A high-throughput functional screen identifies small molecule regulators of temperature- and mechano-sensitive K2P channels.
A high-throughput functional screen identifies small molecule regulators of temperature- and mechano-sensitive K2P channels.
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DOI：
10.1021/cb400289x
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发表时间：
2013-08-16
影响因子：
4
通讯作者：
Minor, Daniel L., Jr.
中科院分区：
文献类型：
--
作者：
Bagriantsev, Sviatoslav N.;Ang, Kean-Hooi;Gallardo-Godoy, Alejandra;Clark, Kimberly A.;Arkin, Michelle R.;Renslo, Adam R.;Minor, Daniel L., Jr.
K2P(KCNK)钾通道产生“泄漏”钾电流，强烈影响细胞的兴奋性，并导致疼痛、躯体感觉、麻醉和情绪。尽管K2P具有重要的生理作用，但它们缺乏特定的药理作用。K2P电流的泄漏性质给基于电生理学的高通量筛选策略带来了挑战，使解决这一问题变得复杂。在这里，我们提出了一种基于酵母的高通量筛选试验，可以避免这个问题。使用简单的基于生长的功能读数，我们筛选了一个包含106,281个小分子的文库，并确定了哺乳动物K2P通道K2P2.1(KCNK2，Trek-1)的两个新的抑制剂和三个新的激活剂。通过结合生物物理、构效关系和机理分析，我们开发了一种二氢吖啶类似物ML67-33，它可以作为温度和机械敏感的K2P通道的低微摩尔选择性激活剂。生物物理研究表明，ML67-33通过激活基于细胞外选择性过滤器的C型门，可逆地增加通道电流，C型门形成核心门控装置，各种不同的调制输入在其上汇聚。本文提出的新的K2P调节剂，结合基于酵母菌的测定，将使K2P活性的机制和生理研究成为可能，并促进其他K2P小分子调节剂的发现和发展。
K2P (KCNK) potassium channels generate “leak” potassium currents that strongly influence cellular excitability and contribute to pain, somatosensation, anesthesia, and mood. Despite their physiological importance, K2Ps lack specific pharmacology. Addressing this issue has been complicated by the challenges that the leak nature of K2P currents poses for electrophysiology-based high-throughput screening strategies. Here, we present a yeast-based high-throughput screening assay that avoids this problem. Using a simple growth-based functional readout, we screened a library of 106,281 small molecules and identified two new inhibitors and three new activators of the mammalian K2P channel K2P2.1 (KCNK2, TREK-1). By combining biophysical, structure–activity, and mechanistic analysis, we developed a dihydroacridine analogue, ML67-33, that acts as a low micromolar, selective activator of temperature- and mechano-sensitive K2P channels. Biophysical studies show that ML67-33 reversibly increases channel currents by activating the extracellular selectivity filter-based C-type gate that forms the core gating apparatus on which a variety of diverse modulatory inputs converge. The new K2P modulators presented here, together with the yeast-based assay, should enable both mechanistic and physiological studies of K2P activity and facilitate the discovery and development of other K2P small molecule modulators.
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影响因子：
11.4
作者：
Maingret, F;Lauritzen, I;Honoré, E
通讯作者：
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影响因子：
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作者：
Bagriantsev, Sviatoslav N.;Peyronnet, Remi;Clark, Kimberly A.;Honore, Eric;Minor, Daniel L., Jr.
通讯作者：
Minor, Daniel L., Jr.
影响因子：
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通讯作者：
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