited the current. As seen in Figure 3B, the inhibition of Kv4.2 by HNTX-VII is concentration-dependent, with an IC 50 value of 299.6 ± 6.48 nM for HNTX-VII against Kv4.2 potassium current. Inhibitory Effect of HNTX-VII on Kv4.3 (Figure 3C): 100 nM HNTX-VII significantly inhibited Kv4.3, while 500 nM HNTX-VII completely inhibited the current. As evident from Figure 3D, the inhibition of Kv4.3 by HNTX-VII is also concentration-dependent, with an IC 50 value of 114.5 5.36 nM for HNTX-VII against Kv4.3 potassium current. 3.3 Voltage-dependent Inhibition of Kv4.2 and Kv4.3 by HNTX-VII As seen in Figure 4A, the inhibition of Kv4.2 potassium channels by HNTX-VII exhibits voltage dependence. At the same concentration of the toxin (300 nM), the inhibited current decreases as the voltage increases, indicating that high voltages can dissociate the binding of the toxin to the channel. Figure 4B demonstrates that the inhibition of Kv4.3 potassium channels by HNTX-VII also exhibits voltage dependence. 3.4 Effect of HNTX-VII on Activation and Inactivation of Kv4.2 and Kv 4.3. The impact of HNTX-VII on the activation and inactivation properties of Kv4.2 potassium currents is illustrated in Figures 4C and 4D. Figure 4C depicts the activation kinetics of Kv4.2. As shown in the figure, after inhibition by HNTX-VII, the activation time constants increase correspondingly at all voltages, indicating that the HNTX-VII delays channel activation. Furthermore, as the voltage increases, the time constants decrease correspondingly, suggesting that higher voltages facilitate faster peak current attainment compared to lower voltages. Figure 4D presents the inactivation kinetics of Kv4.2 potassium channels. It can be observed from the figure that HNTX-VII delays channel inactivation, resulting in inactivation time constants that are larger than the control values at all voltages. The effect of HNTX-VII on the activation and inactivation properties of Kv4.3 potassium currents is illustrated in Figures 4E and 4F. Figure 4E represents the activation kinetics of Kv4.3 potassium currents. As shown in the figure, the time constants decrease with increasing voltage, indicating that higher voltages accelerate channel activation. Under low voltages (less than 30mV), HNTX-VII delays channel activation, whereas at high voltages (greater than 30mV), HNTX-VII has no significant effect on channel activation. This suggests that high voltages can overcome the subtle inhibitory effect of HNTX-VII on activation. Figure 4F depicts the inactivation kinetics of Kv4.3 potassium currents. It can be observed from the figure that HNTX-VII delays channel inactivation, resulting in inactivation time constants that are larger than the control values at all voltages. 3.5 Effect of HNTX-VII on Steady-State Activation and Inactivation Curves of Kv4.2 Figure 5A illustrates the steady-state activation curve. The half-activation voltage of cells without HNTX-VII is 20.69 mV, which shifts to 32.9 mV upon addition of 300 nM HNTX-VII, and further shifts to 45.55 mV when 1μM HNTX-VII is added. The half-activation voltage increases with the concentration of the HNTX-VII, indicating that the inhibitory effect of HNTX-VII enhances the difficulty of channel opening, causing the steady-state activation curve to shift towards depolarization in a concentration-dependent manner. Additionally, as the HNTX-VII concentration increases, the slope decreases, suggesting that HNTX-VII modifies the steady-state activation curve of the channel. As the voltage increases, the range of channel opening expands from mostly closed to mostly open, demonstrating that an increase in depolarization voltage can activate channels bound to HNTX-VII. Figure 5B shows the steady-state inactivation curve. When the channel is inhibited by 1μM HNTX-VII, the half-steady-state inactivation voltage increases by approximately 14mV, indicating that the steady-state inactivation curve shifts towards depolarization by 14mV. Confirming that the inhibitory effect of the toxin alters the inactivation curve characteristics of the channel. 3.6 Effect of HNTX-VII on Steady-State Activation and Inactivation Curves of Kv4.3 Figure 5C depicts the steady-state activation curve. The inhibitory effect of 200 nM HNTX-VII on the channel current increases the half-steady-state activation voltage by 13 mV, causing the steady-state activation curve to shift towards depolarization. This indicates that HNTX-VII alters the characteristics of the channel, making it more difficult for the channel to open. Figure 5D shows the steady-state inactivation curve. Under three conditions: control, 200 nM HNTX-VII, and 500 nM HNTX-VII, there are minimal changes observed in the curve, with negligible fluctuations in the half-steady-state inactivation voltage and slope K value. Therefore, HNTX-VII does not significantly affect the inactivation characteristics of the Kv4.3 channel and does not exhibit concentration-dependence in this regard. 3.7 Effect of HNTX-VII on Inactivation Recovery Time Constants of Kv4.2 and Kv4.3 Channels Figure 5E presents the inactivation recovery curve for the Kv4.2 channel. The recovery curves under control conditions and with inhibition by 500 nM HNTX-VII are similar, with calculated recovery time constants (τ values) of approximately 117 ms for both. This suggests that HNTX-VII has no effect on the recovery of Kv4.2 channels following inactivation. Figure 5F shows the inactivation recovery curve for the Kv4.3 channel. Under control conditions, the recovery time constant of the channel is 43.5 ± 1.98 ms. However, when the channel is inhibited by 200 nM HNTX-VII, the calculated recovery time constant increases to 72.5 ± 3.02 ms, an increase of 29 ms. Therefore, the inhibitory effect of HNTX-VII makes it more difficult for the channel to recover from the inactivated state, altering the inactivation recovery kinetics. 3.8 Molecular Mechanism of Kv4.3 Binding to HNTX-VII As shown in Table 1, the IC 50 values of HNTX-VII acting on the wild-type Kv4.3 channel and its various mutants, as well as their ratios to the wild-type IC 50 , reveal that the V282A mutant on the extracellular loop between S3 and S4 exhibits the highest IC 50 value, which is more than five times that of the wild-type. This indicates that the 282nd amino acid V (valine) on the extracellular loop between S3 and S4 is the most critical residue affecting the interaction between HNTX-VII and the Kv4.3 channel. Additionally, residues such as E280, F286, L274, and G273 also have significant impacts(Figure 6). 