Structural and Functional Diversity of Peptide Toxins from Tarantula Haplopelma hainanum (Ornithoctonus hainana) Venom Revealed by Transcriptomic, Peptidomic, and Patch Clamp Approaches.

Spider venom is a complex mixture of bioactive peptides to subdue their prey. Early estimates suggested that over 400 venom peptides are produced per species. In order to investigate the mechanisms responsible for this impressive diversity, transcriptomics based on second generation high throughput sequencing was combined with peptidomic assays to characterize the venom of the tarantula Haplopelma hainanum. The genes expressed in the venom glands were identified, and the bioactivity of their protein products was analyzed using the patch clamp technique. A total of 1,136 potential toxin precursors were identified that clustered into 90 toxin groups, of which 72 were novel. The toxin peptides clustered into 20 cysteine scaffolds that included between 4 and 12 cysteines, and 14 of these groups were newly identified in this spider. Highly abundant toxin peptide transcripts were present and resulted from hypermutation and/or fragment insertion/deletion. In combination with variable post-translational modifications, this genetic variability explained how a limited set of genes can generate hundreds of toxin peptides in venom glands. Furthermore, the intraspecies venom variability illustrated the dynamic nature of spider venom and revealed how complex components work together to generate diverse bioactivities that facilitate adaptation to changing environments, types of prey, and milking regimes in captivity.

Molecular mechanisms of two novel and selective TRPV1 channel activators.

Venomous toxins hold immense value as tools in elucidating the intricate structure and underlying mechanisms of ion channels. In this article, we identified of two novel toxins, Hainantoxin-XXI (HNTX-XXI) and Hainantoxin-XXII (HNTX-XXII), derived from the venom of the Chinese spider Ornithoctonus hainana. HNTX-XXI, boasting a molecular weight of 6869.095 Da, comprises 64 amino acid residues and contains 8 cysteines. Meanwhile, HNTX-XXII, with a molecular weight of 8623.732 Da, comprises 77 amino acid residues and contains 12 cysteines. Remarkably, we discovered that both HNTX-XXI and HNTX-XXII possess the ability to activate TRPV1. They activated TRPV1 with EC50 values of 3.6 ± 0.19 µM and 862 ± 56 nM, respectively. Furthermore, the current generated by the activation of TRPV1 by these toxins can be rapidly blocked by ruthenium red. Intriguingly, our analysis revealed that the interaction between HNTX-XXI and TRPV1 is mediated by three key amino acid residues: L465, V469, and D471. Similarly, the interaction between HNTX-XXII and TRPV1 is facilitated by four key amino acid residues: A657, F659, E600, and R601. These findings provide profound insights into the molecular basis of toxin-TRPV1 interactions and pave the way for future research exploring the therapeutic potential of these toxic peptides.

Quantum Neural Network Inspired Hardware Adaptable Ansatz for Efficient Quantum Simulation of Chemical Systems.

The variational quantum eigensolver is a promising way to solve the Schrödinger equation on a noisy intermediate-scale quantum (NISQ) computer, while its success relies on a well-designed wave function ansatz. Inspired by the quantum neural network, we propose a new hardware heuristic ansatz where its expressibility can be improved by increasing either the depth or the width of the circuit. Such a character makes this ansatz adaptable to different hardware environments. More importantly, it provides a general framework to improve the efficiency of the quantum resource utilization. For example, on a superconducting quantum computer where circuit depth is usually the bottleneck and the qubits thus cannot be fully used, circuit depth can be significantly reduced by introducing ancilla qubits. Ancilla qubits also make the circuit less sensitive to noises in practical application. These results open a new avenue to develop practical applications of quantum computation in the NISQ era.

Molecular mechanism by which spider-driving peptide potentiates coagulation factors.

Hemostasis is a crucial process that quickly forms clots at injury sites to prevent bleeding and infections. Dysfunctions in this process can lead to hemorrhagic disorders, such as hemophilia and thrombocytopenia purpura. While hemostatic agents are used in clinical treatments, there is still limited knowledge about potentiators targeting coagulation factors. Recently, LCTx-F2, a procoagulant spider-derived peptide, was discovered. This study employed various methods, including chromogenic substrate analysis and dynamic simulation, to investigate how LCTx-F2 enhances the activity of thrombin and FXIIa. Our findings revealed that LCTx-F2 binds to thrombin and FXIIa in a similar manner, with the N-terminal penetrating the active-site cleft of the enzymes and the intermediate section reinforcing the peptide-enzyme connection. Interestingly, the C-terminal remained at a considerable distance from the enzymes, as evidenced by the retention of affinity for both enzymes using truncated peptide T-F2. Furthermore, results indicated differences in the bonding relationship of critical residues between thrombin and FXIIa, with His13 facilitating binding to thrombin and Arg7 being required for binding to FXIIa. Overall, our study sheds light on the molecular mechanism by which LCTx-F2 potentiates coagulation factors, providing valuable insights that may assist in designing drugs targeting procoagulation factors.

