Characterization of a new Kv1.3 channel-specific blocker, J123, from the scorpion Buthus martensii Karsch

The potassium channel Kv1.3 is an attractive pharmacological target for T-cell-mediated autoimmune diseases, and specific and selective peptidic blockers of Kv1.3 channels have served as valuable therapeutic leads for treating these diseases. Here, we found a new peptide toxin, J123, with 43 amino acids including six cysteine residues by screening the venomous cDNA library of scorpion Buthus martensii Karsch, which has been used as traditional medicine in China for more than 2000 years. The sequence analysis suggested that peptide J123 constituted a new member of the alpha-KTx toxins. The electrophysiological experiments further indicated that peptide J123 has a novel pharmacological profiles: it blocked Kv1.3 channel with high potency (IC(50)=0.79nM), and exhibited good selectivity on Kv1.3 over Kv1.1 (>1000-fold) and Kv1.2 (about 30-fold), respectively. Furthermore, peptide J123 had no activity on SKCa2 and SKCa3 channels at micromolar concentration level. Based on the pharmacological activities, the possible channel-interacting surface of peptide J123 was also predicted by molecular modeling and docking. All these data not only enrich the knowledge of the structure-function relationship of the new Kv1.3-speicific peptide but also present a potential drug candidate for selectively targeting Kv1.3 channels.     

Nuclear localization and dominant-negative suppression by a mutant SKCa3 N-terminal channel fragment identified in a patient with schizophrenia

The small conductance calcium-activated K+ channel gene SKCa3/KCNN3 maps to 1q21, a region strongly linked to schizophrenia. Recently, a 4-base pair deletion in SKCa3 was reported in a patient with schizophrenia, which truncates the protein at the end of the N-terminal cytoplasmic region (SKCa3Delta). We generated a green fluorescent protein-SKCa3 N-terminal construct (SKCa3-1/285) that is identical to SKCa3Delta except for the last two residues. Using confocal microscopy we demonstrate that SKCa3-1/285 localizes rapidly and exclusively to the nucleus of mammalian cells like several other pathogenic polyglutamine-containing proteins. This nuclear targeting is mediated in part by two polybasic sequences present at the C-terminal end of SKCa3-1/285. In contrast, full-length SKCa3, SKCa2, and IKCa1 polypeptides are all excluded from the nucleus and express as functional channels. When overexpressed in human Jurkat T cells, SKCa3-1/285 can suppress endogenous SKCa2 currents but not voltage-gated K+ currents. This dominant-negative suppression is most likely mediated through the co-assembly of SKCa3-1/285 with native subunits and the formation of non-functional tetramers. The nuclear localization of SKCa3-1/285 may alter neuronal architecture, and its ability to dominantly suppress endogenous small conductance K(Ca) currents may affect patterns of neuronal firing. Together, these two effects may play a part in the pathogenesis of schizophrenia and other neuropsychiatric disorders.     

Characterisation of a novel toxin active on potassium apanaine channels, purified from Buthus occitanus tunetanus scorpion venom

A new peptidyl inhibitor of the small-conductance Ca(2+)-activated K+ channels (SKca) was purified to homogeneity from the venom of the Tunisian scorpion Buthus occitanus tunetanus. The molecular mass determined by SDS-PAGE, shows that it's a short peptide (3300 Da). The primary sequence of this toxin shows that it is a 31-residue polypeptide cross-linked by three disulfide bridges and structurally related to subfamily 5 of short scorpion toxins. This molecule shows similar pharmacological properties with this group of peptides inducing high toxicity in mice after intracerebro-ventricular injection, and competing with iodinated apamin for binding to its receptor site from rat brain synaptosomes (K0.5 = 4 nM).     

