Laboratory for Molecular Biophysics, Physiology and Pharmacology, University of Antwerp, 2610 Antwerp, Belgium.
J Gen Physiol. 2013 Mar;141(3):359-69. doi: 10.1085/jgp.201210890. Epub 2013 Feb 11.
Voltage-gated potassium (Kv) and sodium (Nav) channels are key determinants of cellular excitability and serve as targets of neurotoxins. Most marine ciguatoxins potentiate Nav channels and cause ciguatera seafood poisoning. Several ciguatoxins have also been shown to affect Kv channels, and we showed previously that the ladder-shaped polyether toxin gambierol is a potent Kv channel inhibitor. Most likely, gambierol acts via a lipid-exposed binding site, located outside the K(+) permeation pathway. However, the mechanism by which gambierol inhibits Kv channels remained unknown. Using gating and ionic current analysis to investigate how gambierol affected S6 gate opening and voltage-sensing domain (VSD) movements, we show that the resting (closed) channel conformation forms the high-affinity state for gambierol. The voltage dependence of activation was shifted by >120 mV in the depolarizing direction, precluding channel opening in the physiological voltage range. The (early) transitions between the resting and the open state were monitored with gating currents, and provided evidence that strong depolarizations allowed VSD movement up to the activated-not-open state. However, for transition to the fully open (ion-conducting) state, the toxin first needed to dissociate. These dissociation kinetics were markedly accelerated in the activated-not-open state, presumably because this state displayed a much lower affinity for gambierol. A tetrameric concatemer with only one high-affinity binding site still displayed high toxin sensitivity, suggesting that interaction with a single binding site prevented the concerted step required for channel opening. We propose a mechanism whereby gambierol anchors the channel's gating machinery in the resting state, requiring more work from the VSD to open the channel. This mechanism is quite different from the action of classical gating modifier peptides (e.g., hanatoxin). Therefore, polyether toxins open new opportunities in structure-function relationship studies in Kv channels and in drug design to modulate channel function.
电压门控钾 (Kv) 和钠 (Nav) 通道是细胞兴奋性的关键决定因素，也是神经毒素的靶标。大多数海洋雪卡毒素增强 Nav 通道并导致雪卡鱼中毒。一些雪卡毒素也已被证明会影响 Kv 通道，我们之前曾表明，梯状聚醚毒素 Gambierol 是一种有效的 Kv 通道抑制剂。很可能， Gambierol 通过位于 K(+)渗透途径之外的暴露脂质的结合位点起作用。然而， Gambierol 抑制 Kv 通道的机制尚不清楚。我们使用门控和离子电流分析来研究 Gambierol 如何影响 S6 门控开放和电压感应域 (VSD) 运动，结果表明，处于静息（关闭）状态的通道构象形成了 Gambierol 的高亲和力状态。激活的电压依赖性向去极化方向偏移超过 120 mV，使通道在生理电压范围内无法打开。通过门控电流监测到从静止状态到开放状态的（早期）转变，为 VSD 运动到激活但未打开状态提供了证据。然而，对于完全打开（离子传导）状态的转变，毒素首先需要解离。在激活但未打开状态下，这些解离动力学明显加快，推测这是因为该状态对 Gambierol 的亲和力降低。尽管只有一个高亲和力结合位点的四聚体串联体仍显示出对毒素的高敏感性，但这表明与单个结合位点的相互作用阻止了打开通道所需的协同步骤。我们提出了一种机制，即 Gambierol 将通道的门控机制锚定在静止状态，使 VSD 打开通道需要更多的功。这种机制与经典门控修饰肽（例如，汉坦毒素）的作用非常不同。因此，聚醚毒素为 Kv 通道的结构-功能关系研究和调节通道功能的药物设计开辟了新的机会。
