Hossein Vatanpour

Potent modulation of the voltage-gated sodium channel Nav1.7 by OD1, a toxin from the scorpion Odonthobuthus doriae.

Voltage-gated sodium channels are essential for the propagation of action potentials in nociceptive neurons. Nav1.7 is found in peripheral sensory and sympathetic neurons and involved in short-term and inflammatory pain. Nav1.8 and Nav1.3 are major players in nociception and neuropathic pain, respectively. In our effort to identify isoform-specific and high-affinity ligands for these channels, we investigated the effects of OD1, a scorpion toxin isolated from the venom of the scorpion Odonthobuthus doriae. Nav1.3, Nav1.7, and Nav1.8 channels were coexpressed with β1-subunits in Xenopus laevis oocytes. Na+ currents were recorded with the two-electrode voltage-clamp technique. OD1 modulates Nav1.7 at low nanomolar concentrations: 1) fast inactivation is dramatically impaired, with an EC50 value of 4.5 nM; 2) OD1 substantially increases the peak current at all voltages; and 3) OD1 induces a substantial persistent current. Nav1.8 was not affected by concentrations up to 2 μM, whereas Nav1.3 was sensitive only to concentrations higher than 100 nM. OD1 impairs the inactivation process of Nav1.3 with an EC50 value of 1127 nM. Finally, the effects of OD1 were compared with a classic α-toxin, AahII from Androctonus australis Hector and a classic α-like toxin, BmK M1 from Buthus martensii Karsch. At a concentration of 50 nM, both toxins affected Nav1.7. Nav1.3 was sensitive to AahII but not to BmK M1, whereas Nav1.8 was affected by neither toxin. In conclusion, the present study shows that the scorpion toxin OD1 is a potent modulator of Nav1.7, with a unique selectivity pattern.

OD1, the first toxin isolated from the venom of the scorpion Odonthobuthus doriae active on voltage-gated Na+ channels

In this study, we isolated and pharmacologically characterized the first α‐like toxin from the venom of the scarcely studied Iranian scorpion Odonthobuthus doriae. The toxin was termed OD1 and its primary sequence was determined: GVRDAYIADDKNCVYTCASNGYCNTECTKNGAESGYCQWIGRYGNACWCIKLPDEVPIRIPGKCR. Using the two‐electrode voltage clamp technique, the pharmacological effects of OD1 were studied on three cloned voltage‐gated Na+ channels expressed in Xenopus laevis oocytes (Nav1.2/β1, Nav1.5/β1, para/tipE). The inactivation process of the insect channel, para/tipE, was severely hampered by 200 nM of OD1 (EC50 = 80 ± 14 nM) while Nav1.2/β1 still was not affected at concentrations up to 5 μM. Nav1.5/β1 was influenced at micromolar concentrations.

Structure-activity studies on scorpion toxins that block potassium channels

Scorpion venoms contain toxins that block different types of potassium channels. Some of these toxins have affinity for high conductance Ca(2+)-activated K+ channels and for dendrotoxin-sensitive voltage-dependent K+ channels. The structural features that determine the specificity of binding to different channel types are not known. We investigated this using natural and synthetic scorpion toxins. We have tested the effects of charybdotoxin (CTX) and two homologues (Lqh 15-1 and Lqh 18-2), iberiotoxin (IbTX), and kaliotoxin (KTX) from the scorpions Leiurus quinquestriatus hebreus, Buthus tamulus and Androctonus mauretanicus mauretanicus, respectively, and synthetic variants of CTX, namely CTX2-37, CTX3-37, CTX4-37, and CTX7-37, on a Ca(2+)-activated K+ current (IK-Ca) at a mammalian motor nerve terminal, and on the binding of a radiolabelled dendrotoxin, 125I-DpI, to voltage-dependent K+ channels on rat brain synaptosomal membranes. The native toxins contain 37-38 amino acid residues, they are over 30% identical in sequence (CTX and IbTX are 68% identical), and they have similar three-dimensional conformations. All toxins, except IbTX, displaced 125I-DpI from its synaptosomal binding sites: Lqh 18-2 (Ki = 0.25 nM), KTX (Ki = 2.1 nM), CTX (Ki = 3.8 nM), CTX2-37, (Ki = 30 nM), Lqg 15-1 (Ki = 50 nM), CTX3-37 (Ki = 60 nM), CTX4-37 (Ki = 50 nM), CTX7-37 (Ki = 105 nM). IbTX had no effect at 3 microM. When variants of CTX with deletions at the N-terminal portion were tested for their activity on IK-Ca on motor nerve terminals in mouse triangularis sterni nerve-muscle preparations, CTX3-37 and CTX4-37 were ineffective at 100 nM; and CTX7-37 was ineffective at up to 1 microM. IbTX and CTX (100 nM) completely blocked IK-Ca, but KTX (100 nM) did not affect the nerve terminal IK-Ca. Different residues appear to be important for interactions of the toxins with different K+ channels. IbTX did not displace dendrotoxin binding, but it did block IK-Ca, whereas KTX was as active as CTX against dendrotoxin binding but it did not affect the IK-Ca of the motor nerve terminals. The N-terminal section of the toxins appears to be particularly involved in block of IK-Ca at the motor nerve terminal: it is truncated in the inactive synthetic CTX variants; and it is positively charged at lysine-6 in KTX (which is inactive), but negatively charged in IbTX and neutral in CTX.(ABSTRACT TRUNCATED AT 400 WORDS)