Molecular basis of the tarantula toxin jingzhaotoxin-III (ß-TRTX-Cj1α) interacting with voltage sensors in sodium channel subtype Nav1.5.

With conserved structural scaffold and divergent electrophysiological functions, animal toxins are considered powerful tools for investigating the basic structure-function relationship of voltage-gated sodium channels. Jingzhaotoxin-III (ß-TRTX-Cj1α) is a unique sodium channel gating modifier from the tarantula Chilobrachys jingzhao, because the toxin can selectively inhibit the activation of cardiac sodium channel but not neuronal subtypes. However, the molecular basis of JZTX-III interaction with sodium channels remains unknown. In this study, we showed that JZTX-III was efficiently expressed by the secretory pathway in yeast. Alanine-scanning analysis indicated that 2 acidic residues (Asp1, Glu3) and an exposed hydrophobic patch, formed by 4 Trp residues (residues 8, 9, 28 and 30), play important roles in the binding of JZTX-III to Nav1.5. JZTX-III docked to the Nav1.5 DIIS3-S4 linker. Mutations S799A, R800A, and L804A could additively reduce toxin sensitivity of Nav1.5. We also demonstrated that the unique Arg800, not emerging in other sodium channel subtypes, is responsible for JZTX-III selectively interacting with Nav1.5. The reverse mutation D816R in Nav1.7 greatly increased the sensitivity of the neuronal subtype to JZTX-III. Conversely, the mutation R800D in Nav1.5 decreased JZTX-III's IC50 by 72-fold. Therefore, our results indicated that JZTX-III is a site 4 toxin, but does not possess the same critical residues on sodium channels as other site 4 toxins. Our data also revealed the underlying mechanism for JZTX-III to be highly specific for the cardiac sodium channel.

Understanding High-Temperature Chemical Reactions on Metal Surfaces: A Case Study on Equilibrium Concentration and Diffusivity of C x H y on a Cu(111) Surface.

Chemical reactions on metal surfaces are important in various processes such as heterogeneous catalysis and nanostructure growth. At moderate or lower temperatures, these reactions generally follow the minimum energy path, and temperature effects can be reasonably described by a harmonic oscillator model. At a high temperature approaching the melting point of the substrate, general behaviors of surface reactions remain elusive. In this study, by taking hydrocarbon species adsorbed on Cu(111) as a model system and performing extensive molecular dynamics simulations powered by machine learning potentials, we identify several important high-temperature effects, including local chemical environment, surface atom mobility, and substrate thermal expansion. They affect different aspects of a high-temperature surface reaction in different ways. These results deepen our understanding of high-temperature reactions.

Exploring Accurate Potential Energy Surfaces via Integrating Variational Quantum Eigensolver with Machine Learning.

The potential energy surface (PES) is crucial for interpreting a variety of chemical reaction processes. However, predicting accurate PESs with high-level electronic structure methods is a challenging task due to the high computational cost. As an appealing application of quantum computing, we show in this work that variational quantum algorithms can be integrated with machine learning (ML) techniques as a promising scheme for exploring accurate PESs. Different from using a ML model to represent the potential energy, we encode the molecular geometry information into a deep neural network (DNN) to represent parameters of the variational quantum eigensolver (VQE), leaving the PES to the wave function ansatz. Once the DNN model is trained, the variational optimization procedure that hinders the application of the VQE to complex systems is avoided, and thus the evaluation of PESs is significantly accelerated. Numerical results demonstrate that a simple DNN model is able to reproduce accurate PESs for small molecules.

Toward Epitaxial Growth of Misorientation-Free Graphene on Cu(111) Foils.

