Development of charybdotoxin Q18F variant as a selective peptide blocker of neuronal BK(α + ß4) channel for the treatment of epileptic seizures.

Epilepsy is the results from the imbalance between inhibition and excitation in neural circuits, which is mainly treated by some chemical drugs with side effects. Gain-of-function of BK channels or knockout of its ß4 subunit associates with spontaneous epilepsy. Currently, few reports were published about the efficacy of BK(α + ß4) channel modulators in epilepsy prevention. Charybdotoxin is a non-specific inhibitor of BK and other K+ channels. Here, by nuclear magnetic resonance (NMR) and other biochemical techniques, we found that charybdotoxin might interact with the extracellular loop of human ß4 subunit (i.e., hß4-loop) of BK(α + ß4) channel at a molar ratio 4:1 (hß4-loop vs. charybdotoxin). Charybdotoxin enhanced its ability to prevent K+ current of BK(α + ß4 H101Y) channel. The charybdotoxin Q18F variant selectively reduced the neuronal spiking frequency and increased interspike intervals of BK(α + ß4) channel by π-π stacking interactions between its residue Phe18 and residue His101 of hß4-loop. Moreover, intrahippocampal infusion of charybdotoxin Q18F variant significantly increased latency time of seizure, reduced seizure duration and seizure numbers on pentylenetetrazole-induced pre-sensitized rats, inhibited hippocampal hyperexcitability and c-Fos expression, and displayed neuroprotective effects on hippocampal neurons. These results implied that charybdotoxin Q18F variant could be potentially used for intractable epilepsy treatment by therapeutically targeting BK(α + ß4) channel.

Chlorotoxin targets ERα/VASP signaling pathway to combat breast cancer.

Breast cancer is one of the most common malignant tumors among women worldwide. About 70-75% of primary breast cancers belong to estrogen receptor (ER)-positive breast cancer. In the development of ER-positive breast cancer, abnormal activation of the ERα pathway plays an important role and is also a key point leading to the failure of clinical endocrine therapy. In this study, we found that the small molecule peptide chlorotoxin (CTX) can significantly inhibit the proliferation, migration and invasion of breast cancer cells. In in vitro study, CTX inhibits the expression of ERα in breast cancer cells. Further studies showed that CTX can directly bind to ERα and change the protein secondary structure of its LBD domain, thereby inhibiting the ERα signaling pathway. In addition, we also found that vasodilator stimulated phosphoprotein (VASP) is a target gene of ERα signaling pathway, and CTX can inhibit breast cancer cell proliferation, migration, and invasion through ERα/VASP signaling pathway. In in vivo study, CTX significantly inhibits growth of ER overexpressing breast tumor and, more importantly, based on the mechanism of CTX interacting with ERα, we found that CTX can target ER overexpressing breast tumors in vivo. Our study reveals a new mechanism of CTX anti-ER-positive breast cancer, which also provides an important reference for the study of CTX anti-ER-related tumors.

Solution structure of extracellular loop of human ß4 subunit of BK channel and its biological implication on ChTX sensitivity.

Large-conductance Ca2+- and voltage-dependent K+ (BK) channels display diverse biological functions while their pore-forming α subunit is coded by a single Slo1 gene. The variety of BK channels is correlated with the effects of BKα coexpression with auxiliary ß (ß1-ß4) subunits, as well as newly defined γ subunits. Charybdotoxin (ChTX) blocks BK channel through physically occluding the K+-conduction pore. Human brain enriched ß4 subunit (hß4) alters the conductance-voltage curve, slows activation and deactivation time courses of BK channels. Its extracellular loop (hß4-loop) specifically impedes ChTX to bind BK channel pore. However, the structure of ß4 subunit's extracellular loop and the molecular mechanism for gating kinetics, toxin sensitivity of BK channels regulated by ß4 are still unclear. To address them, here, we first identified four disulfide bonds in hß4-loop by mass spectroscopy and NMR techniques. Then we determined its three-dimensional solution structure, performed NMR titration and electrophysiological analysis, and found that residue Asn123 of ß4 subunit regulated the gating and pharmacological characteristics of BK channel. Finally, by constructing structure models of BKα/ß4 and thermodynamic double-mutant cycle analysis, we proposed that BKα subunit might interact with ß4 subunit through the conserved residue Glu264(BKα) coupling with residue Asn123(ß4).

