Characterization and Chemical Synthesis of Cm39 (α-KTx 4.8): A Scorpion Toxin That Inhibits Voltage-Gated K+ Channel KV1.2 and Small- and Intermediate-Conductance Ca2+-Activated K+ Channels KCa2.2 and KCa3.1.

A novel peptide, Cm39, was identified in the venom of the scorpion Centruroides margaritatus. Its primary structure was determined. It consists of 37 amino acid residues with a MW of 3980.2 Da. The full chemical synthesis and proper folding of Cm39 was obtained. Based on amino acid sequence alignment with different K+ channel inhibitor scorpion toxin (KTx) families and phylogenetic analysis, Cm39 belongs to the α-KTx 4 family and was registered with the systematic number of α-KTx 4.8. Synthetic Cm39 inhibits the voltage-gated K+ channel hKV1.2 with high affinity (Kd = 65 nM). The conductance-voltage relationship of KV1.2 was not altered in the presence of Cm39, and the analysis of the toxin binding kinetics was consistent with a bimolecular interaction between the peptide and the channel; therefore, the pore blocking mechanism is proposed for the toxin-channel interaction. Cm39 also inhibits the Ca2+-activated KCa2.2 and KCa3.1 channels, with Kd = 502 nM, and Kd = 58 nM, respectively. However, the peptide does not inhibit hKV1.1, hKV1.3, hKV1.4, hKV1.5, hKV1.6, hKV11.1, mKCa1.1 K+ channels or the hNaV1.5 and hNaV1.4 Na+ channels at 1 µM concentrations. Understanding the unusual selectivity profile of Cm39 motivates further experiments to reveal novel interactions with the vestibule of toxin-sensitive channels.

Recombinant Expression in Pichia pastoris System of Three Potent Kv1.3 Channel Blockers: Vm24, Anuroctoxin, and Ts6.

The Kv1.3 channel has become a therapeutic target for the treatment of various diseases. Several Kv1.3 channel blockers have been characterized from scorpion venom; however, extensive studies require amounts of toxin that cannot be readily obtained directly from venoms. The Pichia pastoris expression system provides a cost-effective approach to overcoming the limitations of chemical synthesis and E. coli recombinant expression. In this work, we developed an efficient system for the production of three potent Kv1.3 channel blockers from different scorpion venoms: Vm24, AnTx, and Ts6. Using the Pichia system, these toxins could be obtained in sufficient quantities (Vm24 1.6 mg/L, AnTx 46 mg/L, and Ts6 7.5 mg/L) to characterize their biological activity. A comparison was made between the activity of tagged and untagged recombinant peptides. Tagged Vm24 and untagged AnTx are nearly equivalent to native toxins in blocking Kv1.3 (Kd = 4.4 pM and Kd = 0.72 nM, respectively), whereas untagged Ts6 exhibits a 53-fold increase in Kd (Kd = 29.1 nM) as compared to the native peptide. The approach described here provides a method that can be optimized for toxin production to develop more selective and effective Kv1.3 blockers with therapeutic potential.

Multiple mechanisms contribute to fluorometry signals from the voltage-gated proton channel.

Voltage-clamp fluorometry (VCF) supplies information about the conformational changes of voltage-gated proteins. Changes in the fluorescence intensity of the dye attached to a part of the protein that undergoes a conformational rearrangement upon the alteration of the membrane potential by electrodes constitute the signal. The VCF signal is generated by quenching and dequenching of the fluorescence as the dye traverses various local environments. Here we studied the VCF signal generation, using the Hv1 voltage-gated proton channel as a tool, which shares a similar voltage-sensor structure with voltage-gated ion channels but lacks an ion-conducting pore. Using mutagenesis and lipids added to the extracellular solution we found that the signal is generated by the combined effects of lipids during movement of the dye relative to the plane of the membrane and by quenching amino acids. Our 3-state model recapitulates the VCF signals of the various mutants and is compatible with the accepted model of two major voltage-sensor movements.

Cm28, a scorpion toxin having a unique primary structure, inhibits KV1.2 and KV1.3 with high affinity.

