Purification, molecular cloning and functional characterization of HelaTx1 (Heterometrus laoticus): the first member of a new κ-KTX subfamily.
Given their medical importance, most attention has been paid toward the venom composition of scorpions of the Buthidae family. Nevertheless, research has shown that the venom of scorpions of other families is also a remarkable source of unique peptidyl toxins. The κ-KTx family of voltage-gated potassium channel (VGPC) scorpion toxins is hereof an example. From the telson of the scorpion Heterometrus laoticus (Scorpionidae), a peptide, HelaTx1, with unique primary sequence was purified through HPLC and sequenced by Edman degradation. Based on the amino acid sequence, the peptide could be cloned and the cDNA sequence revealed. HelaTx1 was chemically synthesized and functionally characterized on VGPCs of the Shaker-related, Shab-related, Shaw-related and Shal-related subfamilies. Furthermore, the toxin was also tested on small- and intermediate conductance Ca(2+)-activated K(+) channels. From the channels studied, K(v)1.1 and K(v)1.6 were found to be the most sensitive (K(v)1.1 EC(50)=9.9±1.6 μM). The toxin did not alter the activation of the channels. Competition experiments with TEA showed that the toxin is a pore blocker. Mutational studies showed that the residues E353 and Y379 in the pore of K(v)1.1 act as major interaction points for binding of the toxin. Given the amino acid sequence, the predicted secondary structure and the biological activity on VGPCs, HelaTx1 should be included in the κ-KTX family. Based on a phylogenetic study, we rearranged this family of VGPC toxins into five subfamilies and suggest that HelaTx1 is the first member of the new κ-KTx5 subfamily.
Laboratory of Toxicology, Katholieke Universiteit Leuven, Onderwijs & Navorsing II, Herestraat 49, Leuven, Belgium.
Thomas
T
Vandendriessche
Ivan
I
Kopljar
David Paul
DP
Jenkins
Elia
E
Diego-Garcia
Yousra
Y
Abdel-Mottaleb
Elke
E
Vermassen
Elke
E
Clynen
Liliane
L
Schoofs
Heike
H
Wulff
Dirk
D
Snyders
Jan
J
Tytgat
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D051662:Kv1.1 Potassium Channel / Q000378:metabolism; D051667:Kv1.6 Potassium Channel / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D009865:Oocytes / Q000187:drug effects / Q000502:physiology; D018408:Patch-Clamp Techniques; D010455:Peptides / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism / Q000494:pharmacology*; D010802:Phylogeny; D024642:Potassium Channels, Voltage-Gated / Q000378:metabolism; D017433:Protein Structure, Secondary; D012605:Scorpions / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D013111:Spider Venoms / Q000737:chemistry*; D029867:Xenopus Proteins / Q000378:metabolism; D014982:Xenopus laevis
D051667:Kv1.6 Potassium Channel; D010455:Peptides; D024642:Potassium Channels, Voltage-Gated; D013111:Spider Venoms; D029867:Xenopus Proteins; C084069:kcna1 protein, Xenopus; D051662:Kv1.1 Potassium Channel