人电压门控K+通道Kv1.1的功能障碍与癫痫有关,多发性硬化症,偶发性共济失调,Myokymia,和心肺功能失调.我们在这里报道AETX-K,我们从噬菌体展示库中分离的海葵I型(SAK1)肽毒素,阻断Kv1.1具有高亲和力(Ki~1.6pM)和显著特异性,抑制其他KV通道,我们测试的效果差了一百万倍。核磁共振(NMR)用于确定AETX-K的三维结构,显示它采用经典的SAK1支架,同时表现出独特的静电势表面,并观察与嵌入脂蛋白纳米盘中的Kv1.1孔结构域结合的AETX-K。使用电生理学研究具有AETX-K和点变体的非洲爪的卵母细胞中的Kv1.1证明了阻断机制采用我们之前描述的毒素通道构型,其中AETX-KLys23,毒素相互作用表面上远离经典阻断残基的两个位置,进入孔足够深以与从膜的相对侧穿过路径的K+离子相互作用。突变通道Kv1.1-L296F与婴儿的药物抗性多灶性癫痫相关,因为它通过促进通道的打开和减慢关闭而显着增加K电流。与AETX-K对Kv1.1功能获得相关疾病的治疗潜力一致,4pM的AETX-K将Kv1.1-L296F电流降低至野生型水平;此外,由Kv1.1和Kv1.2共表达形成的异聚通道群体,如在许多神经元中发现的,显示~10nM的Ki,即使同源Kv1.2通道对毒素不敏感(Ki>2000nM)。
Dysfunction of the human voltage-gated K+ channel Kv1.1 has been associated with epilepsy, multiple sclerosis, episodic ataxia, myokymia, and cardiorespiratory dysregulation. We report here that AETX-K, a sea anemone type I (SAK1) peptide toxin we isolated from a phage display library, blocks Kv1.1 with high affinity (Ki ~ 1.6 pM) and notable specificity, inhibiting other Kv channels we tested a million-fold less well. Nuclear magnetic resonance (NMR) was employed both to determine the three-dimensional structure of AETX-K, showing it to employ a classic SAK1 scaffold while exhibiting a unique electrostatic potential surface, and to visualize AETX-K bound to the Kv1.1 pore domain embedded in lipoprotein nanodiscs. Study of Kv1.1 in Xenopus oocytes with AETX-K and point variants using electrophysiology demonstrated the blocking mechanism to employ a toxin-channel configuration we have described before whereby AETX-K Lys23 , two positions away on the toxin interaction surface from the classical blocking residue, enters the pore deeply enough to interact with K+ ions traversing the pathway from the opposite side of the membrane. The mutant channel Kv1.1-L296 F is associated with pharmaco-resistant multifocal epilepsy in infants because it significantly increases K+ currents by facilitating opening and slowing closure of the channels. Consistent with the therapeutic potential of AETX-K for Kv1.1 gain-of-function-associated diseases, AETX-K at 4 pM decreased Kv1.1-L296 F currents to wild-type levels; further, populations of heteromeric channels formed by co-expression Kv1.1 and Kv1.2, as found in many neurons, showed a Ki of ~10 nM even though homomeric Kv1.2 channels were insensitive to the toxin (Ki > 2000 nM).