A High-Throughput Functional Screen Identifies Small Molecule Regulators of Temperature- and Mechano-Sensitive K2P Channels

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• 作者：Sviatoslav N. Bagriantsev ; Kean-Hooi Ang ; Alejandra Gallardo-Godoy ; Kimberly A. Clark ; Michelle R. Arkin ; Adam R. Renslo ; Daniel L. Minor ; Jr.
• 刊名：ACS Chemical Biology
• 出版年：2013
• 出版时间：August 16, 2013
• 年：2013
• 卷：8
• 期：8
• 页码：1841-1851
• 全文大小：589K
• 年卷期：v.8,no.8(August 16, 2013)
• ISSN：1554-8937

文摘

K_2P (KCNK) potassium channels generate 鈥渓eak鈥?potassium currents that strongly influence cellular excitability and contribute to pain, somatosensation, anesthesia, and mood. Despite their physiological importance, K_2Ps lack specific pharmacology. Addressing this issue has been complicated by the challenges that the leak nature of K_2P 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 K_2P channel K_2P2.1 (KCNK2, TREK-1). By combining biophysical, structure鈥揳ctivity, and mechanistic analysis, we developed a dihydroacridine analogue, ML67-33, that acts as a low micromolar, selective activator of temperature- and mechano-sensitive K_2P 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 K_2P modulators presented here, together with the yeast-based assay, should enable both mechanistic and physiological studies of K_2P activity and facilitate the discovery and development of other K_2P small molecule modulators.