4. Discussion The binding sites of toxins with voltage-gated potassium channels are either located in the pore region or in the voltage-sensitive extracellular loop region. Depending on the binding site, toxins are classified into pore-blocking and gating-modulatory types[15,16]. In recent decades, most peptides isolated and purified from animal venoms belong to the gating-modulatory type, which either rely on charge-charge interactions, hydrophobic interactions, or both to bind to the extracellular loop of the voltage-sensitive region of the channel, thereby affecting the gating properties of the channel[17,18]. Kv4.3 is a typical voltage-dependent potassium channel belonging to the transient outward potassium channel kv4 (Shal) family, which is highly expressed in cardiomyocytes and has a significant impact on the duration and amplitude of the cardiac action potential[19]. Both activation and inactivation of this channel exhibit time and voltage dependence. HpTx-2, isolated from spider venom, specifically acts on Kv4.2 and Kv4.3 potassium channels in Ito, inhibiting them in a voltage-dependent manner. HpTx-2 shifts the activation curve of the Kv4.3 potassium channel towards depolarization, accelerates channel deactivation, and delays inactivation. It is speculated that this molecule alters the gating properties of the channel by binding to the extracellular portion of the voltage-sensitive region of the Kv4.3 potassium channel[20]. Christopher V et al. further investigated the mechanism of interaction between HpTx-2 and the Kv4.3 potassium channel. Using site-directed mutagenesis, mutants on the S3b segment were constructed, and it was found that the V276A and L275A mutants weakened the binding strength between the toxin and the channel. When both sites were mutated simultaneously, HpTx-2 basically did not bind to the Kv4.3 channel, suggesting that these two hydrophobic amino acid residues are crucial for toxin-channel binding[20]. These amino acids share similar characteristics with the key residues (IF) of hanatoxin (HaTX) binding to the Kv2.1 potassium channel, which are located in the same region as the S3b segment of the Kv4.3 potassium channel[21]. When the LV in the S3b segment of the Kv4.3 potassium channel was mutated to IF, the binding between HpTx-2 and the Kv4.3 channel was strengthened, further confirming the importance of hydrophobic amino acids at this position for HpTx-2 binding to the Kv4.3 channel[20,22]. Literature has reported other polypeptides isolated from spider venoms that specifically inhibit the Kv4.3 potassium channel, including phrixotoxins and JZTX-V[23,24]. These toxins are believed to have a similar hydrophobic surface surrounded by charged amino acid residues. They all alter the kinetic properties of the Kv4.3 channel and are thought to do so by binding the hydrophobic surface of the toxin to the extracellular loop of the voltage-sensitive region of the Kv4.3 potassium channel, thereby modifying the channel's gating properties. HNTX-VII is a newly discovered spider toxin that specifically acts on Kv4.3. Its inhibition of the Kv4.3 potassium channel is voltage-dependent, and the toxin dissociates from the channel under higher voltages. Previous experiments have demonstrated that it can alter the gating properties of the channel, shifting the steady-state activation curve of the Kv4.3 potassium channel towards depolarization, increasing the difficulty of channel opening, and delaying the recovery after channel inactivation. Therefore, it is speculated that, similar to other ICK toxins, HNTX-VII is also a gating modulatory toxin that controls the opening and closing of the channel through the interaction between the hydrophobic amino acids and charged amino acid domains of the toxin and the extracellular loop region of the channel's voltage-sensitive domain. To delve into the molecular mechanism of the interaction between HNTX-VII and Kv4.3, this experiment utilized site-directed mutagenesis to construct 19 mutants in the S1-S2 extracellular loop and S3b-S4 extracellular loop and examined the inhibitory activity of HNTX-VII against each mutant. It was found that hydrophobic amino acids and a charged amino acid had significant impacts on the inhibitory activity of the channel, specifically G273, L274, V275 on the S3b segment, and E280, V282, F286 in the S3-S4 extracellular loop . Interestingly, mutations with substantial effects, such as the hydrophobic amino acids (G273, L274, V275) and the charged E280, occupy similar positions in the extracellular loop as the key residues for hanatoxin binding to the Kv2.1 potassium channel, and are also identical to the crucial amino acid significantly influencing the binding of HpTx-2 to the Kv4.3 channel. This leads to the inference that HNTX-VII may function similarly to these toxins in modulating the Kv4.3 channel, specifically by acting on the channel through the hydrophobic amino acid residues of the toxin and the surrounding charged amino acids. V282A is the mutant that has the most significant impact on the binding of HNTX-VII to the Kv4.3 potassium channel, increasing the half-maximal inhibitory concentration (IC50) of HNTX-VII by 5.4-fold. Hence, V282A is a crucial residue for the interaction between HNTX-VII and Kv4.3. Furthermore, being a non-polar amino acid, it reinforces the importance of hydrophobic interactions in the binding of HNTX-VII to this channel. It is noteworthy that during the conduct of this experiment, it was discovered that the V282AF286A double mutant exhibited no detectable current under voltage stimulation. However, upon adding HNTX-VII and applying voltage stimulation, a current was detected, which increased with the concentration of the toxin. In this scenario, HNTX-VII transformed from an inhibitor into an activator for the double mutant, with the underlying molecular mechanism remaining unclear. Nevertheless, studying this phenomenon will undoubtedly have significant implications for unraveling the interactions between toxins and channels. Declarations CRediT authorship contribution statement C and XZ.Z initiated the research concept, devised the experimental framework, and outlined the structure of the paper. They also wrote the majority of the paper's content and oversaw the revisions. Additionally, B.C, ZT.H, HB.S, RZ.C and J.H conducted the majority of the experiments and performed the data analyses. All authors participated in the discussion of the results, provided feedback on the manuscript, and gave their final approval for submission. Competing Interests The authors declare no competing interests. Ethical approval Not applicable Funding This work was supported by the Hunan Provincial Natural Science Foundation (NO.2021JJ30540). The Hunan Education Department Project (23A0552). Huaihua University key projects (HHUY2022-10, HHUY2019-07). References Luo A, Wang A, Kamau PM, Lai R, Luo L. Centipede Venom: A Potential Source of Ion Channel Modulators. Int J Mol Sci. 2022 Jun 26;23(13):7105. Shaikh NY, Sunagar K. The deep-rooted origin of disulfide-rich spider venom toxins. Elife. 2023 Feb 9;12:e83761. doi: 10.7554/eLife.83761. Billen B, Bosmans F, Tytgat J. 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S3b amino acid substitutions and ancillary subunits alter the affinity of Heteropoda venatoria toxin 2 for Kv4.3. Mol Pharmacol. 