Discovery and characterisation of a novel toxin from Dendroaspis angusticeps, named Tx7335, that activates the potassium channel KcsA.

Due to their central role in essential physiological processes, potassium channels are common targets for animal toxins. These toxins in turn are of great value as tools for studying channel function and as lead compounds for drug development. Here, we used a direct toxin pull-down assay with immobilised KcsA potassium channel to isolate a novel KcsA-binding toxin (called Tx7335) from eastern green mamba snake (Dendroaspis angusticeps) venom. Sequencing of the toxin by Edman degradation and mass spectrometry revealed a 63 amino acid residue peptide with 4 disulphide bonds that belongs to the three-finger toxin family, but with a unique modification of its disulphide-bridge scaffold. The toxin induces a dose-dependent increase in both open probabilities and mean open times on KcsA in artificial bilayers. Thus, it unexpectedly behaves as a channel activator rather than an inhibitor. A charybdotoxin-sensitive mutant of KcsA exhibits similar susceptibility to Tx7335 as wild-type, indicating that the binding site for Tx7335 is distinct from that of canonical pore-blocker toxins. Based on the extracellular location of the toxin binding site (far away from the intracellular pH gate), we propose that Tx7335 increases potassium flow through KcsA by allosterically reducing inactivation of the channel.

In silico analysis of potential inhibitors of Ca(2+) activated K(+) channel blocker, Charybdotoxin-C from Leiurus quinquestriatus hebraeus through molecular docking and dynamics studies.

OBJECTIVE: Charybdotoxin-C (ChTx-C), from the scorpion Leiurus, quinquestriatus hebraeus blocks the calcium-activated potassium channels and causes hyper excitability of the nervous system. Detailed understanding the structure of ChTx-C, conformational stability, and intermolecular interactions are required to select the potential inhibitors of the toxin. MATERIALS AND METHODS: The structure of ChTx-C was modeled using Modeller 9v7. The amino acid residues lining the binding site were predicted and used for toxin-ligand docking studies, further, selected toxin-inhibitor complexes were studied using molecular dynamics (MD) simulations. RESULTS: The predicted structure has 91.7% of amino acids in the core and allowed regions of Ramachandran plot. A total of 133 analog compounds of existing drugs for scorpion bites were used for docking. As a result of docking, a list of compounds was shown good inhibiting properties with target protein. By analyzing the interactions, Ser 15, Lys 32 had significant interactions with selected ligand molecules and Val5, which may have hydrophobic interaction with the cyclic group of the ligand. MD simulation studies revealed that the conformation and intermolecular interactions of all selected toxin-inhibitor complexes were stable. CONCLUSION: The interactions of the ligand and active site amino acids were found out for the best-docked poses in turn helpful in designing potential antitoxins which may further be exploited in toxin based therapies.

Large conductance voltage- and Ca2+-gated potassium (BK) channel ß4 subunit influences sensitivity and tolerance to alcohol by altering its response to kinases.

Tolerance is a well described component of alcohol abuse and addiction. The large conductance voltage- and Ca(2+)-gated potassium channel (BK) has been very useful for studying molecular tolerance. The influence of association with the ß4 subunit can be observed at the level of individual channels, action potentials in brain slices, and finally, drinking behavior in the mouse. Previously, we showed that 50 mm alcohol increases both α and αß4 BK channel open probability, but only α BK develops acute tolerance to this effect. Currently, we explore the possibility that the influence of the ß4 subunit on tolerance may result from a striking effect of ß4 on kinase modulation of the BK channel. We examine the influence of the ß4 subunit on PKA, CaMKII, and phosphatase modulation of channel activity, and on molecular tolerance to alcohol. We record from human BK channels heterologously expressed in HEK 293 cells composed of its core subunit, α alone (Insertless), or co-expressed with the ß4 BK auxiliary subunit, as well as, acutely dissociated nucleus accumbens neurons using the cell-attached patch clamp configuration. Our results indicate that BK channels are strongly modulated by activation of specific kinases (PKA and CaMKII) and phosphatases. The presence of the ß4 subunit greatly influences this modulation, allowing a variety of outcomes for BK channel activity in response to acute alcohol.

