Abstract
We have isolated a cardiotoxin, denoted jingzhaotoxin-III (JZTX-III), from the venom of the Chinese spider Chilobrachys jingzhao. The toxin contains 36 residues stabilized by three intracellular disulfide bridges (I-IV, II-V, and III-VI), assigned by a chemical strategy of partial reduction and sequence analysis. Cloned and sequenced using 3'-rapid amplification of cDNA ends and 5'-rapid amplification of cDNA ends, the full-length cDNA encoded a 63-residue precursor of JZTX-III. Different from other spider peptides, it contains an uncommon endoproteolytic site (-X-Ser-) anterior to mature protein and the intervening regions of 5 residues, which is the smallest in spider toxin cDNAs identified to date. Under whole cell recording, JZTX-III showed no effects on voltage-gated sodium channels (VGSCs) or calcium channels in dorsal root ganglion neurons, whereas it significantly inhibited tetrodotoxin-resistant VGSCs with an IC(50) value of 0.38 microm in rat cardiac myocytes. Different from scorpion beta-toxins, it caused a 10-mV depolarizing shift in the channel activation threshold. The binding site for JZTX-III on VGSCs is further suggested to be site 4 with a simple competitive assay, which at 10 microm eliminated the slowing currents induced by Buthus martensi Karsch I (BMK-I, scorpion alpha-like toxin) completely. JZTX-III shows higher selectivity for VGSC isoforms than other spider toxins affecting VGSCs, and the toxin hopefully represents an important ligand for discriminating cardiac VGSC subtype.
Highlights
Voltage-gated sodium channels (VGSCs)1 are trans-membrane proteins distributed widely in the most excitable tissue
JZTX-III showed no effects on voltage-gated sodium channels (VGSCs) or calcium channels in dorsal root ganglion neurons, whereas it significantly inhibited tetrodotoxin-resistant VGSCs with an IC50 value of 0.38 M in rat cardiac myocytes
JZTX-III, a TTX-R Sodium Channel Cardiotoxin jingzhaotoxin-III (JZTX-III), is composed of 36 amino acid residues including 6 cysteines cross-linked in a pattern of I-IV, II-V, and III-VI
Summary
Voltage-gated sodium channels (VGSCs) are trans-membrane proteins distributed widely in the most excitable tissue. NMR and homology modeling techniques indicate that, irrespective of the different composition of amino acids, most of them adopt a typical inhibitor cystine knot (ICK) fold distinct from the ␣/ scaffold emerging in scorpion toxins [9, 10] Most residues in their primary structure are believed to support the peptide framework, whereas only a few charged residues situated at the loop domains of ICK motifs are critical to interact with sodium channels. The toxin shows no effect on voltage-gated potassium channels (Kv1.1–1.3) expressed in Xenopus laevis oocytes or VGSCs and voltage-gated calcium channels (VGCCs) distributed in DRG neurons It can selectively inhibit activation of TTX-R VGSCs in cardiac myocytes followed by shifting activated voltage in a depolarizing direction. We further assume that JZTX-III binds to site 4 on sodium channel proteins, which is formed by amino acid residues in the extracellular linker between domain II-S3 and domain II-S4
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