2009 Jul;76(1):125-33. Diochot S, Drici MD, Moinier D, Fink M, Lazdunski M. Effects of phrixotoxins on the Kv4 family of potassium channels and implications for the role of Ito1 in cardiac electrogenesis. Br J Pharmacol. 1999 Jan;126(1):251-63. Dehong X, Wenmei W, Siqin H, Peng Z, Xianchun W, Xiongzhi Z. Effects of JZTX-V on the wild type Kv4.3 Expressed in HEK293T and Molecular Determinants in the Voltage-sensing Domains of Kv4.3 Interacting with JZTX-V. Channels (Austin). 2022 Dec;16(1):72-83. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4866716","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":341996853,"identity":"73788ff7-4a1c-45cc-886a-f33719e16cfc","order_by":0,"name":"Bo Chen","email":"","orcid":"","institution":"Huaihua University","correspondingAuthor":false,"prefix":"","firstName":"Bo","middleName":"","lastName":"Chen","suffix":""},{"id":341996856,"identity":"55f6ea07-1eee-46b8-b856-546a5911fec5","order_by":1,"name":"Zhaotun Hu","email":"","orcid":"","institution":"Huaihua University","correspondingAuthor":false,"prefix":"","firstName":"Zhaotun","middleName":"","lastName":"Hu","suffix":""},{"id":341996857,"identity":"daa5201c-ecc0-4ba5-b4ac-10aa97fbb1dc","order_by":2,"name":"Chen Renzhong","email":"","orcid":"","institution":"Hunan Normal University","correspondingAuthor":false,"prefix":"","firstName":"Chen","middleName":"","lastName":"Renzhong","suffix":""},{"id":341996858,"identity":"e5b7ce8b-be56-4360-a685-b3b8b5c2d85b","order_by":3,"name":"He Juan","email":"","orcid":"","institution":"Hunan Normal University","correspondingAuthor":false,"prefix":"","firstName":"He","middleName":"","lastName":"Juan","suffix":""},{"id":341996861,"identity":"84cdffd9-81ad-4c7e-84c3-d16df46d2725","order_by":4,"name":"Xiongzhi Zeng","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAzUlEQVRIiWNgGAWjYPACmwQog5loLWmkazlMghb59t7Dr3n+nM8zuN17TIKhwjqxgf3sAbxaGHvOpVnO4LldbHDnXJoEw5n0xAaevAS8WpglcswMPkjcTtxwI8dMgrHtcGKDBI8BXi1s8m/MDBIMzkG1/CNCC48Ej/GDDwkHoFoaiNAiwZNjxjjjQHLizBs5xhYJx9KN23hy8GuRbz9j/Jnnj11i340cwxsfaqxl+9nP4NcC8o4EnJkA4hJSDwTMH4hQNApGwSgYBSMZAABi1ENV2xUUAwAAAABJRU5ErkJggg==","orcid":"","institution":"Hunan Normal University","correspondingAuthor":true,"prefix":"","firstName":"Xiongzhi","middleName":"","lastName":"Zeng","suffix":""}],"badges":[],"createdAt":"2024-08-06 08:12:56","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4866716/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4866716/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":64030715,"identity":"c37f34bc-3569-44fa-8208-5ea090c728b2","added_by":"auto","created_at":"2024-09-05 09:02:35","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":189216,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePurification and Identification of HNTX-VII. \u003c/strong\u003eA. reversed-phase chromatography was performed on a reversed-phase column. Asterisked peak in panel A. The typical MALDI-TOF mass spectrometry analysis of HNTX-VII.C. The oligo-nucleotide sequence of HNTX-VII cDNA. The amino acid composition of the precursor reading from the cDNA is suggested below the nucleotide sequence. The cDNA encoding the mature peptide is underlined. The C terminal amidation signals of GK are in shadow. The two arrows show the position of signal peptide and pro-peptide. The polyadenylations signal, aataaa, is double underlined. D.The sequence alignment of HNTX-VII and other high identity toxins. The cysteine residue was highlighted with black. The predicted disulfide linkage is indicated below as lines.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4866716/v1/bc51d3d91ffac0d0c089b56d.png"},{"id":64031199,"identity":"bd7d0682-5a2a-4933-b6a7-859f3f4397a4","added_by":"auto","created_at":"2024-09-05 09:10:35","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":97372,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of HNTX-VII in rat DRG neurons,Kv1.4 and Kv 4.1 channels. \u003c/strong\u003eA.The typical traces of adding 1 mM HNTX-VII to TTX-R sodium current on rat DRG neurons. B.The typical traces of adding 1 mM HNTX-VII to TTX-S sodium current on rat DRG neurons. C. Representative concentration-response curves for HNTX-VII on TTX-S sodium current (n=8). D.The typical traces of adding 1 mM HNTX-VII to delayed rectification current on rat DRG neurons.E and F. 1 and 10 mM HNTX-VII acts on Kv 1.4 and Kv 4.1.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4866716/v1/a2c64b0cd5b686b289317bea.png"},{"id":64031538,"identity":"09d34b5b-af32-4829-8854-a25f330da7b8","added_by":"auto","created_at":"2024-09-05 09:18:35","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":131583,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of HNTX-VII in Kv 4.2 and Kv 4.3. \u003c/strong\u003e(A) The typical traces of HNTX-VII to Kv 4.2. (B) Representative concentration-response curves for HNTX-VII on Kv 4.2 (n=8). (C) The typical traces of HNTX-VII to Kv 4.3. (D) Representative concentration-response curves for HNTX-VII on Kv 4.3 (n=8).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4866716/v1/d3d2c12ee396cfbec8d3ce56.png"},{"id":64030719,"identity":"a5087bcc-74a1-4103-addf-4d55e829d860","added_by":"auto","created_at":"2024-09-05 09:02:35","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":164803,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHNTX-VII dynamic curve analysis of Kv4.2 and Kv4.3. \u003c/strong\u003eA and B.Voltage dependent suppression of Kv4.2 and Kv4.3 by HNTX-VII. C and D.Effect of HNTX-VII on activation and deactivation characteristics of Kv4.2. E and F.Effect of HNTX-VII on activation and deactivation characteristics of Kv4.3.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4866716/v1/0fc9f0cc62e050bbd7146eb7.png"},{"id":64031200,"identity":"9e487c83-9aa0-4e2c-aece-8707d45220c4","added_by":"auto","created_at":"2024-09-05 09:10:35","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":191416,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of HNTX-VII on steady-state activation and inactivation curves of Kv4.2 and Kv4.3. \u003c/strong\u003eA and B. Effects of HNTX-VII on the steady-state activation and inactivation curves of Kv4.2. C and D.Effects of HNTX-VII on the steady-state activation and inactivation curves of Kv4.3. E and F. Effect of HNTX-VII on recovery from Kv4.2 and Kv4.3 inactivation.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-4866716/v1/49b18b37a989b93571b1feeb.png"},{"id":64030720,"identity":"c806b6fe-5c5b-469d-9663-cdb4edb9816c","added_by":"auto","created_at":"2024-09-05 09:02:35","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":76032,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eComparison of the effects of HNTX-VII on Kv4.3 wild type and various mutants.