On the site by which α‐dendrotoxin binds to voltage‐dependent potassium channels: Site‐directed mutagenesis reveals that the lysine triplet 28–30 is not essential for binding

We constructed a synthetic gene encoding the published amino acid sequence of DTx from Dendroaspis angusticeps, a ligand of voltage‐dependent postassium channels that facilitates neurotransmitter release. We expressed it in Escherichia coli as a fusion protein secreted in the culture medium. The recombinant DTx was generated in vitro by chemical treatment and recovered as two isoforms. One of them (rDTx), like the venom toxin, has an N‐terminal pyroglutamate whereas the other (rQDTx) has a free N‐terminal glutamine. Chromatographic differences between rDTx and natural DTx led us to re‐examine the amino acid sequence of natural DTx. In contrast to what was previously published, position 12 was an Asp and not Asn. Despite this difference, rDTx and DTx had similar toxicity in mice and binding affinity to synaptosomes, suggesting that residue 12 is not important for DTx function. Nor is the N‐terminal residue implicated in DTx function since rDTx and rQDTx also had similar biological activities. We also synthesized and expressed a mutant of the DTx gene in which the lysine triplet 28–30 was changed into Ala‐Ala‐Gly. The two resulting recombinant isoforms exhibited only small decreases in biological activity, excluding the possibility that the positively charged lysine triplet 28–30 of DTx is directly involved in the toxin functional site.

The effects of Indian red scorpion Buthus tamulus venom in vivo and in vitro.

The Indian red scorpion Buthus tamulus (or Mesobuthus tamulus) can cause fatal envenoming, but its mechanism of action is unclear. Venom was tested in vivo in anaesthetized rats and in vitro on isolated cardiac and skeletal muscle preparations. In vivo, the venom caused marked rhythmical fluctuations in blood pressure preceding cardiovascular collapse and death. On sheep Purkinje fibres, venom could induce spontaneous action potentials and cause prolongation of action potential duration. In chick biventer cervicis and mouse triangularis sterni preparations, venom enhanced the release of acetylcholine and induced repetitive firing of nerve action potentials in response to single shock stimulation. High concentrations caused stimulation then block of neuromuscular transmission. The main effects of Buthus tamulus venom are likely to be due to toxins that affect the opening of Na+ channels in nerves and muscles. This will cause an increase in the release of neurotransmitters in the peripheral nervous system, which may produce cardiovascular abnormalities and respiratory paralysis.

OdK2, a Kv1.3 channel-selective toxin from the venom of the Iranian scorpion Odonthobuthus doriae ☆

The first Kv1.3 channel-selective toxin from the venom of the Iranian scorpion Odonthobuthus doriae (OdK2) was purified, sequenced and characterized physiologically. OdK2 consists of 38 amino acids, including six conserved cysteine and a C-terminal lysine residue, as revealed by the unique use of a quadrupole ion cyclotron resonance Fourier-transform mass spectrometer. Based on multiple sequence alignments, OdK2 was classified as alpha-KTX3.11. The pharmacological effects of OdK2 were studied on a panel of eight different cloned K(+) channels (vertebrate Kv1.1-Kv1.6, Shaker IR and hERG) expressed in Xenopus laevis oocytes. Interestingly, OdK2 selectively inhibits the currents through Kv1.3 channels with an IC50 value of 7.2+/-2.7nM.