Scorpion toxins for the reversal of BoNT-induced paralysis.

The botulinum neurotoxins, characterized by their neuromuscular paralytic effects, are the most toxic proteins known to man. Due to their extreme potency, ease of production, and duration of activity, the BoNT proteins have been classified by the Centers for Disease Control as high threat agents for bioterrorism. In an attempt to discover effective BoNT therapeutics, we have pursued a strategy in which we leverage the blockade of K(+) channels that ultimately results in the reversal of neuromuscular paralysis. Towards this end, we utilized peptides derived from scorpion venom that are highly potent K(+) channel blockers. Herein, we report the synthesis of charybdotoxin, a 37 amino acid peptide, and detail its activity, along with iberiotoxin and margatoxin, in a mouse phrenic nerve hemidiaphragm assay in the absence and the presence of BoNT/A.

Molecular dynamics simulations of scorpion toxin recognition by the Ca(2+)-activated potassium channel KCa3.1.

The Ca(2+)-activated channel of intermediate-conductance (KCa3.1) is a target for antisickling and immunosuppressant agents. Many small peptides isolated from animal venoms inhibit KCa3.1 with nanomolar affinities and are promising drug scaffolds. Although the inhibitory effect of peptide toxins on KCa3.1 has been examined extensively, the structural basis of toxin-channel recognition has not been understood in detail. Here, the binding modes of two selected scorpion toxins, charybdotoxin (ChTx) and OSK1, to human KCa3.1 are examined in atomic detail using molecular dynamics (MD) simulations. Employing a homology model of KCa3.1, we first determine conduction properties of the channel using Brownian dynamics and ascertain that the simulated results are in accord with experiment. The model structures of ChTx-KCa3.1 and OSK1-KCa3.1 complexes are then constructed using MD simulations biased with distance restraints. The ChTx-KCa3.1 complex predicted from biased MD is consistent with the crystal structure of ChTx bound to a voltage-gated K(+) channel. The dissociation constants (Kd) for the binding of both ChTx and OSK1 to KCa3.1 determined experimentally are reproduced within fivefold using potential of mean force calculations. Making use of the knowledge we gained by studying the ChTx-KCa3.1 complex, we attempt to enhance the binding affinity of the toxin by carrying out a theoretical mutagenesis. A mutant toxin, in which the positions of two amino acid residues are interchanged, exhibits a 35-fold lower Kd value for KCa3.1 than that of the wild-type. This study provides insight into the key molecular determinants for the high-affinity binding of peptide toxins to KCa3.1, and demonstrates the power of computational methods in the design of novel toxins.

Two conserved arginine residues from the SK3 potassium channel outer vestibule control selectivity of recognition by scorpion toxins.

Potassium channel functions are often deciphered by using selective and potent scorpion toxins. Among these toxins, only a limited subset is capable of selectively blocking small conductance Ca(2+)-activated K(+) (SK) channels. The structural bases of this selective SK channel recognition remain unclear. In this work, we demonstrate the key role of the electric charges of two conserved arginine residues (Arg-485 and Arg-489) from the SK3 channel outer vestibule in the selective recognition by the SK3-blocking BmP05 toxin. Indeed, individually substituting these residues with histidyl or lysyl (maintaining the positive electric charge partially or fully), although decreasing BmP05 affinity, still preserved the toxin sensitivity profile of the SK3 channel (as evidenced by the lack of recognition by many other types of potassium channel-sensitive charybdotoxin). In contrast, when Arg-485 or Arg-489 of the SK3 channel was mutated to an acidic (Glu) or alcoholic (Ser) amino acid residue, the channel lost its sensitivity to BmP05 and became susceptible to the "new" blocking activity by charybdotoxin. In addition to these SK3 channel basic residues important for sensitivity, two acidic residues, Asp-492 and Asp-518, also located in the SK3 channel outer vestibule, were identified as being critical for toxin affinity. Furthermore, molecular modeling data indicate the existence of a compact SK3 channel turret conformation (like a peptide screener), where the basic rings of Arg-485 and Arg-489 are stabilized by strong ionic interactions with Asp-492 and Asp-518. In conclusion, the unique properties of Arg-485 and Arg-489 (spatial orientations and molecular interactions) in the SK3 channel account for its toxin sensitivity profile.