Exploring structural features of the interaction between the scorpion toxinCnErg1 and ERG K+ channels

The gamma-KTx-type scorpion toxins specific for K+ channels were found to interact with ERG channels on the turret region, while alpha-KTx3.2 Agitoxin-2 binds to the pore region of the Shaker K+ channel, and alpha-KTx5.3 BmP05 binds to the intermediate region of the small-conductance calcium-activated K-channel (SK(Ca)). In order to explore the critical residues for gamma-KTx binding, we determined the NMR structure of native gamma-KTx1.1 (CnErg1), a 42 amino acid residues scorpion toxin isolated from the venom of the Mexican scorpion Centruro?des noxius Hoffmann, and we used computational evolutionary trace (ET) analysis to predict possible structural and functional features of interacting surfaces. The 1H-NMR three-dimensional solution structure of native ergtoxin (CnErg1) was solved using a total of 452 distance constraints, 13 3J(NH-Halpha) and 10 hydrogen bonds. The structure is characterized by 2 segments of alpha-helices and a triple-stranded antiparallel beta-sheet stabilized by 4 disulfide bridges. The ET and structural analysis provided indication of the presence of two important amino acid residue clusters, one hydrophobic and the other hydrophilic, that should be involved in the surface contact between the toxin and the channel. Some features of the proposed interacting surface are discussed.     

Charybdotoxin blocks voltage-gated K+ channels in human and murine T lymphocytes

A variety of scorpion venoms and purified toxins were tested for effects on ion channels in human T lymphocytes, a human T leukemia cell line (Jurkat), and murine thymocytes, using the whole-cell patch-clamp method. Nanomolar concentrations of charbdotoxin (CTX), a purified peptide component of Leiurus quinquestriatus venom known to block Ca2+-activated K+ channels from muscle, blocked "type n" voltage-gated K+ channels in human T lymphoid cells. The Na+ channels occasionally expressed in these cells were unaffected by the toxin. From the time course of development and removal of K+ channel block we determined the rates of CTX binding and unbinding. CTX blocks K+ channels in Jurkat cells with a Kd value between 0.5 and 1.5 nM. Of the three types of voltage-gated K+ channels present in murine thymocytes, types n and n' are blocked by CTX at nanomolar concentrations. The third variety of K+ channels, "type l," is unaffected by CTX. Noxiustoxin (NTX), a purified toxin from Centruroides noxius known to block Ca2+-activated K+ channels, also blocked type n K+ channels with a high degree of potency (Kd = 0.2 nM). In addition, several types of crude scorpion venoms from the genera Androctonus, Buthus, Centruroides, and Pandinus blocked type n channels. We conclude that CTX and NTX are not specific for Ca2+ activated K+ channels and that purified scorpion toxins will provide useful probes of voltage-gated K+ channels in T lymphocytes. The existence of high-affinity sites for scorpion toxin binding may help to classify structurally related K+ channels and provide a useful tool for their biochemical purification.     

Design and characterization of a highly selective peptide inhibitor of the small conductance calcium-activated K+ channel, SkCa2

Apamin-sensitive small conductance calcium-activated potassium channels (SKCa1-3) mediate the slow afterhyperpolarization in neurons, but the molecular identity of the channel has not been defined because of the lack of specific inhibitors. Here we describe the structure-based design of a selective inhibitor of SKCa2. Leiurotoxin I (Lei) and PO5, peptide toxins that share the RXCQ motif, potently blocked human SKCa2 and SKCa3 but not SKCa1, whereas maurotoxin, Pi1, Tskappa, and PO1 were ineffective. Lei blocked these channels more potently than PO5 because of the presence of Ala(1), Phe(2), and Met(7). By replacing Met(7) in the RXCQ motif of Lei with the shorter, unnatural, positively charged diaminobutanoic acid (Dab), we generated Lei-Dab(7), a selective SKCa2 inhibitor (K(d) = 3.8 nm) that interacts with residues in the external vestibule of the channel. SKCa3 was rendered sensitive to Lei-Dab(7) by replacing His(521) with the corresponding SKCa2 residue (Asn(367)). Intracerebroventricular injection of Lei-Dab(7) into mice resulted in no gross central nervous system toxicity at concentrations that specifically blocked SKCa2 homotetramers. Lei-Dab(7) will be a useful tool to investigate the functional role of SKCa2 in mammalian tissues.     

