Chemical Synthesis and Characterization of Maurotoxin, a Short Scorpion Toxin with four Disulfide Bridges that Acts on K⁺ Channels

Abstract

Maurotoxin is a toxin isolated from the venom of the Tunisian chactoid scorpion Scorpio maurus. It is a 34-amino-acid peptide cross-linked by four disulfide bridges. Maurotoxin competes with radiolabeled apamin and kaliotoxin for binding to rat-brain synaptosomes. Due to its very low concentration in venom (0.6% of the proteins), maurotoxin was chemically synthesized by means of an optimized solid-phase technique. The synthetic maurotoxin was characterized. It was lethal to mice following intracerebroventricular injection (LD,,, 80 ng/mouse). The synthetic maurotoxin competed with '*'I-apamin and 'Z51-kaliotoxin for binding to rat-brain synaptosomes with half-maximal effects at concentrations of 5 nM and 0.2 nM, respectively. Synthetic maurotoxin was tested on K' channels and was found to block the Kvl.1, Kv1.2, and Kv1.3 currents with half-maximal blockage (I&) at 37, 0.8 and 150 nM, respectively. Thus, maurotoxin is a scorpion toxin with four disulfide bridges that acts on K' channels. The half-cystine pairings of synthetic maurotoxin were identified by enzymatic cleavage. The pairings were Cys3 -Cys24, Cys9-Cys29, Cysl3-Cysl9 and Cys31 -Cys34. This disulfide organization is unique among known scorpion toxins. The physicochemical and pharmacological properties of synthetic maurotoxin were indistinguishable from those of natural maurotoxin, which suggests that natural maurotoxin adopts the same half-cystine pairing pattern. The conformation of synthetic maurotoxin was investigated by means of circular dichroism spectroscopy and molecular modeling. In spite of its unusual half-cystine pairings, the synthetic-maurotoxin conformation appears to be similar to that of other short scorpion toxins.Keywords: maurotoxin; scorpion toxin; half-cystine pairing ; apamin-sensitive K' channels ; voltage-gated K' channels.Because polypeptide animal toxins interact with ion channels and modulate their activities [ 1 -31, these toxins are useful pharmacological probes to investigate ion-specific channel proteins and their function. In recent years, toxins acting on various K+ channels have been isolated from diverse scorpion venoms [4]. Maurotoxin has recently been purified from the venom of the chactoid scorpion Scorpio maurus, and characterized (Kharrat, R., Mansuelle, P., Sampieri, F., Crest, M., Martin-Eauclaire, M. F., Rochat, H. and El Ayeb, M., unpublished results). Maurotoxin is a basic toxin of 34 amino acid residues cross-linked by four disulfide bridges. Maurotoxin was found to compete with radiolabeled apamin and kaliotoxin for binding to rat-brain synaptosomes (Kharrat, R., Mansuelle, P., Sampieri, F., Crest, M., Martin-Eauclaire, M. F., Rochat, H. and El Ayeb, M., unpublished results). Thus, it is a scorpion toxin with four disulfide bridges that acts on K' channels. Due to its sequence, maurotoxin does not belong to any of the four groups of K+-channel Maurotoxin is only 0.6% of the total proteins in a crude venom, which is not readily available. Thus, we performed chemical solid-phase synthesis of this toxin to e...

Chemical synthesis and characterization of maurocalcine, a scorpion toxin that activates Ca2+ release channel/ryanodine receptors

“…It should be noted that the Cys residues are similarly arranged in all these peptides. Stepwise assembly of the peptide chains was achieved on 0.25 mmol HMP resin by means of optimized Fmoc/t-butyl chemistry [10,16]. The amount of target protected peptide linked to the resin was 0.20 mmol, which represents a yield of assembly of 80%.…”

“…In the present work, we have chemically synthesized MCa by the Fmoc/t-butyl strategy [9], and carefully characterized the folded synthetic product sMCa for its physicochemical and electrophysiological properties. The disul¢de bridge organization was formerly established by enzyme-based cleavage of sMCa followed by analyses of the proteolytic peptide fragments using mass spectrometry, amino acid content determination, and Edman sequencing techniques, as described [10,11]. sMCa was tested in vivo for lethal activity to [12], and by electrophysiology in vitro for its putative action on Ca 2 £ux through RyR1 channels incorporated into planar bilayer lipid membranes.…”

K+ Channel Modulators for the Treatment of Neurological Disorders and Autoimmune Diseases

“…386,387 However, MTX has even higher affinity (IC 50 ) 100 pM) for K V 1.2. 386,388 Another scorpion toxin that inhibits K Ca 3.1 is ChTX (IC 50 ) 5 nM), which has been traditionally used to distinguish "charybdotoxin-sensitive" from "apamin-sensitive" K Ca channels despite the fact that ChTX also inhibits K V 1.3 and K Ca 1.1 (see section 2.2.1). Mutational studies by Rauer et al, 321,389 which used ChTX and the sea anemone toxins ShK and BgK as molecular calipers, demonstrated that the outer vestibules of K Ca 3.1 and K V 1.3 have very similar dimensions, which partly explains this cross-reactivity.…”

Mapping of Maurotoxin Binding Sites on hKv1.2, hKv1.3, and hIKCa1 Channels

“…Despite its different disulfide bridge organization, MTX has the same three-dimensional structure of potassium channels toxin blockers formed by one ␣-helix and two ␤-sheets. Moreover, MTX has been reported to block, in Xenopus laevis oocytes, IKCa1, Kv1.2, and ShakerB channels with an affinity lower than 10 nM, whereas its affinity for other potassium channels, such as Kv1.1 and Kv1.3, was higher than 50 nM (Kharrat et al, 1996;Avdonin et al, 2000;Lecomte et al, 2000;Castle et al, 2003).hKv1.2 and hIKCa1 channels are distributed differently in different tissues. hKv1.2 channels are found predominantly in the brain, where they are most likely to be associated with Kv1.1 and Kv1.6 channel subunits accomplishing crucial roles in neuronal signal transmission (Coleman et al, 1999;Monaghan et al, 2001;Dodson et al, 2003).…”

“…1B). It is composed of a 34-amino-acid peptide and has four disulfide bridges with an atypical pattern organization (C1-C5, C2-C6, C3-C4, and C7-C8) compared with other toxins belonging to the same family (C1-C4, C2-C5, C3-C6 for three-disulfide-bridged toxins and C1-C5, C2-C6, C3-C7, and C4-C8 for four-disulfide-bridged toxins) (Kharrat et al, 1996). Despite its different disulfide bridge organization, MTX has the same three-dimensional structure of potassium channels toxin blockers formed by one ␣-helix and two ␤-sheets.…”