Chemical derivatization and purification of peptide-toxins for probing ion channel complexes.

Ion channels function as multi-protein complexes made up of ion-conducting α-subunits and regulatory ß-subunits. To detect, identify, and quantitate the regulatory ß-subunits in functioning K(+) channel complexes, we have chemically derivatized peptide-toxins that specifically react with strategically placed cysteine residues in the channel complex. Two protein labeling approaches have been developed to derivatize the peptide-toxin, charybdotoxin, with hydrophilic and hydrophobic bismaleimides, and other molecular probes. Using these cysteine-reactive peptide-toxins, we have specifically targeted KCNQ1-KCNE1 K(+) channel complexes expressed in both Xenopus oocytes and mammalian cells. The modular design of the reagents should permit this approach to be applied to the many ion channel complexes involved in electrical excitability as well as salt and water homoeostasis.

Fusion expression and purification of four disulfide-rich peptides reveals enterokinase secondary cleavage sites in animal toxins.

Animal toxins are powerful tools for testing the pharmacological, physiological, and structural characteristics of ion channels, proteases, and other receptors. However, most animal toxins are disulfide-rich peptides that are difficult to produce functionally. Here, a glutathione S-transferase (GST) fusion expression strategy was used to produce four recombinant animal toxin peptides, ChTX, StKTx23, BmP01, and ImKTx1, with different isoelectric points from 4.7 to 9.2. GST tags were removed by enterokinase, a widely used and effective commercial protease that cleaves after lysine at the cleavage site DDDDK. Using this strategy, two disulfide-rich animal toxins ChTX and StKTx23 were obtained successfully with a yield of approximately 1-2 mg/l culture. Electrophysiological experiments further showed that these two recombinant toxins showed good bioactivities, indicating that our method was effective in producing large amounts of functional disulfide-rich animal toxins. Interestingly, by analyzing the separated fractions of BmP01, StKTx23, and ImKTx1 using matrix-assisted laser desorption ionization time-of-flight mass spectrometry, four new enterokinase secondary cleavage sites were found, consisting of the sequences "WEYR," "EDK," "QNAR," and "DNDK." To our knowledge, this is the first report of the presence of secondary cleavage sites for commercial enterokinase in animal toxins. These findings will help us use commercial enterokinase appropriately as a cleavage tool in the production of animal toxins.

Scorpion toxins specific for potassium (K+) channels: a historical overview of peptide bioengineering.

Scorpion toxins have been central to the investigation and understanding of the physiological role of potassium (K⁺) channels and their expansive function in membrane biophysics. As highly specific probes, toxins have revealed a great deal about channel structure and the correlation between mutations, altered regulation and a number of human pathologies. Radio- and fluorescently-labeled toxin isoforms have contributed to localization studies of channel subtypes in expressing cells, and have been further used in competitive displacement assays for the identification of additional novel ligands for use in research and medicine. Chimeric toxins have been designed from multiple peptide scaffolds to probe channel isoform specificity, while advanced epitope chimerization has aided in the development of novel molecular therapeutics. Peptide backbone cyclization has been utilized to enhance therapeutic efficiency by augmenting serum stability and toxin half-life in vivo as a number of K⁺-channel isoforms have been identified with essential roles in disease states ranging from HIV, T-cell mediated autoimmune disease and hypertension to various cardiac arrhythmias and Malaria. Bioengineered scorpion toxins have been monumental to the evolution of channel science, and are now serving as templates for the development of invaluable experimental molecular therapeutics.

Rigid body Brownian dynamics as a tool for studying ion channel blockers.

Using a novel rigid body Brownian dynamics algorithm, we investigate how a spherically asymmetrical polyamine molecule, a branched analogue of spermine, interacts with the external vestibule of the voltage-gated potassium channel, Kv1.2. Simulations reveal that the blocker, with a charge of +4e, inserts one of its charged amine groups into the selectivity filter, while another forms a salt bridge with an aspartate residue located just outside the entrance of the pore. This binding mode mimics features of the binding of polypeptides such as the scorpion venom charybdotoxin to the channel. The potential of mean force constructed with Brownian dynamics is a reasonable match to that obtained from molecular dynamics simulations, with dissociation constants of 4.7 and 22 µM, respectively. The current-voltage relationships obtained with and without a blocker in the external reservoir show that the inward current is severely attenuated by the presence of the blocker, whereas the outward current is only moderately reduced. The computational molecular modeling technique we introduce here can provide detailed insights into ligand-channel interactions and can be used for rapidly screening potential blocker molecules.

