Purification, characterization, and synthesis of three novel toxins from the Chinese scorpion Buthus martensi, which act on K+ channels.
Three novel toxins belonging to the scorpion K+ channel-inhibitor family were purified to homogeneity from the venom of the Chinese scorpion Buthus martensi. They have been identified according to their molecular mass (3800-4300 Da) and their neurotoxicity in mice and characterized as 37-amino acid peptides. One of them shows 81-87% sequence identity with members of the kaliotoxin group (named BmKTX), whereas the other two, named BmTX1 and BmTX2, show 65-70% identity with toxins of the charybdotoxin group. Their chemical synthesis by the Fmoc methodology allowed us to show that BmKTX, unlike BmTX1 and BmTX2, possesses an amidated C-terminal extremity. Toxicity assays in vivo established that they are lethal neurotoxic agents in mice (LD50s of 40-95 ng per mouse). Those toxins proved to be potent inhibitors of the voltage-gated K+ channels, as they were able to compete with [125I]kaliotoxin for its binding to rat brain synaptosomes (IC50s of 0.05-1 nM) and to block the cloned voltage-gated K+ channel Kv1.3 from rat brain, expressed in Xenopus oocytes (IC50s of 0.6-1.6 nM). BmTX1 and BmTX2 were also shown to compete with [125I]charybdotoxin for its binding to the high-conductance Ca2+-activated K+ channels present on bovine aorta sarcolemmal membranes (IC50s of 0.3-0.6 nM). These new sequences show multipoint mutations when compared to the other related scorpion K+ channel toxins and should prove to be useful probes for studying the diverse family of K+ channels.
Romi-Lebrun
R
R
Suntory Institute for Bioorganic Research, Mishima-Gun, Shimamoto-Cho, Wakayamadai 1-1-1, Osaka 618, Japan.
Lebrun
B
B
Martin-Eauclaire
M F
MF
Ishiguro
M
M
Escoubas
P
P
Wu
F Q
FQ
Hisada
M
M
Pongs
O
O
Nakajima
T
T
D000595:Amino Acid Sequence; D000818:Animals; D001011:Aorta; D001667:Binding, Competitive; D002417:Cattle; D018999:Charybdotoxin / Q000737:chemistry; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D007425:Intracellular Membranes / Q000187:drug effects; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D009498:Neurotoxins / Q000737:chemistry*; D015221:Potassium Channels / Q000187:drug effects* / Q000378:metabolism; D051381:Rats; D012508:Sarcolemma / Q000187:drug effects; D012604:Scorpion Venoms / Q000138:chemical synthesis / Q000302:isolation & purification* / Q000633:toxicity*
C109066:KTX toxin, Buthus; D009498:Neurotoxins; D015221:Potassium Channels; D012604:Scorpion Venoms; C109067:TX1 toxin, Buthus; C109068:TX2 toxin, Buthus; D018999:Charybdotoxin