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-4866716/v1/ee0868715fae0ea652c4f306.png"},{"id":65029092,"identity":"d16e6e74-c252-4f0f-ac51-b0a04017e7b4","added_by":"auto","created_at":"2024-09-22 19:47:32","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1443906,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4866716/v1/7ee2423f-b0c1-4310-9f95-0e4237593d52.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003e\u003cstrong\u003eEffects of HNTX-VII on Kv4.2 and Kv4.3 and Molecular Determinants of Kv4.3 Interacting with HNTX-VII\u003c/strong\u003e\u003c/p\u003e","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eMany animals in nature can utilize toxins they produce to hunt prey or defend against natural enemies, such as snakes, spiders, puffer fish, ants, cone snails, scorpions, and more[1.4]. They inject venom produced within their bodies into other animals to paralyze or kill their prey. In recent years, as scientists have conducted more research in this field, they have increasingly discovered the abundance and valuable biological resources contained in animal venoms[5-7].\u003c/p\u003e\n\u003cp\u003ePotassium ion channels\u0026nbsp;are the most diverse type of ion channels in the body, existing widely and being among the most complex[8]. Research has demonstrated their involvement in multiple physiological activities. As such, the study of potassium ion channel structure and function is currently a research hotspot in ion channel and even molecular biology[9]. Potassium ion channels are composed of four\u0026nbsp;a-subunits and some auxiliary subunits. Like other ion channels, each subunit consists of six transmembrane domains (s1-s6), including a central pore domain formed by the tetrameric arrangement of S5 and S6 transmembrane helices and a voltage-sensing domain composed of S1 to S4 transmembrane helices[10]. Based on these two functional domains, toxin molecules acting on potassium channels are classified into pore-blocking molecules and gating-modulating molecules. In mammals, potassium ion channels are divided into two major families: voltage-gated channels and inward rectifier channels. Voltage-gated potassium channels are further classified into delayed rectifier potassium channels, A-type transient potassium channels, and calcium-activated potassium channels, which are distributed differently in the body and participate in distinct functions[11].\u003c/p\u003e\n\u003cp\u003eThe Kv4 (Shal) family of voltage-gated potassium ion channels is widely distributed in various tissues of mammals, with high expression levels in the central nervous system and heart. This family belongs to A-type transient potassium channels, characterized by rapid activation and inactivation. Currently, Kv4.1, Kv4.2, Kv4.3, and Kv1.4 have been cloned and belong to this channel family. The transient outward potassium current in cardiomyocytes is primarily encoded by three genes: Kv1.4, Kv4.2, and Kv4.3. These genes, especially Kv4.3, participate in the repolarization of cardiomyocyte membrane potential, playing a crucial role in regulating the amplitude and duration of cardiac action potentials, thereby maintaining normal cardiac function. For this reason, this channel is considered a drug target for arrhythmia, and its specific inhibitors hold promise as anti-arrhythmic drugs[12,13].\u003c/p\u003e\n\u003cp\u003eWe isolated HNTX-VII from the Hainan bird-eating spider. HNTX-VII significantly inhibits Kv4.2 and Kv4.3 potassium channel currents in a concentration- and time-dependent manner, with half-maximal inhibitory concentrations (IC50) of 299.6\u0026nbsp;±\u0026nbsp;6.48 nM and 114.5\u0026nbsp;±\u0026nbsp;5.36 nM, respectively. HNTX-VII also shifts the steady-state activation, steady-state inactivation, and recovery from inactivation curves of Kv4.2 and Kv4.3, altering the kinetic properties of the channels. Further electrophysiological experiments demonstrated that HNTX-VII had no effect on TTX-resistant (TTX-R) currents expressed in dorsal root ganglia (DRG) but slightly inhibited TTX-sensitive (TTX-S) currents, with an IC50 of 1.854 mM.\u003c/p\u003e\n\u003cp\u003eTo further investigate the molecular mechanism of the interaction between HNTX-VII and Kv4.3 channels, we designed and constructed 19 mutants on the extracellular loops of the S1-S2 and S3b-S4 segments of the Kv4.3 channel using site-directed mutagenesis. Electrophysiological assays to detect the inhibitory activity of HNTX-VII on these mutants revealed that the V282A mutant on the S3b-S4 segment exhibited the greatest reduction in inhibitory activity by HNTX-VII, with an IC50 5.37 times higher than that of the wild type. Therefore, we speculate that amino acid 282 on the extracellular loop of S3b-S4 is a critical site for the interaction between HNTX-VII and Kv4.3.\u003c/p\u003e\n\u003cp\u003eIn summary, our findings demonstrate that HNTX-VII is a novel gating modulatory toxin for Kv4.2 and Kv4.3 channels. It interacts with the voltage-sensitive domain and extracellular loop of the channel to alter the gating properties of the channel, thereby determining the opening and closing of the channel. This serves as a good tool reagent for studying Kv4.2 and Kv4.3 channels.\u003c/p\u003e"},{"header":"2. Methods and material ","content":"\u003cp\u003e\u003cstrong\u003e2.1 Experimental Materials and Animal Sources\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study employed Sprague-Dawley rats from Xiangya School of Medicine, Central South University, adhering to NIH ethical guidelines. Approval was granted by Huaihua University\u0026apos;s Animal Care Committee. Venom samples were carefully collected, freeze-dried, and experiments utilized DMEM and Sigma reagents.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2 Toxin Refinement Process\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eVenom for the study originated from both sexes of Ornithoctonus hainana spiders. Venom was collected via electrical stimulation, as documented. Purification of HNTX-VII followed established protocols, utilizing reverse phase HPLC for multiple iterations[14]. All the HNTX-VII used in this study were isolated and purified from natural toxins.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3 Characterization of Toxin via Mass Spectrometry and N-Terminal Sequencing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMolecular mass of HNTX-VII was determined using ABI\u0026apos;s Voyager-DETM STR MALDI-TOF mass spectrometer. Its amino acid sequence was deciphered with an Applied Biosystems/Perkin-Elmer Procise 491-A protein sequencer.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.4 cDNA cloning of HNTX-VII\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ecDNA sequence of HNTX-VII was finished using the method of RACE. Total RNA was extracted from the venom gland which was isolated and frozen in liquid nitrogen. The whole cDNA sequence of HNTX-VII was completed by overlapping 3\u0026rsquo; RACE and 5\u0026rsquo; RACE fragments.