Youtian Hu

The genome of Mesobuthus martensii reveals a unique adaptation model of arthropods

Representing a basal branch of arachnids, scorpions are known as ‘living fossils’ that maintain an ancient anatomy and are adapted to have survived extreme climate changes. Here we report the genome sequence of Mesobuthus martensii, containing 32,016 protein-coding genes, the most among sequenced arthropods. Although M. martensii appears to evolve conservatively, it has a greater gene family turnover than the insects that have undergone diverse morphological and physiological changes, suggesting the decoupling of the molecular and morphological evolution in scorpions. Underlying the long-term adaptation of scorpions is the expansion of the gene families enriched in basic metabolic pathways, signalling pathways, neurotoxins and cytochrome P450, and the different dynamics of expansion between the shared and the scorpion lineage-specific gene families. Genomic and transcriptomic analyses further illustrate the important genetic features associated with prey, nocturnal behaviour, feeding and detoxification. The M. martensii genome reveals a unique adaptation model of arthropods, offering new insights into the genetic bases of the living fossils.

Hg1, Novel Peptide Inhibitor Specific for Kv1.3 Channels from First Scorpion Kunitz-type Potassium Channel Toxin Family

Background: The potassium channel inhibitory activity of scorpion Kunitz-type toxins has not yet been determined. Results: We identified the first scorpion Kunitz-type potassium channel toxin family with three groups and seven members. Conclusion: A novel peptide, Hg1, specific for Kv1.3 channel, was found. Significance: Kunitz-type toxins are a new source to screen and design potential peptides for diagnosing and treating Kv1.3-mediated autoimmune diseases. The potassium channel Kv1.3 is an attractive pharmacological target for autoimmune diseases. Specific peptide inhibitors are key prospects for diagnosing and treating these diseases. Here, we identified the first scorpion Kunitz-type potassium channel toxin family with three groups and seven members. In addition to their function as trypsin inhibitors with dissociation constants of 140 nm for recombinant LmKTT-1a, 160 nm for LmKTT-1b, 124 nm for LmKTT-1c, 136 nm for BmKTT-1, 420 nm for BmKTT-2, 760 nm for BmKTT-3, and 107 nm for Hg1, all seven recombinant scorpion Kunitz-type toxins could block the Kv1.3 channel. Electrophysiological experiments showed that six of seven scorpion toxins inhibited ∼50–80% of Kv1.3 channel currents at a concentration of 1 μm. The exception was rBmKTT-3, which had weak activity. The IC50 values of rBmKTT-1, rBmKTT-2, and rHg1 for Kv1.3 channels were ∼129.7, 371.3, and 6.2 nm, respectively. Further pharmacological experiments indicated that rHg1 was a highly selective Kv1.3 channel inhibitor with weak affinity for other potassium channels. Different from classical Kunitz-type potassium channel toxins with N-terminal regions as the channel-interacting interfaces, the channel-interacting interface of Hg1 was in the C-terminal region. In conclusion, these findings describe the first scorpion Kunitz-type potassium channel toxin family, of which a novel inhibitor, Hg1, is specific for Kv1.3 channels. Their structural and functional diversity strongly suggest that Kunitz-type toxins are a new source to screen and design potential peptides for diagnosing and treating Kv1.3-mediated autoimmune diseases.

Structural and Functional Diversity of Acidic Scorpion Potassium Channel Toxins

Background Although the basic scorpion K+ channel toxins (KTxs) are well-known pharmacological tools and potential drug candidates, characterization the acidic KTxs still has the great significance for their potential selectivity towards different K+ channel subtypes. Unfortunately, research on the acidic KTxs has been ignored for several years and progressed slowly. Principal Findings Here, we describe the identification of nine new acidic KTxs by cDNA cloning and bioinformatic analyses. Seven of these toxins belong to three new α-KTx subfamilies (α-KTx28, α-KTx29, and α-KTx30), and two are new members of the known κ-KTx2 subfamily. ImKTx104 containing three disulfide bridges, the first member of the α-KTx28 subfamily, has a low sequence homology with other known KTxs, and its NMR structure suggests ImKTx104 adopts a modified cystine-stabilized α-helix-loop-β-sheet (CS-α/β) fold motif that has no apparent α-helixs and β-sheets, but still stabilized by three disulfide bridges. These newly described acidic KTxs exhibit differential pharmacological effects on potassium channels. Acidic scorpion toxin ImKTx104 was the first peptide inhibitor found to affect KCNQ1 channel, which is insensitive to the basic KTxs and is strongly associated with human cardiac abnormalities. ImKTx104 selectively inhibited KCNQ1 channel with a Kd of 11.69 µM, but was less effective against the basic KTxs-sensitive potassium channels. In addition to the ImKTx104 toxin, HeTx204 peptide, containing a cystine-stabilized α-helix-loop-helix (CS-α/α) fold scaffold motif, blocked both Kv1.3 and KCNQ1 channels. StKTx23 toxin, with a cystine-stabilized α-helix-loop-β-sheet (CS-α/β) fold motif, could inhibit Kv1.3 channel, but not the KCNQ1 channel. Conclusions/Significance These findings characterize the structural and functional diversity of acidic KTxs, and could accelerate the development and clinical use of acidic KTxs as pharmacological tools and potential drugs.