The epitaxial growth of single-crystal thin films relies on the availability of a single-crystal substrate and a strong interaction between epilayer and substrate. Previous studies have reported the roles of the substrate (e.g., symmetry and lattice constant) in determining the orientations of chemical vapor deposition (CVD)-grown graphene, and Cu(111) is considered as the most promising substrate for epitaxial growth of graphene single crystals. However, the roles of gas-phase reactants and graphene-substrate interaction in determining the graphene orientation are still unclear. Here, we find that trace amounts of oxygen is capable of enhancing the interaction between graphene edges and Cu(111) substrate and, therefore, eliminating the misoriented graphene domains in the nucleation stage. A modified anomalous grain growth method is developed to improve the size of the as-obtained Cu(111) single crystal, relying on strongly textured polycrystalline Cu foils. The batch-to-batch production of A3-size (∼0.42 × 0.3 m2) single-crystal graphene films is achieved on Cu(111) foils relying on a self-designed pilot-scale CVD system. The as-grown graphene exhibits ultrahigh carrier mobilities of 68 000 cm2 V-1 s-1 at room temperature and 210 000 cm2 V-1 s-1 at 2.2 K. The findings and strategies provided in our work would accelerate the mass production of high-quality misorientation-free graphene films.

[Inhibition of Jingzhaotoxin-V on Kv4.3 channel].

Kv4.3 channel is present in many mammalian tissues, predominantly in the heart and central nervous system. Its currents are transient, characterized by rapid activation and inactivation. In the hearts of most mammals, it is responsible for repolarization of the action potential of ventricular myocytes and is important in the regulation of the heart rate. Because of its central role in this important physiological process, Kv4.3 channel is a promising target for anti-arrhythmic drug development. Jingzhaotoxin-V (JZTX-V) is a novel peptide neurotoxin isolated from the venom of the spider Chilobrachys jingzhao. Whole-cell patch clamp recording showed that it partly blocked the transient outward potassium channels in dorsal root ganglion neurons of adult rats with an IC(50) value of 52.3 nmol/L. To investigate the effect of JZTX-V on Kv4.3 channel, JZTX-V was synthesized using the solid-phase chemical synthesis and separated by reverse phase high performance liquid chromatography (HPLC). The purity was tested by matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MOLDI-TOF mass spectrometry). Two-electrode voltage-clamp technique was used to characterize the action of JZTX-V on Kv4.3 channels expressed in Xenopus laevis oocytes. As a result, JZTX-V displayed fast kinetics of inhibition and recovery from inactivation. Furthermore, it could inhibit Kv4.3 channel current in a time- and concentration-dependent manner with an IC(50) value of 425.1 nmol/L. The application of JZTX-V affected the activation and inactivation characteristics of Kv4.3 channel and caused a shift of the current-voltage relationship curve and the steady-state inactivation curve to depolarizing direction by approximately 29 mV and 10 mV, respectively. So we deduced that JZTX-V is a gating modifier toxin of Kv4.3 channel. Present findings should be helpful to develop JZTX-V into a molecular probe and drug candidate targeting to Kv4.3 channel in the myocardium.

JZTX-V Targets the Voltage Sensor in Kv4.2 to Inhibit Ito Potassium Channels in Cardiomyocytes.

Kv4 potassium channels are responsible for transient outward K+ currents in the cardiac action potential (AP). Previous experiments by our group demonstrated that Jingzhaotoxin-V (JZTX-V) selectively inhibits A-type potassium channels. However, the specific effects of JZTX-V on the transient outward (Ito) current of cardiomyocytes and underlying mechanism of action remain unclear. In the current study, 100 nM JZTX-V effectively inhibited the Ito current and extended the action potential duration (APD) of neonatal rat ventricular myocytes (NRVM). We further analyzed the effects of JZTX-V on Kv4.2, a cloned channel believed to underlie the Ito current in rat cardiomyocytes. JZTX-V inhibited the Kv4.2 current with a half-maximal inhibitory concentration (IC50) of 13 ± 1.7 nM. To establish the molecular mechanism underlying the inhibitory action of JZTX-V on Kv4.2, we performed alanine scanning mutagenesis of Kv4.2 and JZTX-V and assessed the effects of the mutations on binding activities of the proteins. Interestingly, the Kv4.2 mutations V285A, F289A, and V290A reduced the affinity for JZTX-V while I275A and L277A increased the affinity for JZTX-V. Moreover, mutation of positively charged residues (R20 and K22) of JZTX-V and the hydrophobic patch (formed by W5, M6, and W7) led to a significant reduction in toxin sensitivity, indicating that the hydrophobic patch and electrostatic interactions played key roles in the binding of JZTX-V with Kv4.2. Data from our study have shed light on the specific roles and molecular mechanisms of JZTX-V in the regulation of Ito potassium channels and supported its utility as a potential novel antiarrhythmic drug.