Charybdotoxin unbinding from the mKv1.3 potassium channel: a combined computational and experimental study.

Charybdotoxin, belonging to the group of so-called scorpion toxins, is a short peptide able to block many voltage-gated potassium channels, such as mKv1.3, with high affinity. We use a reliable homology model based on the high-resolution crystal structure of the 94% sequence identical homologue Kv1.2 for charybdotoxin docking followed by molecular dynamics simulations to investigate the mechanism and energetics of unbinding, tracing the behavior of the channel protein and charybdotoxin during umbrella-sampling simulations as charybdotoxin is moved away from the binding site. The potential of mean force is constructed from the umbrella sampling simulations and combined with K(d) and free energy values gained experimentally using the patch-clamp technique to study the free energy of binding at different ion concentrations and the mechanism of the charybdotoxin-mKv1.3 binding process. A possible charybdotoxin binding mechanism is deduced that includes an initial hydrophobic contact followed by stepwise electrostatic interactions and finally optimization of hydrogen bonds and salt bridges.

Accurate determination of the binding free energy for KcsA-charybdotoxin complex from the potential of mean force calculations with restraints.

Free energy calculations for protein-ligand dissociation have been tested and validated for small ligands (50 atoms or less), but there has been a paucity of studies for larger, peptide-size ligands due to computational limitations. Previously we have studied the energetics of dissociation in a potassium channel-charybdotoxin complex by using umbrella sampling molecular-dynamics simulations, and established the need for carefully chosen coordinates and restraints to maintain the physiological ligand conformation. Here we address the ligand integrity problem further by constructing additional potential of mean forces for dissociation of charybdotoxin using restraints. We show that the large discrepancies in binding free energy arising from simulation artifacts can be avoided by using appropriate restraints on the ligand, which enables determination of the binding free energy within the chemical accuracy. We make several suggestions for optimal choices of harmonic potential parameters and restraints to be used in binding studies of large ligands.

Electro-pharmacological profile of a mitochondrial inner membrane big-potassium channel from rat brain.

Recent studies have indicated a calcium-activated large conductance potassium channel in rat brain mitochondrial inner membrane (mitoBK channel). Accordingly, we have characterized the functional and pharmacological profile of a BK channel from rat brain mitochondria in the present study. Brain mitochondrial inner membrane preparations were subjected to SDS-PAGE analysis and channel protein reconstitution into planar lipid bilayers. Western blotting and antibodies directed against various cellular proteins revealed that mitochondrial inner membrane fractions did not contain specific proteins of the other subcellular compartments except a very small fraction of endoplasmic reticulum. Channel incorporation into planar lipid bilayers revealed a voltage dependent 211 pS potassium channel with a voltage for half activation (V(1/2)) of 11.4±1.1mV and an effective gating charge z(d) of 4.7±0.9. Gating and conducting behaviors of this channel were unaffected by the addition of 2.5mM ATP, and 500 nM charybdotoxin (ChTx), but the channel appeared sensitive to 100 nM iberiotoxin (IbTx). Adding 10mM TEA at positive potentials and 10mM 4-AP at negative or positive voltages inhibited the channel activities. These results demonstrate that the mitoBK channel, present in brain mitochondrial inner membrane, displays different pharmacological properties than those classically described for plasma membrane, especially in regard to its sensitivity to iberiotoxin and charybdotoxin sensitivity.

Modeling the binding of three toxins to the voltage-gated potassium channel (Kv1.3).

