Functional Characterization of a New Degradation Peptide BmTX4-P1 from Traditional Chinese Scorpion Medicinal Material.

Thermally processed Buthus martensii Karsch scorpion is an important traditional Chinese medical material that has been widely used to treat various diseases in China for over one thousand years. Our recent work showed that thermally processed Buthus martensii Karsch scorpions contain many degraded peptides; however, the pharmacological activities of these peptides remain to be studied. Here, a new degraded peptide, BmTX4-P1, was identified from processed Buthus martensii Karsch scorpions. Compared with the venom-derived wild-type toxin peptide BmTX4, BmTX4-P1 missed some amino acids at the N-terminal and C-terminal regions, while containing six conserved cysteine residues, which could be used to form disulfide bond-stabilized α-helical and ß-sheet motifs. Two methods (chemical synthesis and recombinant expression) were used to obtain the BmTX4-P1 peptide, named sBmTX4-P1 and rBmTX4-P1. Electrophysiological experimental results showed that sBmTX4-P1 and rBmTX4-P1 exhibited similar activities to inhibit the currents of hKv1.2 and hKv1.3 channels. In addition, the experimental electrophysiological results of recombinant mutant peptides of BmTX4-P1 indicated that the two residues of BmTX4-P1 (Lys22 and Tyr31) were the key residues for its potassium channel inhibitory activity. In addition to identifying a new degraded peptide, BmTX4-P1, from traditional Chinese scorpion medicinal material with high inhibitory activities against the hKv1.2 and hKv1.3 channels, this study also provided a useful method to obtain the detailed degraded peptides from processed Buthus martensii Karsch scorpions. Thus, the study laid a solid foundation for further research on the medicinal function of these degraded peptides.

BmK86-P1, a New Degradation Peptide with Desirable Thermostability and Kv1.2 Channel-Specific Activity from Traditional Chinese Scorpion Medicinal Material.

Thermally processed Buthus martensii Karsch scorpions are a traditional Chinese medical material for treating various diseases. However, their pharmacological foundation remains unclear. Here, a new degraded peptide of scorpion toxin was identified in Chinese scorpion medicinal material by proteomics. It was named BmK86-P1 and has six conserved cysteine residues. Homology modeling and circular dichroism spectra experiments revealed that BmK86-P1 not only contained representative disulfide bond-stabilized α-helical and ß-sheet motifs but also showed remarkable stability at test temperatures from 20-95 °C. Electrophysiology experiments indicated that BmK86-P1 was a highly potent and selective inhibitor of the hKv1.2 channel with IC50 values of 28.5 ± 6.3 nM. Structural and functional dissection revealed that two residues of BmK86-P1 (i.e., Lys19 and Ile21) were the key residues that interacted with the hKv1.2 channel. In addition, channel chimeras and mutagenesis experiments revealed that three amino acids (i.e., Gln357, Val381 and Thr383) of the hKv1.2 channel were responsible for BmK86-P1 selectivity. This research uncovered a new bioactive peptide from traditional Chinese scorpion medicinal material that has desirable thermostability and Kv1.2 channel-specific activity, which strongly suggests that thermally processed scorpions are novel peptide resources for new drug discovery for the Kv1.2 channel-related ataxia and epilepsy diseases.

Mutagenesis computer experiments in pentameric ligand-gated ion channels: the role of simulation tools with different resolution.

Pentameric ligand-gated ion channels (pLGICs) are an important class of widely expressed membrane neuroreceptors, which play a crucial role in fast synaptic communications and are involved in several neurological conditions. They are activated by the binding of neurotransmitters, which trigger the transmission of an electrical signal via facilitated ion flux. They can also be activated, inhibited or modulated by a number of drugs. Mutagenesis electrophysiology experiments, with natural or unnatural amino acids, have provided a large body of functional data that, together with emerging structural information from X-ray spectroscopy and cryo-electron microscopy, are helping unravel the complex working mechanisms of these neuroreceptors. Computer simulations are complementing these mutagenesis experiments, with insights at various levels of accuracy and resolution. Here, we review how a selection of computational tools, including first principles methods, classical molecular dynamics and enhanced sampling techniques, are contributing to construct a picture of how pLGICs function and can be pharmacologically targeted to treat the disorders they are responsible for.