Characterization of the Actions of Toxins II-9.2.2 and II-10 from the Venom of the Scorpion Centruroides noxius on Transmitter Release from Mouse Brain Synaptosomes

Toxic peptides II-9.2.2 and II-10, purified from Centruroides noxius venom, bear highly homologous N-terminal amino acid sequences, and both toxins are lethal to mice. However, only toxin II-10 is active on the voltage-clamped squid axon, selectively decreasing the voltage-dependent Na+ current. Here, we have tested toxins II-9 and II-10 on synaptosomes from mouse brain: both toxins increased the release of gamma-[3H]aminobutyric acid ([3H]GABA). Their effect was completely blocked by tetrodotoxin or by the absence of external Na+. Also, both toxins increased Na+ permeability in isolated nerve terminals. Besides the observation that toxin II-9 is active on synaptosomes, the effect of toxin II-10 in this preparation is opposite to that observed in the squid axon. Thus, our results reflect functional differences between the populations of Na+ channels in mouse brain synaptosomes and in the squid axon. The release of GABA evoked by these toxins from synaptosomes required external Ca2+ and was blocked by Ca2+ channel blockers (verapamil and Co2+). This latter observation is in sharp contrast to the releasing action of veratrine, which evoked release even in the absence of external Ca2+. Furthermore, the action of both C. noxius toxins was potentiated by veratrine, a result suggesting they have different mechanisms of action. Among drugs that release neurotransmitters by increasing Na+ permeability, it is noteworthy that scorpion toxins are the only ones yet reported to have a strict requirement for external Ca2+.     

Charybdotoxin is a new member of the K+ channel toxin family that includes dendrotoxin I and mast cell degranulating peptide

A polypeptide was identified in the venom of the scorpion Leiurus quinquestriatus hebraeus by its potency to inhibit the high-affinity binding of the radiolabeled snake venom toxin dendrotoxin I (125I-DTX1) to its receptor site. It has been purified, and its properties investigated by different techniques were found to be similar to those of MCD and DTXI, two polypeptide toxins active on a voltage-dependent K+ channel. However, its amino acid sequence was determined, and it was shown that this toxin is in fact charybdotoxin (ChTX), a toxin classically used as a specific tool to block one class of Ca2+-activated K+ channels. ChTX, DTXI, and MCD are potent convulsants and are highly toxic when injected intracerebroventricularly in mice. Their toxicities correlate well with their affinities for their receptors in rat brain. These three structurally different toxins release [3H]GABA from preloaded synaptosomes, the efficiency order being DTXI greater than ChTX greater than MCD. Both binding and cross-linking experiments of ChTX to rat brain membranes and to the purified MCD/DTXI binding protein have shown that the alpha-subunit (Mr = 76K-78K) of the MCD/DTXI-sensitive K+ channel protein also contains the ChTX binding sites. Binding sites for DTXI, MCD, and ChTX are in negative allosteric interaction. Our results show that charybdotoxin belongs to the family of toxins which already includes the dendrotoxins and MCD, which are blockers of voltage-sensitive K+ channels. ChTX is clearly not selective for Ca2+-activated K+ channel.     

New binding site on common molecular scaffold provides HERG channel specificity of scorpion toxin BeKm-1

The scorpion toxin BeKm-1 is unique among a variety of known short scorpion toxins affecting potassium channels in its selective action on ether-a-go-go-related gene (ERG)-type channels. BeKm-1 shares the common molecular scaffold with other short scorpion toxins. The toxin spatial structure resolved by NMR consists of a short alpha-helix and a triple-stranded antiparallel beta-sheet. By toxin mutagenesis study we identified the residues that are important for the binding of BeKm-1 to the human ERG K+ (HERG) channel. The most critical residues (Tyr-11, Lys-18, Arg-20, Lys-23) are located in the alpha-helix and following loop whereas the "traditional" functional site of other short scorpion toxins is formed by residues from the beta-sheet. Thus the unique location of the binding site of BeKm-1 provides its specificity toward the HERG channel.     

Scorpion toxins specific for Na+-channels.