The conduction properties of the voltage-gated potassium channel Kv1.3 and its modes of interaction with several polypeptide venoms are examined using Brownian dynamics simulations and molecular dynamics calculations. Employing an open-state homology model of Kv1.3, we first determine current-voltage and current-concentration curves and ascertain that simulated results accord with experimental measurements. We then investigate, using a molecular docking method and molecular dynamics simulations, the complexes formed between the Kv1.3 channel and several Kv-specific polypeptide toxins that are known to interfere with the conducting mechanisms of several classes of voltage-gated K(+) channels. The depths of potential of mean force encountered by charybdotoxin, α-KTx3.7 (also known as OSK1) and ShK are, respectively, -19, -27, and -25 kT. The dissociation constants calculated from the profiles of potential of mean force correspond closely to the experimentally determined values. We pinpoint the residues in the toxins and the channel that are critical for the formation of the stable venom-channel complexes.

Permeation and block of the Kv1.2 channel examined using brownian and molecular dynamics.

Using both Brownian and molecular dynamics, we replicate many of the salient features of Kv1.2, including the current-voltage-concentration profiles and the binding affinity and binding mechanisms of charybdotoxin, a scorpion venom. We also elucidate how structural differences in the inner vestibule can give rise to significant differences in its permeation characteristics. Current-voltage-concentration profiles are constructed using Brownian dynamics simulations, based on the crystal structure 2A79. The results are compatible with experimental data, showing similar conductance, rectification, and saturation with current. Unlike KcsA, for example, the inner pore of Kv1.2 is mainly hydrophobic and neutral, and to explore the consequences of this, we investigate the effect of mutating neutral proline residues at the mouth of the inner vestibule to charged aspartate residues. We find an increased conductance, less inward rectification, and quicker saturation of the current-voltage profile. Our simulations use modifications to our Brownian dynamics program that extend the range of channels that can be usefully modeled. Using molecular dynamics, we investigate the binding of the charybdotoxin scorpion venom to the outer vestibule of the channel. A potential of mean force is derived using umbrella sampling, giving a dissociation constant within a factor of ∼2 to experimentally derived constants. The residues involved in the toxin binding are in agreement with experimental mutagenesis studies. We thus show that the experimental observations on the voltage-gated channel, including the toxin-channel interaction, can reliably be replicated by using the two widely used computational tools.

Molecular Information of charybdotoxin blockade in the large conductance calcium-activated potassium channel.

The scorpion toxin, charybdotoxin (ChTX), is the first identified peptide inhibitor for the large-conductance Ca2+ and voltage-dependent K+ (BK) channel, and the chemical information of the interaction between ChTX and BK channel remains unclear today. Using combined computational methods, we obtained a ChTX-BK complex structure model, which correlated well with the mutagenesis data. In this complex, ChTX mainly used its beta-sheet domains to associate the BK channel with a conserved pore-blocking Lys27. Another crucial Tyr36 residue of ChTX lied over the loop connecting selectivity filter and S6 helix of BK channel, forming a hydrogen bond with Gly291 of BK channel. Besides, the unique turret region of BK channel was found to be far away from bound ChTX, which could explain the fact that many BK channel blockers show less selectivity over Kv channels. Together, all these information is helpful to reveal the diverse interactions between scorpion toxins and potassium channels and can accelerate the molecular engineering of specific inhibitor design.

Mechanism and energetics of charybdotoxin unbinding from a potassium channel from molecular dynamics simulations.

Ion channel-toxin complexes are ideal systems for computational studies of protein-ligand interactions, because, in most cases, the channel axis provides a natural reaction coordinate for unbinding of a ligand and a wealth of physiological data is available to check the computational results. We use a recently determined structure of a potassium channel-charybdotoxin complex in molecular dynamics simulations to investigate the mechanism and energetics of unbinding. Pairs of residues on the channel protein and charybdotoxin that are involved in the binding are identified, and their behavior is traced during umbrella-sampling simulations as charybdotoxin is moved away from the binding site. The potential of mean force for the unbinding of charybdotoxin is constructed from the umbrella sampling simulations using the weighted histogram analysis method, and barriers observed are correlated with specific breaking of interactions and influx of water molecules into the binding site. Charybdotoxin is found to undergo conformational changes as a result of the reaction coordinate choice--a nontrivial decision for larger ligands--which we explore in detail, and for which we propose solutions. Agreement between the calculated and the experimental binding energies is obtained once the energetic consequences of these conformational changes are included in the calculations.