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.5 Extraction and Cultivation of DRG Neurons\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDorsal root ganglion (DRG) neurons from rats were acutely dissociated and maintained in a primary culture setting, with a focus on short-term cultivation, ensuring the optimal growth and health of the neurons.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.6 Electrophysiological Examinations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUsing an EPC-10 patch clamp amplifier (HEKA Electronics, Germany), voltage-clamp recordings were conducted with whole-cell patch-clamp techniques. Borosilicate glass pipettes, pulled with a PC-10 puller, facilitated the recordings. For sodium current analysis, the internal solution contained CsF, NaCl, Hepes, and EGTA, while the external bath solution comprised NaCl, CsCl, D-glucose, MgCl\u003csub\u003e2\u003c/sub\u003e, CaCl\u003csub\u003e2\u003c/sub\u003e, HEPES, and tetramethylammonium chloride. Neurons of varying diameters were studied to differentiate between TTX-S and TTX-R sodium currents, with 0.2 mM TTX employed to isolate TTX-R currents. Additionally, K\u003csup\u003e+\u003c/sup\u003e currents in DRG neurons and HEK293 cells were recorded using standard pipette and bath solutions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.7 Construction of Kv4.3 channel mutants\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA primer pair tailored for the mutation site\u0026apos;s target genes was designed using Primer Premier 5.0, with both primers around 25-30 nucleotides long. PCR amplification was performed and validated by agarose gel electrophoresis. After template removal, the plasmid was transformed into DH5a\u0026nbsp;(TSC-C14, Tsingke Biotechnology, China) for expression. The mutant plasmid was extracted using PureLink\u0026trade;HiPure Kit (K210004, Thermo Fisher Scientific, USA), following strict protocols. A 3-5\u0026nbsp;mL purified plasmid sample was sent to Changsha Tsingke Biotechnology for comprehensive sequencing to confirm successful mutation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.8 Data analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eExperimental data were acquired and analyzed by the program pulse + pulsefit8.0 (HEKA,Germany). Data analysises were performed using Sigmaplot (Sigma, USA). All data are showed as mean \u0026plusmn; standard error and n is the number of independent experiments. The fitted curves of both concentration-dependent inhibition (inhibition%) and steady-state Na+ channels inactivation (I/Imax) were obtained by using the following form of the Boltzmann equation:\u003c/p\u003e\n\u003cp\u003einhibition%=100/[1+exp(C-IC\u003csub\u003e50\u003c/sub\u003e)/K] \u0026nbsp;(1)\u003c/p\u003e\n\u003cp\u003eI/Imax=1/[1+exp(V-V1/2)/K] \u0026nbsp;(2)\u003c/p\u003e\n\u003cp\u003eIn Eq.(1) where IC\u003csub\u003e50\u003c/sub\u003e\u0026nbsp; is \u0026nbsp;the concentration of toxin at half-maximal inhibition and K is the slope factor, C is the toxin concentration. In Eq.(2) where V1/2 is the voltage of half inactivation and K is the slope factor, V is the test voltage.\u003c/p\u003e"},{"header":"3. Results","content":"\u003cp\u003e\u003cstrong\u003e3.1 Purification, Identification and analysis of amino-acid sequences of HNTX-VII\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe preliminary separation and purification of pretreated crude venom from Hainan were performed using reversed-phase HPLC, resulting in the chromatogram shown in Figure 1A. The target peak, marked with an asterisk, was named HNTX-VII. Subsequent mass spectrometry identified its relative molecular mass as 3830.97 Da (Figure 1B). The sequence of HNTX-VII, determined by Edman degradation, is ECRYWLGTCSKTGDCCSHLSCSPKHGWCVWDWT, consisting of 33 amino acid residues with six cysteines forming three disulfide bonds, suggesting that it is likely a typical inhibitor cystine knot (ICK) motif molecule(Figure 1C). Concurrently, a sequence homology alignment was conducted, with conserved cysteines highlighted in black, revealing a 70% sequence similarity between Protx-1 and HNTX-VII (Figure 1D).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.2 Inhibitory Effects of HNTX-VII on Sodium and Potassium Channels\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe inhibitory effects of HNTX-VII on TTX-resistant (TTX-R) and TTX-sensitive (TTX-S) sodium currents expressed in dorsal root ganglion (DRG) cells of Sprague-Dawley (SD) rats are shown in Figures 2A and 2B: In Figure 2A, 1\u0026nbsp;mM HNTX-VII can only inhibit approximately 20% of the TTX-R sodium current. In Figure 2B, 1\u0026nbsp;mM HNTX-VII inhibits approximately 30% of the TTX-S sodium current, while 5\u0026nbsp;mM HNTX-VII significantly inhibits the TTX-S current. Figure 2C illustrates that the inhibition of TTX-S sodium current by HNTX-VII is concentration-dependent, with an IC50 value of 1.854\u0026nbsp;mM for HNTX-VII against TTX-S sodium current. This suggests that HNTX-VII does not exhibit strong inhibitory activity against sodium currents.The inhibitory effect of HNTX-VII on delayed rectifier potassium currents expressed in SD rat DRG is depicted in Figure 2D: It is evident from the figure that 1\u0026nbsp;mM HNTX-VII has no effect on delayed rectifier potassium currents in rat DRG. Further experiments confirm that HNTX-VII exerts only weak inhibitory effects on Kv1.4 and Kv4.1 transiently expressed in HEK293T cells (Figures 2E and 2F). However, Inhibitory Effect of HNTX-VII on Kv4.2 (Figure 3A): Cells were clamped at -80mV, and a transient outward potassium current was elicited by applying a 300ms, +10mV test pulse. 300nM HNTX-VII significantly inhibited Kv4.2, while 1\u0026nbsp;mM HNTX-VII completely inhibited the current. As seen in Figure 3B, the inhibition of Kv4.2 by HNTX-VII is concentration-dependent, with an IC\u003csub\u003e50\u003c/sub\u003e value of 299.6 ± 6.48 nM for HNTX-VII against Kv4.2 potassium current. Inhibitory Effect of HNTX-VII on Kv4.3 (Figure 3C): 100 nM HNTX-VII significantly inhibited Kv4.3, while 500 nM HNTX-VII completely inhibited the current. As evident from Figure 3D, the inhibition of Kv4.3 by HNTX-VII is also concentration-dependent, with an IC\u003csub\u003e50\u003c/sub\u003e value of 114.5 5.36 nM for HNTX-VII against Kv4.3 potassium current.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.3 Voltage-dependent Inhibition of Kv4.2 and Kv4.3 by HNTX-VII\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAs seen in Figure 4A, the inhibition of Kv4.2 potassium channels by HNTX-VII exhibits voltage dependence. At the same concentration of the toxin (300 nM), the inhibited current decreases as the voltage increases, indicating that high voltages can dissociate the binding of the toxin to the channel. Figure 4B demonstrates that the inhibition of Kv4.3 potassium channels by HNTX-VII also exhibits voltage dependence.