The Cm28 in the venom of Centruroides margaritatus is a short peptide consisting of 27 amino acid residues with a mol wt of 2,820 D. Cm28 has <40% similarity with other known α-KTx from scorpions and lacks the typical functional dyad (lysine-tyrosine) required to block KV channels. However, its unique sequence contains the three disulfide-bond traits of the α-KTx scorpion toxin family. We propose that Cm28 is the first example of a new subfamily of α-KTxs, registered with the systematic number α-KTx32.1. Cm28 inhibited voltage-gated K+ channels KV1.2 and KV1.3 with Kd values of 0.96 and 1.3 nM, respectively. There was no significant shift in the conductance-voltage (G-V) relationship for any of the channels in the presence of toxin. Toxin binding kinetics showed that the association and dissociation rates are consistent with a bimolecular interaction between the peptide and the channel. Based on these, we conclude that Cm28 is not a gating modifier but rather a pore blocker. In a selectivity assay, Cm28 at 150 nM concentration (>100× Kd value for KV1.3) did not inhibit KV1.5, KV11.1, KCa1.1, and KCa3.1 K+ channels; NaV1.5 and NaV1.4 Na+ channels; or the hHV1 H+ channel but blocked ∼27% of the KV1.1 current. In a biological functional assay, Cm28 strongly inhibited the expression of the activation markers interleukin-2 receptor and CD40 ligand in anti-CD3-activated human CD4+ effector memory T lymphocytes. Cm28, due to its unique structure, may serve as a template for the generation of novel peptides targeting KV1.3 in autoimmune diseases.

Peptide Inhibitors of Kv1.5: An Option for the Treatment of Atrial Fibrillation.

The human voltage gated potassium channel Kv1.5 that conducts the IKur current is a key determinant of the atrial action potential. Its mutations have been linked to hereditary forms of atrial fibrillation (AF), and the channel is an attractive target for the management of AF. The development of IKur blockers to treat AF resulted in small molecule Kv1.5 inhibitors. The selectivity of the blocker for the target channel plays an important role in the potential therapeutic application of the drug candidate: the higher the selectivity, the lower the risk of side effects. In this respect, small molecule inhibitors of Kv1.5 are compromised due to their limited selectivity. A wide range of peptide toxins from venomous animals are targeting ion channels, including mammalian channels. These peptides usually have a much larger interacting surface with the ion channel compared to small molecule inhibitors and thus, generally confer higher selectivity to the peptide blockers. We found two peptides in the literature, which inhibited IKur: Ts6 and Osu1. Their affinity and selectivity for Kv1.5 can be improved by rational drug design in which their amino acid sequences could be modified in a targeted way guided by in silico docking experiments.

Optimization of Pichia pastoris Expression System for High-Level Production of Margatoxin.

Margatoxin (MgTx) is a high-affinity blocker of voltage-gated potassium (Kv) channels. It inhibits Kv1.1-Kv1.3 ion channels in picomolar concentrations. This toxin is widely used to study physiological function of Kv ion channels in various cell types, including immune cells. Isolation of native MgTx in large quantities from scorpion venom is not affordable. Chemical synthesis and recombinant production in Escherichia coli need in vitro oxidative refolding for proper disulfide bond formation, resulting in a very low yield of peptide production. The Pichia pastoris expression system offers an economical approach to overcome all these limitations and gives a higher yield of correctly refolded recombinant peptides. In this study, improved heterologous expression of recombinant MgTx (rMgTx) in P. pastoris was obtained by using preferential codons, selecting the hyper-resistant clone against Zeocin, and optimizing the culturing conditions. About 36 ± 4 mg/L of >98% pure His-tagged rMgTx (TrMgTx) was produced, which is a threefold higher yield than has been previously reported. Proteolytic digestion of TrMgTx with factor Xa generated untagged rMgTx (UrMgTx). Both TrMgTx and UrMgTx blocked the Kv1.2 and Kv1.3 currents (patch-clamp) (K d for Kv1.2 were 64 and 14 pM, and for Kv1.3, 86 and 50 pM, respectively) with comparable potency to the native MgTx. The analysis of the binding kinetics showed that TrMgTx had a lower association rate than UrMgTx for both Kv1.2 and Kv1.3. The dissociation rate of both the analogues was the same for Kv1.3. However, in the case of Kv1.2, TrMgTx showed a much higher dissociation rate with full recovery of the block than UrMgTx. Moreover, in a biological functional assay, both peptides significantly downregulated the expression of early activation markers IL2R and CD40L in activated CD4+ TEM lymphocytes whose activation was Kv1.3 dependent. In conclusion, the authors report that the Pichia expression system is a powerful method to produce disulfide-rich peptides, the overexpression of which could be enhanced noticeably through optimization strategies, making it more cost-effective. Since the presence of the His-tag on rMgTx only mildly altered the block equilibrium and binding kinetics, recombinant toxins could be used in ion channel research without removing the tag and could thus reduce the cost and time demand for toxin production.