Na+-channel specific scorpion toxins are peptides of 60-76 amino acid residues in length, tightly bound by four disulfide bridges. The complete amino acid sequence of 85 distinct peptides are presently known. For some toxins, the three-dimensional structure has been solved by X-ray diffraction and NMR spectroscopy. A constant structural motif has been found in all of them, consisting of one or two short segments of alpha-helix plus a triple-stranded beta-sheet, connected by variable regions forming loops (turns). Physiological experiments have shown that these toxins are modifiers of the gating mechanism of the Na+-channel function, affecting either the inactivation (alpha-toxins) or the activation (beta-toxins) kinetics of the channels. Many functional variations of these peptides have been demonstrated, which include not only the classical alpha- and beta-types, but also the species specificity of their action. There are peptides that bind or affect the function of Na+-channels from different species (mammals, insects or crustaceans) or are toxic to more than one group of animals. Based on functional and structural features of the known toxins, a classification containing 10 different groups of toxins is proposed in this review. Attempts have been made to correlate the presence of certain amino acid residues or 'active sites' of these peptides with Na+-channel functions. Segments containing positively charged residues in special locations, such as the five-residue turn, the turn between the second and the third beta-strands, the C-terminal residues and a segment of the N-terminal region from residues 2-11, seems to be implicated in the activity of these toxins. However, the uncertainty, and the limited success obtained in the search for the site through which these peptides bind to the channels, are mainly due to the lack of an easy method for expression of cloned genes to produce a well-folded, active peptide. Many scorpion toxin coding genes have been obtained from cDNA libraries and from polymerase chain reactions using fragments of scorpion DNAs, as templates. The presence of an intron at the DNA level, situated in the middle of the signal peptide, has been demonstrated.     

A maurotoxin with constrained standard disulfide bridging: innovative strategy of chemical synthesis,pharmacology, and docking on K+ channels

Maurotoxin (MTX) is a 34-residue toxin that has been isolated initially from the venom of the scorpion Scorpio maurus palmatus. It presents a large number of pharmacological targets, including small conductance Ca2+-activated and voltage-gated K+ channels. Contrary to other toxins of the alpha-KTx6 family (Pi1, Pi4, Pi7, and HsTx1), MTX exhibits a unique disulfide bridge organization of the type C1-C5, C2-C6, C3-C4, and C7-C8 (instead of the conventional C1-C5, C2-C6, C3-C7, and C4-C8, herein referred to as Pi1-like) that does not prevent its folding along the classic alpha/beta scaffold of scorpion toxins. Here, we developed an innovative strategy of chemical peptide synthesis to produce an MTX variant (MTXPi1) with a conventional pattern of disulfide bridging without any alteration of the toxin chemical structure. This strategy was used solely to address the impact of half-cystine pairings on MTX structural properties and pharmacology. The data indicate that MTXPi1 displays some marked changes in affinities toward the target K+ channels. Computed docking analyses using molecular models of both MTXPi1 and the various voltage-gated K+ channel subtypes (Shaker B, Kv1.2, and Kv1.3) were found to correlate with MTXPi1 pharmacology. A functional map detailing the interaction between MTXPi1 and Shaker B channel was generated in line with docking experiments.     

Synthesis and characterization of Pi4, a scorpion toxin from Pandinus imperator that acts on K+ channels.

Pi4 is a 38-residue toxin cross-linked by four disulfide bridges that has been isolated from the venom of the Chactidae scorpion Pandinus imperator. Together with maurotoxin, Pi1, Pi7 and HsTx1, Pi4 belongs to the alpha KTX6 subfamily of short four-disulfide-bridged scorpion toxins acting on K+ channels. Due to its very low abundance in venom, Pi4 was chemically synthesized in order to better characterize its pharmacology and structural properties. An enzyme-based cleavage of synthetic Pi4 (sPi4) indicated half-cystine pairings between Cys6-Cys27, Cys12-32, Cys16-34 and Cys22-37, which denotes a conventional pattern of scorpion toxin reticulation (Pi1/HsTx1 type). In vivo, sPi4 was lethal after intracerebroventricular injection to mice (LD50 of 0.2 microg per mouse). In vitro, addition of sPi4 onto Xenopus laevis oocytes heterologously expressing various voltage-gated K+ channel subtypes showed potent inhibition of currents from rat Kv1.2 (IC50 of 8 pm) and Shaker B (IC50 of 3 nm) channels, whereas no effect was observed on rat Kv1.1 and Kv1.3 channels. The sPi4 was also found to compete with 125I-labeled apamin for binding to small-conductance Ca(2+)-activated K+ (SK) channels from rat brain synaptosomes (IC50 value of 0.5 microm). sPi4 is a high affinity blocker of the Kv1.2 channel. The toxin was docked (BIGGER program) on the Kv channel using the solution structure of sPi4 and a molecular model of the Kv1.2 channel pore region. The model suggests a key role for residues Arg10, Arg19, Lys26 (dyad), Ile28, Lys30, Lys33 and Tyr35 (dyad) in the interaction and the associated blockage of the Kv1.2 channel.     