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.4 Effect of HNTX-VII on Activation and Inactivation of Kv4.2 and Kv 4.3.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe impact of HNTX-VII on the activation and inactivation properties of Kv4.2 potassium currents is illustrated in Figures 4C and 4D. Figure 4C depicts the activation kinetics of Kv4.2. As shown in the figure, after inhibition by HNTX-VII, the activation time constants increase correspondingly at all voltages, indicating that the HNTX-VII delays channel activation. Furthermore, as the voltage increases, the time constants decrease correspondingly, suggesting that higher voltages facilitate faster peak current attainment compared to lower voltages. Figure 4D presents the inactivation kinetics of Kv4.2 potassium channels. It can be observed from the figure that HNTX-VII delays channel inactivation, resulting in inactivation time constants that are larger than the control values at all voltages.\u003c/p\u003e\n\u003cp\u003eThe effect of HNTX-VII on the activation and inactivation properties of Kv4.3 potassium currents is illustrated in Figures 4E and 4F. Figure 4E represents the activation kinetics of Kv4.3 potassium currents. As shown in the figure, the time constants decrease with increasing voltage, indicating that higher voltages accelerate channel activation. Under low voltages (less than 30mV), HNTX-VII delays channel activation, whereas at high voltages (greater than 30mV), HNTX-VII has no significant effect on channel activation. This suggests that high voltages can overcome the subtle inhibitory effect of HNTX-VII on activation. Figure 4F depicts the inactivation kinetics of Kv4.3 potassium currents. It can be observed from the figure that HNTX-VII delays channel inactivation, resulting in inactivation time constants that are larger than the control values at all voltages.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.5 Effect of HNTX-VII on Steady-State Activation and Inactivation Curves of Kv4.2\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFigure 5A illustrates the steady-state activation curve. The half-activation voltage of cells without HNTX-VII is 20.69 mV, which shifts to 32.9 mV upon addition of 300 nM HNTX-VII, and further shifts to 45.55 mV when 1μM HNTX-VII is added. The half-activation voltage increases with the concentration of the HNTX-VII, indicating that the inhibitory effect of HNTX-VII enhances the difficulty of channel opening, causing the steady-state activation curve to shift towards depolarization in a concentration-dependent manner. Additionally, as the HNTX-VII concentration increases, the slope decreases, suggesting that HNTX-VII modifies the steady-state activation curve of the channel. As the voltage increases, the range of channel opening expands from mostly closed to mostly open, demonstrating that an increase in depolarization voltage can activate channels bound to HNTX-VII. Figure 5B shows the steady-state inactivation curve. When the channel is inhibited by 1μM HNTX-VII, the half-steady-state inactivation voltage increases by approximately 14mV, indicating that the steady-state inactivation curve shifts towards depolarization by 14mV. Confirming that the inhibitory effect of the toxin alters the inactivation curve characteristics of the channel.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.6 Effect of HNTX-VII on Steady-State Activation and Inactivation Curves of Kv4.3\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFigure 5C depicts the steady-state activation curve. The inhibitory effect of 200 nM HNTX-VII on the channel current increases the half-steady-state activation voltage by 13 mV, causing the steady-state activation curve to shift towards depolarization. This indicates that HNTX-VII alters the characteristics of the channel, making it more difficult for the channel to open. Figure 5D shows the steady-state inactivation curve. Under three conditions: control, 200 nM HNTX-VII, and 500 nM HNTX-VII, there are minimal changes observed in the curve, with negligible fluctuations in the half-steady-state inactivation voltage and slope K value. Therefore, HNTX-VII does not significantly affect the inactivation characteristics of the Kv4.3 channel and does not exhibit concentration-dependence in this regard.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.7 Effect of HNTX-VII on Inactivation Recovery Time Constants of Kv4.2 and Kv4.3 Channels\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFigure 5E presents the inactivation recovery curve for the Kv4.2 channel. The recovery curves under control conditions and with inhibition by 500 nM HNTX-VII are similar, with calculated recovery time constants (τ values) of approximately 117 ms for both. This suggests that HNTX-VII has no effect on the recovery of Kv4.2 channels following inactivation. Figure 5F shows the inactivation recovery curve for the Kv4.3 channel. Under control conditions, the recovery time constant of the channel is 43.5 ± 1.98 ms. However, when the channel is inhibited by 200 nM HNTX-VII, the calculated recovery time constant increases to 72.5 ± 3.02 ms, an increase of 29 ms. Therefore, the inhibitory effect of HNTX-VII makes it more difficult for the channel to recover from the inactivated state, altering the inactivation recovery kinetics.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.8 Molecular Mechanism of Kv4.3 Binding to HNTX-VII\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAs shown in Table 1, the IC\u003csub\u003e50\u003c/sub\u003e values of HNTX-VII acting on the wild-type Kv4.3 channel and its various mutants, as well as their ratios to the wild-type IC\u003csub\u003e50\u003c/sub\u003e, reveal that the V282A mutant on the extracellular loop between S3 and S4 exhibits the highest IC\u003csub\u003e50\u003c/sub\u003e value, which is more than five times that of the wild-type. This indicates that the 282nd amino acid V (valine) on the extracellular loop between S3 and S4 is the most critical residue affecting the interaction between HNTX-VII and the Kv4.3 channel. Additionally, residues such as E280, F286, L274, and G273 also have significant impacts(Figure 6).\u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThe binding sites of toxins with voltage-gated potassium channels are either located in the pore region or in the voltage-sensitive extracellular loop region. Depending on the binding site, toxins are classified into pore-blocking and gating-modulatory types[15,16]. In recent decades, most peptides isolated and purified from animal venoms belong to the gating-modulatory type, which either rely on charge-charge interactions, hydrophobic interactions, or both to bind to the extracellular loop of the voltage-sensitive region of the channel, thereby affecting the gating properties of the channel[17,18].