Structure-function relationships in ShKT domain peptides: ShKT-Ts1 from the sea anemone Telmatactis stephensoni.

Diverse structural scaffolds have been described in peptides from sea anemones, with the ShKT domain being a common scaffold first identified in ShK toxin from Stichodactyla helianthus. ShK is a potent blocker of voltage-gated potassium channels (KV 1.x), and an analog, ShK-186 (dalazatide), has completed Phase 1 clinical trials in plaque psoriasis. The ShKT domain has been found in numerous other species, but only a tiny fraction of ShKT domains has been characterized functionally. Despite adopting the canonical ShK fold, some ShKT peptides from sea anemones inhibit KV 1.x, while others do not. Mutagenesis studies have shown that a Lys-Tyr (KY) dyad plays a key role in KV 1.x blockade, although a cationic residue followed by a hydrophobic residue may also suffice. Nevertheless, ShKT peptides displaying an ShK-like fold and containing a KY dyad do not necessarily block potassium channels, so additional criteria are needed to determine whether new ShKT peptides might show activity against potassium channels. In this study, we used a combination of NMR and molecular dynamics (MD) simulations to assess the potential activity of a new ShKT peptide. We determined the structure of ShKT-Ts1, from the sea anemone Telmatactis stephensoni, examined its tissue localization, and investigated its activity against a range of ion channels. As ShKT-Ts1 showed no activity against KV 1.x channels, we used MD simulations to investigate whether solvent exposure of the dyad residues may be informative in rationalizing and potentially predicting the ability of ShKT peptides to block KV 1.x channels. We show that either a buried dyad that does not become exposed during MD simulations, or a partially exposed dyad that becomes buried during MD simulations, correlates with weak or absent activity against KV 1.x channels. Therefore, structure determination coupled with MD simulations, may be used to predict whether new sequences belonging to the ShKT family may act as potassium channel blockers.

Of Seven New K+ Channel Inhibitor Peptides of Centruroides bonito, α-KTx 2.24 Has a Picomolar Affinity for Kv1.2.

Seven new peptides denominated CboK1 to CboK7 were isolated from the venom of the Mexican scorpion Centruroides bonito and their primary structures were determined. The molecular weights ranged between 3760.4 Da and 4357.9 Da, containing 32 to 39 amino acid residues with three putative disulfide bridges. The comparison of amino acid sequences with known potassium scorpion toxins (KTx) and phylogenetic analysis revealed that CboK1 (α-KTx 10.5) and CboK2 (α-KTx 10.6) belong to the α-KTx 10.x subfamily, whereas CboK3 (α-KTx 2.22), CboK4 (α-KTx 2.23), CboK6 (α-KTx 2.21), and CboK7 (α-KTx 2.24) bear > 95% amino acid similarity with members of the α-KTx 2.x subfamily, and CboK5 is identical to Ce3 toxin (α-KTx 2.10). Electrophysiological assays demonstrated that except CboK1, all six other peptides blocked the Kv1.2 channel with Kd values in the picomolar range (24-763 pM) and inhibited the Kv1.3 channel with comparatively less potency (Kd values between 20-171 nM). CboK3 and CboK4 inhibited less than 10% and CboK7 inhibited about 42% of Kv1.1 currents at 100 nM concentration. Among all, CboK7 showed out-standing affinity for Kv1.2 (Kd = 24 pM), as well as high selectivity over Kv1.3 (850-fold) and Kv1.1 (~6000-fold). These characteristics of CboK7 may provide a framework for developing tools to treat Kv1.2-related channelopathies.

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.