Synthesis and characterization of huwentoxin-IV, a neurotoxin inhibiting central neuronal sodium channels.

Our previous work demonstrated that huwentoxin-IV was an inhibitor cystine knot peptide from Chinese tarantula Ornithoctonus huwena venom that blocked tetrodotoxin-sensitive voltage-gated sodium channels from mammalian sensory neurons [Peng, K., Shu, Q., Liu, Z., Liang, S., 2002. Function and solution structure of huwentoxin-IV, a potent neuronal tetrodotoxin (TTX)-sensitive sodium channel antagonist from Chinese bird spider Selenocosmia huwena. J. Biol. Chem. 277(49), 47564-47571]. However, the actions of the neurotoxin on central neuronal sodium channels remain unknown. In this study, we chemically synthesized native huwentoxin-IV and found that sodium channel isoforms from rat hippocampus neurons were also sensitive to native and synthetic toxins, but the toxin-binding affinity (IC(50) approximately 0.4 microM) was 12-fold lower than to peripheral isoforms. The blockade by huwentoxin-IV could be reversed by strong depolarization due to the dissociation of toxin-channel complex as observed for receptor site 3 toxins. Moreover, small unilamellar vesicle-binding assays showed that in contrast to ProTx-II from the tarantula Thrixopelma pruriens, huwentoxin-IV almost lacked the ability to partition into the negatively charged and neutral phospholipid bilayer of artificial membranes. These findings indicated that huwentoxin-IV was a sodium channel antagonist preferentially targeting peripheral isoforms via a mechanism quite different from ProTx-II.

The Venom of Ornithoctonus huwena affect the electrophysiological stability of neonatal rat ventricular myocytes by inhibiting sodium, potassium and calcium current.

Spider venoms are known to contain various toxins that are used as an effective means to capture their prey or to defend themselves against predators. An investigation of the properties of Ornithoctonus huwena (O.huwena) crude venom found that the venom can block neuromuscular transmission of isolated mouse phrenic nerve-diaphragm and sciatic nerve-sartorius preparations. However, little is known about its electrophysiological effects on cardiac myocytes. In this study, electrophysiological activities of ventricular myocytes were detected by 100 µg/mL venom of O.huwena, and whole cell patch-clamp technique was used to study the acute effects of the venom on action potential (AP), sodium current (INa), potassium currents (IKr, IKs, Ito1 and IK1) and L-type calcium current (ICaL). The results indicated that the venom prolongs APD90 in a frequency-dependent manner in isolated neonatal rat ventricular myocytes. 100 µg/mL venom inhibited 72.3 ± 3.6% INa current, 58.3 ± 4.2% summit current and 54 ± 6.1% the end current of IKr, and 65 ± 3.3% ICaL current, yet, didn't have obvious effect on IKs, Ito1 and IK1 currents. In conclusion, the O.huwena venom represented a multifaceted pharmacological profile. It contains abundant of cardiac channel antagonists and might be valuable tools for investigation of both channels and anti- arrhythmic therapy development.

Selective Closed-State Nav1.7 Blocker JZTX-34 Exhibits Analgesic Effects against Pain.

Jingzhaotoxin-34 (JZTX-34) is a selective inhibitor of tetrodotoxin-sensitive (TTX-S) sodium channels. In this study, we found that JZTX-34 selectively acted on Nav1.7 with little effect on other sodium channel subtypes including Nav1.5. If the DIIS3-S4 linker of Nav1.5 is substituted by the correspond linker of Nav1.7, the sensitivity of Nav1.5 to JZTX-34 extremely increases to 1.05 µM. Meanwhile, a mutant D816R in the DIIS3-S4 linker of Nav1.7 decreases binding affinity of Nav1.7 to JZTX-34 about 32-fold. The reverse mutant R800D at the corresponding position in Nav1.5 greatly increased its binding affinity to JZTX-34. This implies that JZTX-34 binds to DIIS3-S4 linker of Nav1.7 and the critical residue of Nav1.7 is D816. Unlike ß-scorpion toxin trapping sodium channel in an open state, activity of JZTX-34 requires the sodium channel to be in a resting state. JZTX-34 exhibits an obvious analgesic effect in a rodent pain model. Especially, it shows a longer duration and is more effective than morphine in hot pain models. In a formalin-induced pain model, JZTX-34 at dose of 2 mg/kg is equipotent with morphine (5 mg/kg) in the first phase and several-fold more effective than morphine in second phase. Taken together, our data indicate that JZTX-34 releases pain by selectively binding to the domain II voltage sensor of Nav1.7 in a closed configuration.

