title: Active Sites of Spinoxin, a Potassium Channel Scorpion Toxin, Elucidated by Systematic Alanine Scanning.
abstract: Peptide toxins from scorpion venoms constitute the largest group of toxins that target the voltage-gated potassium channel (Kv). Spinoxin (SPX) isolated from the venom of scorpion Heterometrus spinifer is a 34-residue peptide neurotoxin cross-linked by four disulfide bridges. SPX is a potent inhibitor of Kv1.3 potassium channels (IC50 = 63 nM), which are considered to be valid molecular targets in the diagnostics and therapy of various autoimmune disorders and cancers. Here we synthesized 25 analogues of SPX and analyzed the role of each amino acid in SPX using alanine scanning to study its structure-function relationships. All synthetic analogues showed similar disulfide bond pairings and secondary structures as native SPX. Alanine replacements at Lys(23), Asn(26), and Lys(30) resulted in loss of activity against Kv1.3 potassium channels, whereas replacements at Arg(7), Met(14), Lys(27), and Tyr(32) also largely reduced inhibitory activity. These results suggest that the side chains of these amino acids in SPX play an important role in its interaction with Kv1.3 channels. In particular, Lys(23) appears to be a key residue that underpins Kv1.3 channel inhibition. Of these seven amino acid residues, four are basic amino acids, suggesting that the positive electrostatic potential on the surface of SPX is likely required for high affinity interaction with Kv1.3 channels. This study provides insight into the structure-function relationships of SPX with implications for the rational design of new lead compounds targeting potassium channels with high potency.
authors.affiliation: Toxicology and Pharmacology, University of Leuven , Campus Gasthuisberg O&N2, Herestraat 49, P.O. Box 922, Leuven 3000, Belgium.
authors.forename: Steve
authors.initials: S
authors.lastname: Peigneur
authors.affiliation: Department of Environment Sciences, Fukuoka Women's University , Fukuoka 813-8529, Japan.
authors.forename: Yoko
authors.initials: Y
authors.lastname: Yamaguchi
authors.affiliation: Department of Environment Sciences, Fukuoka Women's University , Fukuoka 813-8529, Japan.
authors.forename: Chihiro
authors.initials: C
authors.lastname: Kawano
authors.affiliation: Faculty of Arts and Science, Kyushu University , Fukuoka, 819-0395, Japan.
authors.forename: Takeru
authors.initials: T
authors.lastname: Nose
authors.affiliation: Menzies Health Institute Queensland , Gold Coast, Queensland 4222, Australia.
authors.forename: Selvanayagam
authors.initials: S
authors.lastname: Nirthanan
authors.affiliation: Venom and Toxin Research Program, Yong Loo Lin School of Medicine, National University of Singapore , Singapore 117597, Singapore.
authors.forename: Ponnampalam
authors.initials: P
authors.lastname: Gopalakrishnakone
authors.affiliation: Toxicology and Pharmacology, University of Leuven , Campus Gasthuisberg O&N2, Herestraat 49, P.O. Box 922, Leuven 3000, Belgium.
authors.forename: Jan
authors.initials: J
authors.lastname: Tytgat
authors.affiliation: Department of Environment Sciences, Fukuoka Women's University , Fukuoka 813-8529, Japan.
authors.forename: Kazuki
authors.initials: K
authors.lastname: Sato
mesh_terms: D000409:Alanine / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D020134:Catalytic Domain; D002478:Cells, Cultured; D002942:Circular Dichroism; D004594:Electrophysiology; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics; D009865:Oocytes / Q000166:cytology / Q000378:metabolism; D015221:Potassium Channels / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D012604:Scorpion Venoms / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012605:Scorpions / Q000378:metabolism*; D014982:Xenopus laevis / Q000378:metabolism
chemical_list: D015221:Potassium Channels; D012604:Scorpion Venoms; D000409:Alanine