Search

Objective: Scorpion α-neurotoxins (ɑ-NaTx) inhibit the inactivation of voltage-gated sodium channels (Nav) with variable efficiency between organisms and channel isoforms. Based on our previous results, we hypothesized that the derivative of the ɑ-NaTx called BeM9 with two amino acid residues substituted with glycine (A4G and Y17G; BeM9GG) should be more selective for the mammalian channels. Surprisingly, BeM9GG lost its activity on the cardiac isoform Nav1.5. We provide a possible explanation of this effect, taking into account the published structures of ɑ-NaTx–Nav complexes. Methods: We produced BeM9GG in Escherichia coli as a fusion protein with thioredoxin, which was cleaved by cyanogen bromide. We purified BeM9GG using chromatography and measured its activity on Nav isoforms expressed in Xenopus laevis oocytes by the two-electrode voltage clamp technique. We performed computer modeling of ɑ-NaTx–Nav complexes to figure out the peculiarities of toxin interactions with Nav1.5. Results and Discussion: Using electrophysiology, we tested BeM9GG on Nav, and unexpectedly observed compromised activity on Nav1.5. We compared the structures of available ɑ-NaTx–Nav complexes to explain this effect. In case of Nav1.5 the toxin-binding site is immersed deeper in the membrane. We explain this by sequence variations in two positions of the voltage-sensing domain IV of Nav: in Nav1.5 corresponding residues have shorter side chains, permitting the toxin to sit deeper. At the same time, residue Y17 in BeM9, which is missing in BeM9GG, interacts with the hydrophobic core of the membrane and may play a significant role in its activity against Nav1.5. Conclusions: The role of the membrane in ɑ-NaTx interactions with Nav has been overlooked. It is especially important in case of Nav1.5, where the tripartite toxin–ion channel– membrane complex is formed. Our study will be of help for the future design of selective Nav ligands. [ABSTRACT FROM AUTHOR]

Obtaining compounds with specificity for certain isoforms of ion channels is a significant problem of curent physiology and pharmacology. In a series of papers, we have shown that the α-hairpinin fold can serve as a template for the rational design of peptide ligands of potassium channels. Here, we used molecular modeling to optimize the structure of the previously obtained Tk-hefu-10 peptide, a selective KV1.3 channel blocker, with a half-maximal inhibitory concentration (IC50) of ≈150 nM. Molecular dynamics simulation of the Tk-hefu-10–KV1.3 complex provided information on the interaction of individual amino acid residues of the peptide and channel, and the analysis of these interactions made it possible to propose amino acid substitutions in the structure of Tk-hefu-10 to increase its affinity. Novel Tk-hefu-12 peptide is a truncated analog of Tk-hefu-10 by one residue with five substitutions; it is characterized by an IC50 value of ≈70 nM against KV1.3. In addition, there are no methionine residues in the structure of Tk-hefu-12, which makes it possible to obtain Tk-hefu peptides using cyanogen bromide. [ABSTRACT FROM AUTHOR]

The isoform of voltage-gated potassium channels KV1.2 is of interest because mutations in its gene are associated with various diseases, such as ataxia and epilepsy. Selective ligands are needed to study the function of KV1.2 in health and disease. In our work, we obtained such a ligand based on the known scorpion peptide toxin, charybdotoxin (ChTx, α-KTx1.1) from the venom of Leiurus hebraeus, by introducing a single amino acid substitution M29I into its structure. ChTx_M29I peptide was produced in a bacterial expression system. Its pharmacological characterization was carried out in Xenopus laevis frog oocytes expressing a panel of human KV1 channels. We found that, compared to the parent toxin, ChTx_M29I peptide showed lower affinity for KV1.1, 1.3, and 1.6 channels, while its activity against KV1.2 increased manifold. We attribute this effect to the interaction of the peptide with a specific channel residue (V381 in KV1.2). If there is a relatively small residue at this position, then an advantageous contact is formed that increases the affinity. ChTx_M29I peptide studied by us presents one of the highest affinity (with a half-maximal inhibitory concentration IC50 ≈ 6 pM) and selectivity among KV1.2 ligands (affinity for other isoforms is lower by 680 times or more). [ABSTRACT FROM AUTHOR]

Scorpion α-toxins are small proteins inhibiting the inactivation of voltage-gated sodium channels. They can selectively act on either mammalian (mammal toxins) or insect channels (insect toxins), or affect both types of channels (α-like toxins). Currently no model has been proposed that fully explains the dependence of selectivity upon amino acid sequence, but some patterns have already been established. Thus, most mammal toxins have an aspartic acid residue in position 8, which is involved in the formation of the nest motif, but it is still not clear whether this residue interacts directly with channels. The objective of our study was to obtain a derivative of the α-like toxin BeM9 with the replacement of lysine in position 8 by glutamate (K8E), changing the charge, but excluding the formation of the nest motif. In addition, we replaced the tyrosine in position 17 with glycine (Y17G), which is characteristic of mammal toxins. Surprisingly, the double-mutant derivative BeM9EG lost its activity on mammalian channels, becoming an insect toxin. To explain these changes, we constructed models of BeM9 and BeM9EG complexes with channels, and also performed molecular dynamics of isolated toxins. Analysis of intermolecular contacts in the complexes did not explain the reason for the selectivity change. Nevertheless, the structure of intramolecular contacts and data on molecular mobility indicate an important role of residues K8 and Y17 in stabilizing a certain conformation of BeM9 loops. We assume that the replacement of these residues allosterically affects the efficiency of toxin binding to channels. [ABSTRACT FROM AUTHOR]

