M'Barek Sarrah, Mosbah Amor, Sandoz Guillaume, Fajloun Ziad, Olamendi-Portugal Timoteo, Rochat Hervé, Sampieri François, Guijarro J Iñaki, Mansuelle Pascal, Delepierre Muriel, De Waard Michel, Sabatier Jean-Marc
Laboratoire International Associé d'Ingénierie Biomoléculaire et Laboratoire de Biochimie CNRS UMR 6560, IFR Jean Roche, Faculté de Médecine Nord, Marseille, France.
Eur J Biochem. 2003 Sep;270(17):3583-92. doi: 10.1046/j.1432-1033.2003.03743.x.
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.
Pi4是一种由四个二硫键交联的38个氨基酸残基的毒素，已从帝王蝎科（Chactidae）蝎子帝王蝎（Pandinus imperator）的毒液中分离出来。Pi4与马罗毒素、Pi1、Pi7和HsTx1一起，属于作用于钾通道的短链四二硫键桥联蝎子毒素的α-KTX6亚家族。由于其在毒液中的含量极低，为了更好地表征其药理学和结构特性，对Pi4进行了化学合成。基于酶的合成Pi4（sPi4）切割表明，半胱氨酸之间的配对为Cys6-Cys27、Cys12-32、Cys16-34和Cys22-37，这表明蝎子毒素网状结构具有传统模式（Pi1/HsTx1类型）。在体内，向小鼠脑室内注射sPi4后具有致死性（每只小鼠的半数致死量为0.2微克）。在体外，将sPi4添加到异源表达各种电压门控钾通道亚型的非洲爪蟾卵母细胞上，显示出对大鼠Kv1.2（半数抑制浓度为8皮摩尔）和Shaker B（半数抑制浓度为3纳米）通道电流的有效抑制，而对大鼠Kv1.1和Kv1.3通道没有影响。还发现sPi4与125I标记的蜂毒明肽竞争结合大鼠脑突触体中的小电导钙激活钾（SK）通道（半数抑制浓度值为0.5微米）。sPi4是Kv1.2通道的高亲和力阻断剂。利用sPi4的溶液结构和Kv1.2通道孔区域的分子模型，将该毒素对接（BIGGER程序）到钾通道上。该模型表明，Arg10、Arg19、Lys26（二元组）、Ile28、Lys30、Lys33和Tyr35（二元组）残基在与Kv1.2通道的相互作用及相关阻断中起关键作用。