\u003c/p\u003e\n\u003cp\u003eKv4.3 is a typical voltage-dependent potassium channel belonging to the transient outward potassium channel kv4 (Shal) family, which is highly expressed in cardiomyocytes and has a significant impact on the duration and amplitude of the cardiac action potential[19]. Both activation and inactivation of this channel exhibit time and voltage dependence. HpTx-2, isolated from spider venom, specifically acts on Kv4.2 and Kv4.3 potassium channels in Ito, inhibiting them in a voltage-dependent manner. HpTx-2 shifts the activation curve of the Kv4.3 potassium channel towards depolarization, accelerates channel deactivation, and delays inactivation. It is speculated that this molecule alters the gating properties of the channel by binding to the extracellular portion of the voltage-sensitive region of the Kv4.3 potassium channel[20].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eChristopher V et al. further investigated the mechanism of interaction between HpTx-2 and the Kv4.3 potassium channel. Using site-directed mutagenesis, mutants on the S3b segment were constructed, and it was found that the V276A and L275A mutants weakened the binding strength between the toxin and the channel. When both sites were mutated simultaneously, HpTx-2 basically did not bind to the Kv4.3 channel, suggesting that these two hydrophobic amino acid residues are crucial for toxin-channel binding[20]. These amino acids share similar characteristics with the key residues (IF) of hanatoxin (HaTX) binding to the Kv2.1 potassium channel, which are located in the same region as the S3b segment of the Kv4.3 potassium channel[21]. When the LV in the S3b segment of the Kv4.3 potassium channel was mutated to IF, the binding between HpTx-2 and the Kv4.3 channel was strengthened, further confirming the importance of hydrophobic amino acids at this position for HpTx-2 binding to the Kv4.3 channel[20,22].\u003c/p\u003e\n\u003cp\u003eLiterature has reported other polypeptides isolated from spider venoms that specifically inhibit the Kv4.3 potassium channel, including phrixotoxins and JZTX-V[23,24]. These toxins are believed to have a similar hydrophobic surface surrounded by charged amino acid residues. They all alter the kinetic properties of the Kv4.3 channel and are thought to do so by binding the hydrophobic surface of the toxin to the extracellular loop of the voltage-sensitive region of the Kv4.3 potassium channel, thereby modifying the channel's gating properties.\u003c/p\u003e\n\u003cp\u003eHNTX-VII is a newly discovered spider toxin that specifically acts on Kv4.3. Its inhibition of the Kv4.3 potassium channel is voltage-dependent, and the toxin dissociates from the channel under higher voltages. Previous experiments have demonstrated that it can alter the gating properties of the channel, shifting the steady-state activation curve of the Kv4.3 potassium channel towards depolarization, increasing the difficulty of channel opening, and delaying the recovery after channel inactivation. Therefore, it is speculated that, similar to other ICK toxins, HNTX-VII is also a gating modulatory toxin that controls the opening and closing of the channel through the interaction between the hydrophobic amino acids and charged amino acid domains of the toxin and the extracellular loop region of the channel's voltage-sensitive domain.\u003c/p\u003e\n\u003cp\u003eTo delve into the molecular mechanism of the interaction between HNTX-VII and Kv4.3, this experiment utilized site-directed mutagenesis to construct 19 mutants in the S1-S2 extracellular loop and S3b-S4 extracellular loop and examined the inhibitory activity of HNTX-VII against each mutant. It was found that hydrophobic amino acids and a charged amino acid had significant impacts on the inhibitory activity of the channel, specifically G273, L274, V275 on the S3b segment, and E280, V282, F286 in the S3-S4 extracellular loop .\u003c/p\u003e\n\u003cp\u003eInterestingly, mutations with substantial effects, such as the hydrophobic amino acids (G273, L274, V275) and the charged E280, occupy similar positions in the extracellular loop as the key residues for hanatoxin binding to the Kv2.1 potassium channel, and are also identical to the crucial amino acid significantly influencing the binding of HpTx-2 to the Kv4.3 channel. This leads to the inference that HNTX-VII may function similarly to these toxins in modulating the Kv4.3 channel, specifically by acting on the channel through the hydrophobic amino acid residues of the toxin and the surrounding charged amino acids.\u003c/p\u003e\n\u003cp\u003eV282A is the mutant that has the most significant impact on the binding of HNTX-VII to the Kv4.3 potassium channel, increasing the half-maximal inhibitory concentration (IC50) of HNTX-VII by 5.4-fold. Hence, V282A is a crucial residue for the interaction between HNTX-VII and Kv4.3. Furthermore, being a non-polar amino acid, it reinforces the importance of hydrophobic interactions in the binding of HNTX-VII to this channel.\u003c/p\u003e\n\u003cp\u003eIt is noteworthy that during the conduct of this experiment, it was discovered that the V282AF286A double mutant exhibited no detectable current under voltage stimulation. However, upon adding HNTX-VII and applying voltage stimulation, a current was detected, which increased with the concentration of the toxin. In this scenario, HNTX-VII transformed from an inhibitor into an activator for the double mutant, with the underlying molecular mechanism remaining unclear. Nevertheless, studying this phenomenon will undoubtedly have significant implications for unraveling the interactions between toxins and channels.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eCRediT authorship contribution statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eC and XZ.Z initiated the research concept, devised the experimental framework, and outlined the structure of the paper. They also wrote the majority of the paper\u0026apos;s content and oversaw the revisions. Additionally, B.C, ZT.H, HB.S, RZ.C and J.H conducted the majority of the experiments and performed the data analyses. All authors participated in the discussion of the results, provided feedback on the manuscript, and gave their final approval for submission.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e The authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval\u003c/strong\u003e Not applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Hunan Provincial Natural Science Foundation (NO.2021JJ30540). The Hunan Education Department Project (23A0552). Huaihua University key projects (HHUY2022-10, HHUY2019-07).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eLuo A, Wang A, Kamau PM, Lai R, Luo L. Centipede Venom: A Potential Source of Ion Channel Modulators. Int J Mol Sci. 2022 Jun 26;23(13):7105. \u003c/li\u003e\n\u003cli\u003eShaikh NY, Sunagar K. The deep-rooted origin of disulfide-rich spider venom toxins. Elife. 