Characterization of Kbot21 Reveals Novel Side Chain Interactions of Scorpion Toxins Inhibiting Voltage-Gated Potassium Channels

Scorpion toxins are important pharmacological tools for probing the physiological roles of ion channels which are involved in many physiological processes and as such have significant therapeutic potential. The discovery of new scorpion toxins with different specificities and affinities is needed to further characterize the physiology of ion channels. In this regard, a new short polypeptide called Kbot21 has been purified to homogeneity from the venom of Buthus occitanus tunetanus scorpion. Kbot21 is structurally related to BmBKTx1 from the venom of the Asian scorpion Buthus martensii Karsch. These two toxins differ by only two residues at position 13 (R /V) and 24 (D/N).Despite their very similar sequences, Kbot21 and BmBKTx1 differ in their electrophysiological activities. Kbot21 targets K_V channel subtypes whereas BmBKTx1 is active on both big conductance (BK) and small conductance (SK) Ca²⁺-activated K⁺ channel subtypes, but has no effects on Kv channel subtypes. The docking model of Kbot21 with the Kv1.2 channel shows that the D24 and R13 side-chain of Kbot21 are critical for its interaction with K_V channels.     

Synthesis, 3-D structure, and pharmacology of a reticulated chimeric peptide derived from maurotoxin and Tsk scorpion toxins

Maurotoxin (MTX) is a 34-mer scorpion toxin cross-linked by four disulfide bridges that acts on both Ca(2+)-activated (SK) and voltage-gated (Kv) K(+) channels. A 38-mer chimera of MTX, Tsk-MTX, has been synthesized by the solid-phase method. It encompasses residues from 1 to 6 of Tsk at N-terminal, and residues from 3 to 34 of MTX at C-terminal. As established by enzyme cleavage, Tsk-MTX displays half-cystine pairings of the type C1-C5, C2-C6, C3-C7 and C4-C8 which, contrary to MTX, correspond to a disulfide bridge pattern common to known scorpion toxins. The 3-D structure of Tsk-MTX, solved by (1)H NMR, demonstrates that it adopts the alpha/beta scaffold of scorpion toxins. In vivo, Tsk-MTX is lethal by intracerebroventricular injection in mice (LD(50) value of 0.2 microg/mouse). In vitro, Tsk-MTX is as potent as MTX, or Tsk, to interact with apamin-sensitive SK channels of rat brain synaptosomes (IC(50) value of 2.5 nM). It also blocks voltage-gated K(+) channels expressed in Xenopus oocytes, but is inactive on rat Kv1.3 contrary to MTX.     

Hemitoxin, the first potassium channel toxin from the venom of the Iranian scorpion Hemiscorpius lepturus

Hemitoxin (HTX) is a new K+ channel blocker isolated from the venom of the Iranian scorpion Hemiscorpius lepturus. It represents only 0.1% of the venom proteins, and displaces [125 I]alpha-dendrotoxin from its site on rat brain synaptosomes with an IC50 value of 16 nm. The amino acid sequence of HTX shows that it is a 35-mer basic peptide with eight cysteine residues, sharing 29-69% sequence identity with other K+ channel toxins, especially with those of the alphaKTX6 family. A homology-based molecular model generated for HTX shows the characteristic alpha/beta-scaffold of scorpion toxins. The pairing of its disulfide bridges, deduced from MS of trypsin-digested peptide, is similar to that of classical four disulfide bridged scorpion toxins (Cys1-Cys5, Cys2-Cys6, Cys3-Cys7 and Cys4-Cys8). Although it shows the highest sequence similarity with maurotoxin, HTX displays different affinities for Kv1 channel subtypes. It blocks rat Kv1.1, Kv1.2 and Kv1.3 channels expressed in Xenopus oocytes with IC50 values of 13, 16 and 2 nM, respectively. As previous studies have shown the critical role played by the beta-sheet in Kv1.3 blockers, we suggest that Arg231 is also important for Kv1.3 versus Kv1.2 HTX positive discrimination. This article gives information on the structure-function relationships of Kv1.2 and Kv1.3 inhibitors targeting developing peptidic inhibitors for the rational design of new toxins targeting given K+ channels with high selectivity.     

