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 hK_V1.2 with high affinity (K_d = 65 nM). The conductance–voltage relationship of K_V1.2 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 K_Ca2.2 and K_Ca3.1 channels, with K_d = 502 nM, and K_d = 58 nM, respectively. However, the peptide does not inhibit hK_V1.1, hK_V1.3, hK_V1.4, hK_V1.5, hK_V1.6, hK_V11.1, mK_Ca1.1 K⁺ channels or the hNa_V1.5 and hNa_V1.4 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.

The human voltage gated potassium channel Kv1.5 that conducts the I_Kur 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 I_Kur 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 I_Kur: 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.

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.

The voltage-gated proton channel (H_v1) selectively transports protons (H⁺) across biological membranes in response to membrane potential changes. H_v1 is assembled as a dimer, and unlike most voltage-gated ion channels, it lacks a traditional central pore domain; instead, the voltage-sensing domain (VSD) of each monomer facilitates proton conduction via a hydrogen-bond network. H_v1 is widely expressed in various human cell types (e.g., immune cells, sperm, etc.) including tumor cells. In tumor cells, the accumulation of acidic intermediates generated by glycolysis under hypoxic conditions or ROS production leads to significant cytosolic acidification. H_v1 can remove protons from the cytosol rapidly, contributing to the adaptation of the cells to the tumor microenvironment, which may have significant consequences in tumor cell survival, proliferation, and progression. Therefore, H_v1 may be very promising not only as a tumor marker but also as a potential therapeutic target in oncology. Molecules that modulate the proton flux through H_v1 can be divided into two broad groups: inhibitors and activators. H_v1 inhibitors can be simple ions, small molecules, lipids, and peptides. In contrast, fewer H_v1 activators are known, including albumin, NH29, quercetin, and arachidonic acid. The mechanism of action of some inhibitors is well described, but not all. H_v1 modulation has profound effects on cellular physiology, especially under stress or pathological conditions, like cancer and inflammation. The therapeutic application of selective H_v1 inhibitors or activators could be a very promising strategy in the treatment of several serious diseases.