Unusual binding mode of scorpion toxin BmKTX onto potassium channels relies on its distribution of acidic residues

Besides classical scorpion toxin-potassium channel binding modes, novel modes remain unknown. Here, we report a novel binding mode of native toxin BmKTX towards Kv1.3 channel. The combined experimental and computational data indicated that BmKTX-D33H analog used the classical anti-parallel β-sheet domain as the channel-interacting interface together with the conserved channel pore-blocking Lys(26). However, the wild-type BmKTX was found to use Arg(23) rather than Lys(26) as the new pore-blocking residue, and mainly adopt the turn motif between the α-helix and antiparallel β-sheet domains to recognize Kv1.3 channel. Together, these findings not only reveal that scorpion toxin-potassium channel interaction modes are more diverse than thought, but also highlight the functional role of toxin acidic residues in mediating diverse toxin-potassium channel binding modes.

Two Conserved Arginine Residues from the SK3 Potassium Channel Outer Vestibule Control Selectivity of Recognition by Scorpion Toxins

Background: The SK channel mechanism that controls scorpion toxin recognition remains unknown. Results: Two conserved arginine residues in the SK3 channel outer vestibule were found to control toxin recognition. Conclusion: SK3 channel selectively controls toxin recognition through differential electrostatic repulsion forces between the channel vestibule and toxins. Significance: These findings expand our understanding of the diverse channel structures and functions and unlock the pharmacological potential of toxins. Potassium channel functions are often deciphered by using selective and potent scorpion toxins. Among these toxins, only a limited subset is capable of selectively blocking small conductance Ca2+-activated K+ (SK) channels. The structural bases of this selective SK channel recognition remain unclear. In this work, we demonstrate the key role of the electric charges of two conserved arginine residues (Arg-485 and Arg-489) from the SK3 channel outer vestibule in the selective recognition by the SK3-blocking BmP05 toxin. Indeed, individually substituting these residues with histidyl or lysyl (maintaining the positive electric charge partially or fully), although decreasing BmP05 affinity, still preserved the toxin sensitivity profile of the SK3 channel (as evidenced by the lack of recognition by many other types of potassium channel-sensitive charybdotoxin). In contrast, when Arg-485 or Arg-489 of the SK3 channel was mutated to an acidic (Glu) or alcoholic (Ser) amino acid residue, the channel lost its sensitivity to BmP05 and became susceptible to the “new” blocking activity by charybdotoxin. In addition to these SK3 channel basic residues important for sensitivity, two acidic residues, Asp-492 and Asp-518, also located in the SK3 channel outer vestibule, were identified as being critical for toxin affinity. Furthermore, molecular modeling data indicate the existence of a compact SK3 channel turret conformation (like a peptide screener), where the basic rings of Arg-485 and Arg-489 are stabilized by strong ionic interactions with Asp-492 and Asp-518. In conclusion, the unique properties of Arg-485 and Arg-489 (spatial orientations and molecular interactions) in the SK3 channel account for its toxin sensitivity profile.

ImKTx1, a new Kv1.3 channel blocker with a unique primary structure†

Toxins from the venoms of scorpion, snake, and spider are valuable tools to probe the structure–function relationship of ion channels. In this investigation, a new toxin gene encoding the peptide ImKTx1 was isolated from the venom gland of the scorpion Isometrus maculates by constructing cDNA library method, and the recombinant ImKTx1 peptide was characterized physiologically. The mature peptide of ImKTx1 has 39 amino acid residues including six cross‐linked cysteines. The electrophysiological experiments showed that the recombinant ImKTx1 peptide had a pharmacological profile where it inhibited Kv1.3 channel currents with IC50 of 1.70 n± 1.35 µM, whereas 10 µM rImKTx1 peptide inhibited about 40% Kv1.1 and 42% Kv1.2 channel currents, respectively. In addition, 10 µM rImKTx1 had no effect on the Nav1.2 and Nav1.4 channel currents. Multiple sequence alignments showed that ImKTx1 had no homologous toxin peptide, but it was similar with Ca2+ channel toxins from scorpion and spider in the arrangement of cysteine residues. These results indicate that ImKTx1 is a new Kv1.3 channel blocker with a unique primary structure. Our results indicate the diversity of K+ channel toxins from scorpion venoms and also provide a new molecular template targeting Kv1.3 channel.

Toxin acidic residue evolutionary function-guided design of de novo peptide drugs for the immunotherapeutic target, the Kv1.3 channel.

