Function and Solution Structure of Huwentoxin-X, a Specific Blocker of N-type Calcium Channels, from the Chinese Bird Spider Ornithoctonus huwena

Longjun Dai,Jie Dai,Zhonghua Liu,Zhe Hu,Weijun Hu,Meichun Deng,Songping Liang

doi:10.1074/jbc.m513542200

Longjun Dai, Jie Dai + Show 5 more

Open Access

https://doi.org/10.1074/jbc.m513542200

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Abstract

Huwentoxin-X (HWTX-X) is a novel peptide toxin, purified from the venom of the spider Ornithoctonus huwena. It comprises 28 amino acid residues including six cysteine residues as disulfide bridges linked in the pattern of I-IV, II-V, and III-VI. Its cDNA, determined by rapid amplification of 3' and 5' cDNA ends, encodes a 65-residue prepropeptide. HWTX-X shares low sequence homology with omega-conotoxins GVIA and MVIIA, two well known blockers of N-type Ca2+ channels. Nevertheless, whole cell studies indicate that it can block N-type Ca2+ channels in rat dorsal root ganglion cells (IC50 40 nm) and the blockage by HWTX-X is completely reversible. The rank order of specificity for N-type Ca2+ channels is GVIA approximately HWTX-X > MVIIA. In contrast to GVIA and MVIIA, HWTX-X had no detectable effect on the twitch response of rat vas deferens to low frequency electrical stimulation, indicating that HWTX-X has different selectivity for isoforms of N-type Ca2+ channels, compared with GVIA or MVIIA. A comparison of the structures of HWTX-X and GVIA reveals that they not only adopt a common structural motif (inhibitor cystine knot), but also have a similar functional motif, a binding surface formed by the critical residue Tyr, and several basic residues. However, the dissimilarities of their binding surfaces provide some insights into their different selectivities for isoforms of N-type Ca2+ channels.

Highlights

Sal root ganglion (DRG)2 cells, and as potential targets for the development of analgesic drugs [9]
We report the isolation and characterization of huwentoxin-X (HWTX-X) from the venom of O. huwena, a novel specific blocker of N-type Ca2ϩ channels in rat DRG cells. cDNA sequencing by rapid amplification of the 3Ј and 5Ј cDNA ends (RACE) method indicates HWTX-X is initially expressed as a prepropeptide, an expression pattern similar to other spider peptide toxins and conotoxins
Purification Characterization and Synthesis of HWTX-X—Crude venom from the spider O. huwena was fractionated by reverse-phase high performance liquid chromatography (RP-HPLC) on a Vydac C18 column (Fig. 1A)

Summary

Introduction

Sal root ganglion (DRG)2 cells, and as potential targets for the development of analgesic drugs [9]. We report the isolation and characterization of huwentoxin-X (HWTX-X) from the venom of O. huwena, a novel specific blocker of N-type Ca2ϩ channels in rat DRG cells. The sharp peak labeled HWTX-X, eluted at 13 min at a point in the gradient of about 18% acetonitrile, 0.1% trifluoroacetic acid, was found to block N-type Ca2ϩ channels in rat DRG cells.

Results

Conclusion