In mammals, about 40 isoforms of voltage-gated potassium channels (KVs) have been found. To study such a variety of KVs, substances are needed that are able to selectively bind to them and change their properties. We have previously reported on the isolation and pharmacological characterization of MeKTx13-3, a peptide toxin from the venom of the scorpion Mesobuthus eupeus. The toxin has shown high affinity to a number of KVs, with little selectivity for the KV1.1 isoform. In this paper, we describe the production of an artificial derivative of MeKTx13-3, named MeKTx13-3_RMRH, using rational design. The selectivity of MeKTx13-3_RMRH in relation to KV1.1 is increased by an order of magnitude making it one of the most specific ligands of this KV isoform. Finally, using computer simulations, we demonstrate that the preference of the new ligand to KV1.1 can be realized through a specific positioning of the toxin in complex with the channel. [ABSTRACT FROM AUTHOR]

A new neurotoxin RTX-VI that modulates the voltage-gated sodium channels (NaV) was isolated from the ethanolic extract of the sea anemone Heteractis crispa. Its amino acid sequence was determined using the combination of Edman degradation and tandem mass spectrometry. RTX-VI turned out to be an unusual natural analogue of the previously described sea anemone toxin RTX-III. The RTX-VI molecule consists of two disulfide-linked peptide chains and is devoid of Arg13, which is important for the selectivity and affinity of such peptides for the NaV channels. Electrophysiological screening of RTV-VI on NaV channel subtypes showed its selective interaction with the central nervous system (NaV1.2, NaV1.6) and insect (BgNaV1, VdNaV1) sodium channels. [ABSTRACT FROM AUTHOR]

An effective bacterial system for the production of β-toxin Ts1, the main component of the Brazilian scorpion Tityus serrulatus venom, was developed. Recombinant toxin and its 15N-labeled analogue were obtained via direct expression of synthetic gene in Escherichia coli with subsequent folding from the inclusion bodies. According to NMR spectroscopy data, the recombinant toxin is structured in an aqueous solution and contains a significant fraction of β-structure. The formation of a stable disulfide-bond isomer of Ts1, having a disordered structure, has also been observed during folding. Recombinant Ts1 blocks Na+ current through NaV1.5 channels without affecting the processes of activation and inactivation. At the same time, the effect upon NaV1.4 channels is associated with a shift of the activation curve towards more negative membrane potentials. [ABSTRACT FROM AUTHOR]

The HMIQ3c1 recombinant peptide (6434 Da), the Kunitz-type protease inhibitor, which belonged to the new group of the IQ-peptides from the Heteractis magnifica sea anemone was prepared. The inhibitory constant of trypsin by this peptide proved to be 5.0 × 10-8 M. rHMIQ3c1 was shown to have no interaction with eight isoforms of Kv1 channels. This peptide exhibited neuroprotective activity in a concentration of 10 μM, and the peptide increased the viability of cells of the Neuro2a murine neuroblastoma by 39.4 ± 6.6% in the presence of β-amyloid. However, the peptide did not influence the viability of the cells during their incubation with 6-hydroxydofamine. [ABSTRACT FROM AUTHOR]

Introduction and Objectives: Potassium channel play a key role in cellular excitability and signal transduction pathways. Most pharmacological and structural characteristics of K+ channels have been elucidated by toxins isolated from scorpion venoms that modulate the flow of K+ ions while interacting with the channel pore. The compact small size and the strong specificity of scorpion toxins make them the ideal support for the analysis of protein folds as well as the study of the mechanisms involved in the toxin-channel interaction. Material and Methods: Buthus occitanus tunetanus scorpion venom was provided by electric stimulation of the scorpion's post-abdomen. Sephadex G50 fractionization was followed by an HPLC purification step using a Hewlett-Packard Series II 1100 chromatograph with diode array UV detector. Molecular weight of Kbot55 was first estimated by SDS-PAGE analysis under non-reducing conditions and confirmed by MALDI-TOF mass spectrometry. Amino-acid sequence was identified by automated Edman degradation in an applied Biosystems 476A protein sequence. Kbot55 has been subjected to a screening on a wide range of 7 cloned voltage-gated potassium channels Kv1.1-Kv1.6, Shaker IR insect channel and Nav1.4, expressed in Xenopus laevis ovocytes in order to evaluate its electrophysiological properties. Results and Conclusion: Herein, we isolated, identified and characterized a new peptide from Buthus occitanus tunetanus scorpion venom. The 39 amino acid sequence of Kbot55is crosslinked by 3 disulfide bridges and has a molecular mass of 4128.65 Da. Kbot55 is very weakly represented in the venom and remains a challenge for biochemical characterization. This molecule targets voltage-gated potassium channels with high affinity. In term of sequence homology, Kbot55 shows very low amino acid similarity with other scorpion potassium toxins and therefore was considered as novel type of scorpion toxins. It is thus considered as the first representative toxin of the new a-Ktx31 subfamily and therefore was classified as a-Ktx31.1. [ABSTRACT FROM AUTHOR]