Jingzhaotoxin-XII, a gating modifier specific for Kv4.1 channels.

Jingzhaotoxin-XII (JZTX-XII), a 29-residue polypeptide, was purified from the venom of the Chinese tarantula Chilobrachys jingzhao. Electrophysiological recordings carried out in Xenopus laevis oocytes showed that JZTX-XII is specific for Kv4.1 channels, with the IC50 value of 0.363 microM. It interacts with the channels by modifying the gating behavior. JZTX-XII shares 80% sequence identity with phrixotoxin1, a potent inhibitor for Kv4.2 and Kv4.3 channels. Structure analysis indicates that the difference of the charge distribution in the interactive surface perhaps influences the specific pharmacology of the toxins. JZTX-XII should be a valuable tool for the investigation of the Kv4.1 channels.

Isolation and characterization of Jingzhaotoxin-V, a novel neurotoxin from the venom of the spider Chilobrachys jingzhao.

Jingzhaotoxin-V (JZTX-V), a 29-residue polypeptide, is derived from the venom of the spider Chilobrachys jingzhao. Its cDNA determined by rapid amplification of 3' and 5'-cDNA ends encoded an 83-residue precursor with a pro-region of 16 residues. JZTX-V inhibits tetrodotoxin-resistant and tetrodotoxin-sensitive sodium currents in rat dorsal root ganglion neurons with IC50 values of 27.6 and 30.2 nM, respectively. Moreover, the toxin exhibits high affinity to the resting closed states of the channels. JZTX-V also inhibits Kv4.2 potassium currents expressed in Xenpus Laevis oocytes (IC50=604.2 nM), but has no effects on outward delay-rectified potassium channels expressed in Xenopus laevis oocytes. JZTX-V alters the gating properties of sodium channels by shifting the activation curves to the depolarizing direction and the inactivation curves to the hyperpolarizing direction. Small unilamellar vesicles binding assays show that the partitioning of JZTX-V into lipid bilayer requires negatively charged phospholipids. The phospholipid membrane binding activity of JZTX-V is also verified using intrinsic tryptophan fluorescence analysis as well as acrylamide-quenching assays. Importantly, human multiple sodium channel subtypes are attractive targets for treatment of pain, highlighting the importance of JZTX-V as potential lead for drug development.

Molecular determinant for the tarantula toxin Jingzhaotoxin-I slowing the fast inactivation of voltage-gated sodium channels.

Peptide toxins often have divergent pharmacological functions and are powerful tools for a deep review on the current understanding of the structure-function relationships of voltage-gated sodium channels (VGSCs). However, knowing about the interaction of site 3 toxins from tarantula venoms with VGSCs is not sufficient. In the present study, using whole-cell patch clamp technique, we determined the effects of Jingzhaotoxin-I (JZTX-I) on five VGSC subtypes expressed in HEK293 cells. The results showed that JZTX-I could inhibit the inactivation of rNav1.2, rNav1.3, rNav1.4, hNav1.5 and hNav1.7 channels with the IC50 of 870 ± 8 nM, 845 ± 4 nM, 339 ± 5 nM, 335 ± 9 nM, and 348 ± 6 nM, respectively. The affinity of the toxin interaction with subtypes (rNav1.4, hNav1.5, and hNav1.7) was only 2-fold higher than that for subtypes (rNav1.2 and rNav1.3). The toxin delayed the inactivation of VGSCs without affecting the activation and steady-state inactivation kinetics in the physiological range of voltages. Site-directed mutagenesis indicated that the toxin interacted with site 3 located at the extracellular S3-S4 linker of domain IV, and the acidic residue Asp at the position1609 in hNav1.5 was crucial for JZTX-I activity. Our results provide new insights in single key residue that allows toxins to recognize distinct ion channels with similar potency and enhance our understanding of the structure-function relationships of toxin-channel interactions.

Purification and characterization of raventoxin-I and raventoxin-III, two neurotoxic peptides from the venom of the spider Macrothele raveni.