Gene expression, mutation, and structure-function relationship of scorpion toxin BmP05 active on SK(Ca) channels

Four peptide inhibitors of small-conductance Ca(2+)-activated, apamin-sensitive K(+) channels (SK(Ca)) have been isolated from the venom of the Chinese scorpion Buthus martensi, named BmP01, BmP02, BmP03, and BmP05, respectively [Romi-Lebrun, R. (1997) Eur. J. Biochem. 245, 457-464]. Among them BmP05 with 31 amino acid residues has been intensively studied due to its most potent toxicity. To investigate the structure-function relationship of BmP05, its wild type and seven mutants (their C-termini unamidated) were successfully expressed in the yeast secretion system and purified with a high yield over 8 mg/L. Their toxicity to mice and electrophysiological activity on the K(+) currents (SK(Ca) and Kv) in rat adrenal chromaffin cells were measured and compared. The results indicated the following: (1) As a selective antagonist against SK(Ca), 1 microM rBmP05 is equivalent to 0.2 microM apamin, and its IC(50) is 0.92 microM. (2) The basic residues Lys and Arg located at positions 6 and 13 in the N-terminal alpha-helix region are essential and synergetic in the interaction of the toxin with SK(Ca). (3) Disruption of the alpha-helix by mutation of Gln at position 9 with Pro results in almost total loss of toxicity. (4) The C-terminal residue His31 plays an auxiliary role in the interaction of the toxin with SK(Ca). (5) The beta-turn connecting two beta-sheets near the C-terminal part is responsible for the specificity of the toxin to the different subtypes of K(+) channels.     

Stimulation of sodium and calcium uptake by scorpion toxin in chick embryo heart cells.

Scorpion toxins, the basic miniproteins of scorpion venom, stimulated the passive uptake of Na+ and Ca2+ in chick embryo heart cells. Half-maximum stimulation was obtained for 20-30 nM Na+ and 40-50 nM Ca2+. Scorpion toxin-activated Na+ and Ca2+ uptakes were fully inhibited by tetrodotoxin, a specific inhibitor of the action potential Na+ ionophore in excitable membranes. Half-maximum inhibition was obtained with the same concentration of tetrodotoxin (10 nM) for both Na+ and Ca2+. Scorpion toxin-stimulated Ca2+ uptake was dependent on extracellular Na+ concentration and was not inhibited by Ca2+ channel blocking drugs which are inactive on heart cell action potential. Thus, in heart cells scorpion toxin affects the passive Ca2+ transport, which is coupled to passive Na+ ionphore. Other results suggest that (1) tetrodotoxin and scorpion toxin bind to different sites of the sarcolemma and (2) binding of scorpion toxin to its specific sites may unmask latent tetrodotoxin - sensitive fast channels.     

Purification, characterization and biosynthesis of parabutoxin 3, a component of Parabuthus transvaalicus venom.

A novel peptidyl inhibitor of voltage-gated K+ channels, named parabutoxin 3 (PBTx3), has been purified to homogeneity from the venom of Parabuthus transvaalicus. This scorpion toxin contains 37 residues, has a mass of 4274 Da and displays 41% identity with charybdotoxin (ChTx, also called 'alpha-KTx1.1'). PBTx3 is the tenth member (called 'alpha-KTx1.10') of subfamily 1 of K+ channel-blocking peptides known thus far. Electrophysiological experiments using Xenopus laevis oocytes indicate that PBTx3 is an inhibitor of Kv1 channels (Kv1.1, Kv1.2, Kv1.3), but has no detectable effects on Kir-type and ERG-type channels. The dissociation constants (Kd) for Kv1.1, Kv1.2 and Kv1.3 channels are, respectively, 79 microm, 547 nm and 492 nm. A synthetic gene encoding a PBTx3 homologue was designed and expressed as a fusion protein with the maltose-binding protein (MBP) in Escherichia coli. The recombinant protein was purified from the bacterial periplasm compartment using an amylose affinity resin column, followed by a gel filtration purification step and cleavage by factor Xa (fXa) to release the recombinant toxin peptide (rPBTx3). After final purification and refolding, rPBTx3 was shown to be identical to the native PBTx3 with respect to HPLC retention time, mass spectrometric analysis and functional properties. The three-dimensional structure of PBTx3 is proposed by homology modelling to contain a double-stranded antiparallel beta sheet and a single alpha-helix, connected by three disulfide bridges. The scaffold of PBTx3 is homologous to most other alpha-KTx scorpion toxins.