Synthesis and characterization of Pi4, a scorpion toxin from Pandinus imperator that acts on K+ channels.
Pi4 is a 38-residue toxin cross-linked by four disulfide bridges that has been isolated from the venom of the Chactidae scorpion Pandinus imperator. Together with maurotoxin, Pi1, Pi7 and HsTx1, Pi4 belongs to the alpha KTX6 subfamily of short four-disulfide-bridged scorpion toxins acting on K+ channels. Due to its very low abundance in venom, Pi4 was chemically synthesized in order to better characterize its pharmacology and structural properties. An enzyme-based cleavage of synthetic Pi4 (sPi4) indicated half-cystine pairings between Cys6-Cys27, Cys12-32, Cys16-34 and Cys22-37, which denotes a conventional pattern of scorpion toxin reticulation (Pi1/HsTx1 type). In vivo, sPi4 was lethal after intracerebroventricular injection to mice (LD50 of 0.2 microg per mouse). In vitro, addition of sPi4 onto Xenopus laevis oocytes heterologously expressing various voltage-gated K+ channel subtypes showed potent inhibition of currents from rat Kv1.2 (IC50 of 8 pm) and Shaker B (IC50 of 3 nm) channels, whereas no effect was observed on rat Kv1.1 and Kv1.3 channels. The sPi4 was also found to compete with 125I-labeled apamin for binding to small-conductance Ca(2+)-activated K+ (SK) channels from rat brain synaptosomes (IC50 value of 0.5 microm). sPi4 is a high affinity blocker of the Kv1.2 channel. The toxin was docked (BIGGER program) on the Kv channel using the solution structure of sPi4 and a molecular model of the Kv1.2 channel pore region. The model suggests a key role for residues Arg10, Arg19, Lys26 (dyad), Ile28, Lys30, Lys33 and Tyr35 (dyad) in the interaction and the associated blockage of the Kv1.2 channel.
M'Barek
Sarrah
S
Laboratoire International Associé d'Ingénierie Biomoléculaire et Laboratoire de Biochimie CNRS UMR 6560, IFR Jean Roche, Faculté de Médecine Nord, Marseille, France.
Mosbah
Amor
A
Sandoz
Guillaume
G
Fajloun
Ziad
Z
Olamendi-Portugal
Timoteo
T
Rochat
Hervé
H
Sampieri
François
F
Guijarro
J Iñaki
JI
Mansuelle
Pascal
P
Delepierre
Muriel
M
De Waard
Michel
M
Sabatier
Jean-Marc
JM
D000595:Amino Acid Sequence; D000818:Animals; D001030:Apamin / Q000737:chemistry / Q000378:metabolism; D002118:Calcium / Q000737:chemistry / Q000378:metabolism; D003545:Cysteine / Q000737:chemistry / Q000235:genetics; D004220:Disulfides / Q000737:chemistry; D004594:Electrophysiology; D007928:Lethal Dose 50; D051379:Mice; D008810:Mice, Inbred C57BL; D008958:Models, Molecular; D008969:Molecular Sequence Data; D026902:Potassium Channel Blockers / Q000138:chemical synthesis* / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D015221:Potassium Channels / Q000187:drug effects* / Q000502:physiology; D011485:Protein Binding; D051381:Rats; D012604:Scorpion Venoms / Q000138:chemical synthesis* / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D017386:Sequence Homology, Amino Acid; D013574:Synaptosomes / Q000378:metabolism; D014982:Xenopus laevis
D004220:Disulfides; C114245:Pi4 peptide; D026902:Potassium Channel Blockers; D015221:Potassium Channels; D012604:Scorpion Venoms; D001030:Apamin; D003545:Cysteine; D002118:Calcium