2023 Feb 9;12:e83761. doi: 10.7554/eLife.83761. \u003c/li\u003e\n\u003cli\u003eBillen B, Bosmans F, Tytgat J. Animal peptides targeting voltage-activated sodium channels. Curr Pharm Des. 2008;14(24):2492-502.\u003c/li\u003e\n\u003cli\u003eTouchard A, Aili SR, Fox EG, Escoubas P, Orivel J, Nicholson GM, Dejean A. The Biochemical Toxin Arsenal from Ant Venoms. Toxins (Basel). 2016 Jan 20;8(1):30. \u003c/li\u003e\n\u003cli\u003eZainal Abidin SA, Liew AKY, Othman I, Shaikh F. Animal Venoms as Potential Source of Anticonvulsants. F1000Res. 2024 Mar 27;13:225. \u003c/li\u003e\n\u003cli\u003eSaez NJ, Senff S, Jensen JE, Er SY, Herzig V, Rash LD, King GF. Spider-venom peptides as therapeutics. Toxins (Basel). 2010 Dec;2(12):2851-71. \u003c/li\u003e\n\u003cli\u003eMendes LC, Viana GMM, Nencioni ALA, Pimenta DC, Beraldo-Neto E. Scorpion Peptides and Ion Channels: An Insightful Review of Mechanisms and Drug Development. Toxins (Basel). 2023 Mar 24;15(4):238. \u003c/li\u003e\n\u003cli\u003eBednenko J, Colussi P, Hussain S, Zhang Y, Clark T. Therapeutic Antibodies Targeting Potassium Ion Channels. Handb Exp Pharmacol. 2021;267:507-545. \u003c/li\u003e\n\u003cli\u003eCoates L. Ion permeation in potassium ion channels. Acta Crystallogr D Struct Biol. 2020 Apr 1;76(Pt 4):326-331.\u003c/li\u003e\n\u003cli\u003eKuang Q, Purhonen P, Hebert H. Structure of potassium channels. Cell Mol Life Sci. 2015 Oct;72(19):3677-93. \u003c/li\u003e\n\u003cli\u003eHibino H, Inanobe A, Furutani K, Murakami S, Findlay I, Kurachi Y. Inwardly rectifying potassium channels: their structure, function, and physiological roles. Physiol Rev. 2010 Jan;90(1):291-366. \u003c/li\u003e\n\u003cli\u003eBougis PE, Martin-Eauclaire MF. Shal-type (Kv4.x) potassium channel pore blockers from scorpion venoms. Sheng Li Xue Bao. 2015 Jun 25;67(3):248-54. \u003c/li\u003e\n\u003cli\u003eKise Y, Kasuya G, Okamoto HH, Yamanouchi D, Kobayashi K, Kusakizako T, Nishizawa T, Nakajo K, Nureki O. Structural basis of gating modulation of Kv4 channel complexes. Nature. 2021 Nov;599(7883):158-164. \u003c/li\u003e\n\u003cli\u003eChen B, Hu Z, Chen X, Zeng X. Molecular mechanisms of two novel and selective TRPV1 channel activators. Int J Biol Macromol. 2024 Jul 3;275(Pt 1):133658. \u003c/li\u003e\n\u003cli\u003eDilly S, Lamy C, Marrion NV, Li\u0026eacute;geois JF, Seutin V. Ion-channel modulators: more diversity than previously thought. Chembiochem. 2011 Aug 16;12(12):1808-12. \u003c/li\u003e\n\u003cli\u003eLee AG. Interfacial Binding Sites for Cholesterol on Kir, Kv, K2P, and Related Potassium Channels. Biophys J. 2020 Jul 7;119(1):35-47. \u003c/li\u003e\n\u003cli\u003eMouhat S, Andreotti N, Jouirou B, Sabatier JM. Animal toxins acting on voltage-gated potassium channels. Curr Pharm Des. 2008;14(24):2503-18. \u003c/li\u003e\n\u003cli\u003eOliva C, Gonz\u0026aacute;lez V, Naranjo D. Slow inactivation in voltage gated potassium channels is insensitive to the binding of pore occluding peptide toxins. Biophys J. 2005 Aug;89(2):1009-19. \u003c/li\u003e\n\u003cli\u003eHuo R, Sheng Y, Guo WT, Dong DL. The potential role of Kv4.3 K\u003csup\u003e+\u003c/sup\u003e channel in heart hypertrophy. Channels (Austin). 2014;8(3):203-9. \u003c/li\u003e\n\u003cli\u003eDeSimone CV, Zarayskiy VV, Bondarenko VE, Morales MJ. Heteropoda toxin 2 interaction with Kv4.3 and Kv4.1 reveals differences in gating modification. Mol Pharmacol. 2011 Aug;80(2):345-55. \u003c/li\u003e\n\u003cli\u003eSwartz KJ, MacKinnon R. An inhibitor of the Kv2.1 potassium channel isolated from the venom of a Chilean tarantula. Neuron. 1995 Oct;15(4):941-9. \u003c/li\u003e\n\u003cli\u003eDeSimone CV, Lu Y, Bondarenko VE, Morales MJ. S3b amino acid substitutions and ancillary subunits alter the affinity of Heteropoda venatoria toxin 2 for Kv4.3. Mol Pharmacol. 2009 Jul;76(1):125-33. \u003c/li\u003e\n\u003cli\u003eDiochot S, Drici MD, Moinier D, Fink M, Lazdunski M. Effects of phrixotoxins on the Kv4 family of potassium channels and implications for the role of Ito1 in cardiac electrogenesis. Br J Pharmacol. 1999 Jan;126(1):251-63. \u003c/li\u003e\n\u003cli\u003eDehong X, Wenmei W, Siqin H, Peng Z, Xianchun W, Xiongzhi Z. Effects of JZTX-V on the wild type Kv4.3 Expressed in HEK293T and Molecular Determinants in the Voltage-sensing Domains of Kv4.3 Interacting with JZTX-V. Channels (Austin). 2022 Dec;16(1):72-83.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"HNTX-VII, Kv 4.2, Kv 4.3","lastPublishedDoi":"10.21203/rs.3.rs-4866716/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4866716/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eHNTX-VII is a novel peptide isolated and purified from the venom of the Chinese spider \u003cem\u003eOrnithoctonus hainana\u003c/em\u003e, with a relative molecular mass of 3830.973 Da. Electrophysiological experiments have demonstrated that HNTX-VII exhibits minimal effects on TTX-S, TTX-R, and delayed rectifier potassium channels on dorsal root ganglia (DRG), as well as on Kv1.4 and Kv4.1. However, it significantly inhibits Kv4.2 and Kv4.3 currents, with IC50 values of 299.6 ± 6.48 nM and 114.5 ± 5.36 nM, respectively, for Kv4.2 and Kv4.3. The sequence of HNTX-VII, determined by Edman degradation, is ECRYWLGTCSKTGDCCSHLSCSPKHGWCVWDWT. Composed of 33 amino acids and containing 3 pairs of disulfide bonds, this molecule represents a typical inhibitor cystine knot (ICK) motif. Furthermore, HNTX-VII alters the kinetic properties of Kv4.2 and Kv4.3 channels by causing corresponding shifts in their steady-state activation, steady-state inactivation, and inactivation recovery curves. To further investigate the molecular mechanism underlying the interaction between HNTX-VII and Kv4.3 channels, 19 mutants in the extracellular loops of the S1-S2 and S3b-S4 segments of the Kv4.3 channel were designed and constructed using site-directed mutagenesis. Electrophysiological techniques were then employed to assess the inhibitory activity of HNTX-VII against these mutants. Notably, the V282A mutant in the S3b-S4 loop exhibited the most significant reduction in sensitivity to HNTX-VII, with an IC50 value 5.37 times that of the wild-type channel. Therefore, it is inferred that the 282nd amino acid in the extracellular loop of S3b-S4 serves as a crucial site for the interaction between HNTX-VII and Kv4.3.\u003c/p\u003e","manuscriptTitle":"Effects of HNTX-VII on Kv4.2 and Kv4.3 and Molecular Determinants of Kv4.3 Interacting with HNTX-VII","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-09-05 09:02:30","doi":"10.21203/rs.3.rs-4866716/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"735925a4-47bf-41fd-92c3-9df212b007a7","owner":[],"postedDate":"September 5th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-09-22T19:39:25+00:00","versionOfRecord":[],"versionCreatedAt":"2024-09-05 09:02:30","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4866716","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4866716","identity":"rs-4866716","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}