Key amino acid residues involved in mammalian and insecticidal activities of Magi4 and Hv1b, cysteine-rich spider peptides from the δ-atracotoxin family.

δ-Atracotoxins, also known as δ-hexatoxins, are spider neurotoxic peptides, lethal to both vertebrates and insects. Their mechanism of action involves the binding to of the S3/S4 loop of the domain IV of the voltage-gated sodium channels (Nav). Because of the chemical difficulties of synthesizing folded synthetic δ-atracotoxins correctly, here we explore an expression system that is designed to produce biologically active recombinant δ-atracotoxins, and a number of variants, in order to establish certain amino acids implicated in the pharmacophore of this lethal neurotoxin. In order to elucidate and verify which amino acid residues play a key role that is toxic to vertebrates and insects, amino acid substitutes were produced by aligning the primary structures of several lethal δ-atracotoxins with those of δ-atracotoxins-Hv1b; a member of the δ-atracotoxin family that has low impact on vertebrates and is not toxic to insects. Our findings corroborate that the substitutions of the amino acid residue Y22 from δ-atracotoxin-Mg1a (Magi4) to K22 in δ-atracotoxin-Hv1b reduces its mammalian activity. Moreover, the substitutions of the amino acid residues Y22 and N26 from δ-atracotoxin-Mg1a (Magi4) to K22 and N26 in δ-atracotoxin-Hv1b reduces its insecticidal activity. Also, the basic residues K4 and R5 are important for keeping such insecticidal activity. Structural models suggest that such residues are clustered onto two bioactive surfaces, which share similar areas, previously reported as bioactive surfaces for scorpion α-toxins. Furthermore, these bioactive surfaces were also found to be similar to those found in related spider and anemone toxins, which affect the same Nav receptor, indicating that these motifs are important not only for scorpion but may be also for animal toxins that affect the S3/S4 loop of the domain IV of the Nav.

A Novel Insecticidal Spider Peptide that Affects the Mammalian Voltage-Gated Ion Channel hKv1.5.

Spider venoms include various peptide toxins that modify the ion currents, mainly of excitable insect cells. Consequently, scientific research on spider venoms has revealed a broad range of peptide toxins with different pharmacological properties, even for mammal species. In this work, thirty animal venoms were screened against hKv1.5, a potential target for atrial fibrillation therapy. The whole venom of the spider Oculicosa supermirabilis, which is also insecticidal to house crickets, caused voltage-gated potassium ion channel modulation in hKv1.5. Therefore, a peptide from the spider O. supermirabilis venom, named Osu1, was identified through HPLC reverse-phase fractionation. Osu1 displayed similar biological properties as the whole venom; so, the primary sequence of Osu1 was elucidated by both of N-terminal degradation and endoproteolytic cleavage. Based on its primary structure, a gene that codifies for Osu1 was constructed de novo from protein to DNA by reverse translation. A recombinant Osu1 was expressed using a pQE30 vector inside the E. coli SHuffle expression system. recombinant Osu1 had voltage-gated potassium ion channel modulation of human hKv1.5, and it was also as insecticidal as the native toxin. Due to its novel primary structure, and hypothesized disulfide pairing motif, Osu1 may represent a new family of spider toxins.