The spider Macrothele raveni was recently identified as a new species of Genus Macrothele. The crude venom from M. raveni was found to be neurotoxic to mice and the LD(50) of the crude venom in mice was 2.852mg/kg. Two neurotoxic peptides, raventoxin-I and raventoxin-III, were isolated from the crude venom by ion-exchange and reverse phase high performance liquid chromatography. Raventoxin-I was the most abundant toxic component in the venom, while raventoxin-III was a lower abundant component. Both toxins can kill mice and block neuromuscular transmission in an isolated mouse phrenic nerve diaphragm preparation, but have no effect on cockroaches. The LD(50) of raventoxin-I in mice is 0.772mg/kg. The complete amino acid sequences of raventoxin-I and raventoxin-III were determined and found to consist of 43 and 29 amino acid residues, respectively. It was determined by mass spectrometry that all Cys residues from raventoxin-I and raventoxin-III are involved in disulphide bonds. raventoxin-III showed no significant sequence homology with any presently known neurotoxins in the protein/DNA databases, while raventoxin-I has limited sequence identity with delta-AcTx-Hv1 and delta-AcTx-Ar1, which target both mammalian and insect sodium channels. Both raventoxin-I and raventoxin-III only work on vertebrates, but not on insects. Moreover, raventoxin-I could exert an effect of first exciting and then inhibiting the contraction of mouse diaphragm muscle caused by electrically stimulating the phrenic nerve, but raventoxin-III could not.

Molecular surface of JZTX-V (ß-Theraphotoxin-Cj2a) interacting with voltage-gated sodium channel subtype NaV1.4.

Voltage-gated sodium channels (VGSCs; NaV1.1-NaV1.9) have been proven to be critical in controlling the function of excitable cells, and human genetic evidence shows that aberrant function of these channels causes channelopathies, including epilepsy, arrhythmia, paralytic myotonia, and pain. The effects of peptide toxins, especially those isolated from spider venom, have shed light on the structure-function relationship of these channels. However, most of these toxins have not been analyzed in detail. In particular, the bioactive faces of these toxins have not been determined. Jingzhaotoxin (JZTX)-V (also known as ß-theraphotoxin-Cj2a) is a 29-amino acid peptide toxin isolated from the venom of the spider Chilobrachys jingzhao. JZTX-V adopts an inhibitory cysteine knot (ICK) motif and has an inhibitory effect on voltage-gated sodium and potassium channels. Previous experiments have shown that JZTX-V has an inhibitory effect on TTX-S and TTX-R sodium currents on rat DRG cells with IC50 values of 27.6 and 30.2 nM, respectively, and is able to shift the activation and inactivation curves to the depolarizing and the hyperpolarizing direction, respectively. Here, we show that JZTX-V has a much stronger inhibitory effect on NaV1.4, the isoform of voltage-gated sodium channels predominantly expressed in skeletal muscle cells, with an IC50 value of 5.12 nM, compared with IC50 values of 61.7-2700 nM for other heterologously expressed NaV1 subtypes. Furthermore, we investigated the bioactive surface of JZTX-V by alanine-scanning the effect of toxin on NaV1.4 and demonstrate that the bioactive face of JZTX-V is composed of three hydrophobic (W5, M6, and W7) and two cationic (R20 and K22) residues. Our results establish that, consistent with previous assumptions, JZTX-V is a Janus-faced toxin which may be a useful tool for the further investigation of the structure and function of sodium channels.

The activation effect of hainantoxin-I, a peptide toxin from the Chinese spider, Ornithoctonus hainana, on intermediate-conductance Ca2+-activated K+ channels.

Intermediate-conductance Ca2+-activated K+ (IK) channels are calcium/calmodulin-regulated voltage-independent K+ channels. Activation of IK currents is important in vessel and respiratory tissues, rendering the channels potential drug targets. A variety of small organic molecules have been synthesized and found to be potent activators of IK channels. However, the poor selectivity of these molecules limits their therapeutic value. Venom-derived peptides usually block their targets with high specificity. Therefore, we searched for novel peptide activators of IK channels by testing a series of toxins from spiders. Using electrophysiological experiments, we identified hainantoxin-I (HNTX-I) as an IK-channel activator. HNTX-I has little effect on voltage-gated Na+ and Ca2+ channels from rat dorsal root ganglion neurons and on the heterologous expression of voltage-gated rapidly activating delayed rectifier K+ channels (human ether-à-go-go-related gene; human ERG) in HEK293T cells. Only 35.2% ± 0.4% of the currents were activated in SK channels, and there was no effect on BK channels. We demonstrated that HNTX-I was not a phrenic nerve conduction blocker or acutely toxic. This is believed to be the first report of a peptide activator effect on IK channels. Our study suggests that the activity and selectivity of HNTX-I on IK channels make HNTX-I a promising template for designing new drugs for cardiovascular diseases.