During the long-term evolution of animal toxins acting on potassium channels, the acidic residues can orientate the toxin binding interfaces by adjusting the molecular polarity. Based on the evolutionary function of toxin acidic residues, de novo peptide drugs with distinct binding interfaces were designed for the immunotherapeutic target, the Kv1.3 channel. Using a natural basic toxin, BmKTX, as a template, which contains 2 acidic residues (Asp19 and Asp33), we engineered two new peptides BmKTX-19 with 1 acidic residue (Asp33), and BmKTX-196 with 2 acidic residues (Asp6 and Asp33) through only adjusting acidic residue distribution for reorientation of BmKTX binding interface. Pharmacological experiments indicated that BmKTX-19 and BmKTX-196 peptides were specific inhibitors of the Kv1.3 channel and effectively suppressed cytokine secretion. In addition to the structural similarity between the designed and native peptides, both experimental alanine-scanning mutagenesis and computational simulation further indicated that the binding interface of wild-type BmKTX was successfully reoriented in BmKTX-19 and BmKTX-196, which adopted distinct toxin surfaces as binding interfaces. Together, these findings indicate not only the promising prospect of BmKTX-19 and BmKTX-196 as drug candidates but also the desirable feasibility of the evolution-guided peptide drug design for discovering numerous peptide drugs for the Kv1.3 channel.

ImKTx88, a novel selective Kv1.3 channel blocker derived from the scorpion Isometrus maculates

Scorpion toxins are useful in the structure-function research of ion channels and valuable resources for drug design. The Kv1.3 channel is an important pharmacological target for the therapy of T cell-mediated autoimmune diseases, and many toxin peptides targeting Kv1.3 have been identified as good drug candidates in recent years. In this study, a novel toxin gene ImKTx88 was isolated from the venom of the scorpion Isometrus maculates through the construction of the cDNA library method, and the recombinant toxin peptide was purified and characterized physiologically. The mature peptide of ImKTx88 contained 39 amino acid residues including six cysteines and was predicted to be a new member of α-KTx scorpion family by sequence analysis. The electrophysiological experiments further indicated that the rImKTx88 peptide had a novel pharmacological profile: it inhibited Kv1.3 channel current with an IC₅₀ of 91 ± 42 pM, and exhibited very good selectivity for Kv1.3 over Kv1.1 (4200-fold) and Kv1.2 (93000-fold) channels, respectively. All these results suggested that, as a new selective Kv1.3 channel blocker, the ImKTx88 peptide may serve as a potential drug candidate in the therapy of autoimmune diseases.

Molecular cloning and functional identification of a new K(+) channel blocker, LmKTx10, from the scorpion Lychas mucronatus.

Scorpions have a venom gland which is an important determinant in contributing to their successful survival for more than 400 million years. Their venoms contain a diversity of neurotoxins, which represent a tremendous hitherto partially unexplored resource not only for understanding ion channels but also for use in drug design and development. In this investigation, LmKTx10, a new toxin gene was identified from the venom of the scorpion Lychas mucronatus by constructing cDNA library method, and its product was expressed and characterized physiologically. The mature peptide has 38 residues including six conserved cysteines. The electrophysiological experiments further indicated that the recombinant LmKTx10 peptide has an interesting pharmacological profile: it blocks Kv1.3 channel with IC(50)=28nM which is moderate Kv1.3 channel blocking activity compared to the other a-KTxs toxins, and exhibits good selectivity on Kv1.3 over Kv1.1 and Kv1.2, about 60 folds and 450 folds, respectively. These data not only enrich the family of K(+) channel toxins from scorpion venoms but also present a potential drug template for selectively targeting the Kv1.3 channel.

hERG Potassium Channel Blockage by Scorpion Toxin BmKKx2 Enhances Erythroid Differentiation of Human Leukemia Cells K562

Background The hERG potassium channel can modulate the proliferation of the chronic myelogenous leukemic K562 cells, and its role in the erythroid differentiation of K562 cells still remains unclear. Principal Findings The hERG potassium channel blockage by a new 36-residue scorpion toxin BmKKx2, a potent hERG channel blocker with IC50 of 6.7±1.7 nM, enhanced the erythroid differentiation of K562 cells. The mean values of GPA (CD235a) fluorescence intensity in the group of K562 cells pretreated by the toxin for 24 h and followed by cytosine arabinoside (Ara-C) treatment for 72 h were about 2-fold stronger than those of K562 cells induced by Ara-C alone. Such unique role of hERG potassium channel was also supported by the evidence that the effect of the toxin BmKKx2 on cell differentiation was nullified in hERG-deficient cell lines. During the K562 cell differentiation, BmKKx2 could also suppress the expression of hERG channels at both mRNA and protein levels. Besides the function of differentiation enhancement, BmKKx2 was also found to promote the differentiation-dependent apoptosis during the differentiation process of K562 cells. In addition, the blockage of hERG potassium channel by toxin BmKKx2 was able to decrease the intracellular Ca2+ concentration during the K562 cell differentiation, providing an insight into the mechanism of hERG potassium channel regulating this cellular process. Conclusions/Significance Our results revealed scorpion toxin BmKKx2 could enhance the erythroid differentiation of leukemic K562 cells via inhibiting hERG potassium channel currents. These findings would not only accelerate the functional research of hERG channel in different leukemic cells, but also present the prospects of natural scorpion toxins as anti-leukemic drugs.