Neuromuscular effects of some potassium channel blocking toxins from the venom of the scorpion Leiurus quinquestriatus hebreus

The scorpion venom Leiurus quinquestriatus hebreus was fractionated by chromatography in order to isolate toxins that affected binding of radiolabelled dendrotoxin to K+ channel proteins on synaptosomal membranes and that facilitated acetylcholine release in chick biventer cervicis nerve-muscle preparations. In addition to the previously characterized charybdotoxin, three toxins were isolated: 14-2, 15-1 and 18-2. Toxin 14-2 has a blocked N-terminus and because of low quantities, it has not been sequenced; 15-1 is a newly sequenced toxin of 36 residues with some overall homology to charybdotoxin and noxiustoxin; 18-2 is identical to charybdotoxin-2. The apparent Ki against dendrotoxin binding were: charybdotoxin, 3.8 nM; 14-2, 150 nM; 15-1, 50 nM; and 18-2, 0.25 nM. Toxin 14-2 (75 nM-1.5 microM) had a presynaptic facilitatory effect on neuromuscular preparations. Toxin 15-1 augmented responses to direct muscle stimulation, probably because it blocked Ca(2+)-activated K+ currents in muscle fibres. Toxin 18-2 (charybdotoxin-2) had a potent presynaptic facilitatory action, with less effect on direct muscle stimulation. This contrasts with the relatively weak neuromuscular effects of the highly homologous charybdotoxin. On a Ca(2+)-activated K+ current in mouse motor nerve endings, charybdotoxin and toxin 18-2 produced maximal block at around 100 nM, whereas 15-1 was inactive at 300 nM. Charybdotoxin can increase quantal content, but this is more likely to result from block of voltage-dependent K+ channels than Ca(2+)-activated channels: the increase in transmitter release occurred in conditions in which little IKCa would be present; higher concentration of charybdotoxin and longer exposure times were required to increase transmitter release than those needed to block IKCa, and the facilitatory effects of charybdotoxin and toxin 18-2 correlated more with their effects on dendrotoxin binding than on block of IKCa.

The first potassium channel toxin from the venom of the Iranian scorpion Odonthobuthus doriae

OdK1 has 29 amino acids, six conserved cysteines and a pI value of 4.95. Based on multiple sequence alignments, OdK1 was classified as α‐KTx 8.5. The pharmacological effects of OdK1 were studied on six different cloned K+ channels (vertebrate Kv1.1‐Kv1.5 and Shaker IR) expressed in Xenopus laevis oocytes. Interestingly, OdK1 selectively inhibited the currents through Kv1.2 channels with an IC50 value of 183 ± 3 nM but did not affect any of the other channels.

Modulation of acetylcholine release at mouse neuromuscular junctions by interaction of three homologous scorpion toxins with K+ channels

1 The effects of three scorpion toxins, charybdotoxin (CTX), iberiotoxin (IbTX), and noxiustoxin (NTX) have been studied on acetylcholine release and on K+ channels by means of twitch tension and electrophysiological recording techniques using isolated skeletal muscle preparations and by a radioligand binding assay using 125I‐labelled dendrotoxin I (DpI) and rat brain synaptosomal membranes.

Changes to biological activity following acetylation of dendrotoxin I from Dendroaspis polylepis (black mamba).

The potassium channel blocker dendrotoxin I was acetylated with acetic anhydride. Mono-acetyl derivatives of all seven lysine residues (N-terminus blocked) and a di-derivative were isolated by chromatography on the cation-exchanger Bio-Rex 70 and reversed-phase high-performance liquid chromatography. The derivative acetyl-Lys 29 and the di-derivative of Tyr 24 and Lys 28 had more than 1000 times lower affinity than the native toxin as determined by inhibition of the 125I-dendrotoxin binding to synaptosomal membranes from rat brain. Lys 29 is part of the triplet Lys-Lys-Lys (28-30) which also occurs in the homologous alpha-dendrotoxin where the triplet is not in the functional site, as shown by site-directed mutagenesis. Acetylation of Lys 29 may have produced large structural perturbations that inactivated the toxin. Acetylation of Lys 28 alone had little effect, but the toxin became almost inactive when both Lys 28 and Tyr 24 were modified. Ten experiments were conducted under similar conditions, but a derivative of Tyr 24 was obtained only three times. In these cases the toxin apparently had a different structure, with Tyr 24 accessible to the reagent. This may depend on freeze-drying, which can alter the structure of proteins. The third derivative with low activity was acetyl-Lys 5, with affinity decreased 20-fold. Lys 5 has a protruding side-chain that does not interact with any other group in the toxin molecule. Therefore, Lys 5 is probably part of the functional site for dendrotoxins binding to the voltage-dependent K+ channels.