CAS Key Laboratory of Receptor Research, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China (F.T., Y.Q., X.L., Z.G.); University of Chinese Academy of Sciences, Beijing, China (F.T., Y.Q., X.L., Z.G.); Department of Neuroscience, High Throughput Biology Center and Johns Hopkins Ion Channel Center, School of Medicine, Johns Hopkins University, Baltimore, Maryland (M.L.); and Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences, School of Life Sciences, East China Normal University, Shanghai, China (H.Y.).
CAS Key Laboratory of Receptor Research, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China (F.T., Y.Q., X.L., Z.G.); University of Chinese Academy of Sciences, Beijing, China (F.T., Y.Q., X.L., Z.G.); Department of Neuroscience, High Throughput Biology Center and Johns Hopkins Ion Channel Center, School of Medicine, Johns Hopkins University, Baltimore, Maryland (M.L.); and Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences, School of Life Sciences, East China Normal University, Shanghai, China (H.Y.)
Mol Pharmacol. 2019 Jul;96(1):26-35. doi: 10.1124/mol.118.115303. Epub 2019 Apr 23.
The TASK-3 channel is a member of the K2P family that is important for the maintenance of the resting membrane potential. Previous studies have demonstrated that the TASK-3 channel is involved in several physiologic and pathologic processes, including sleep/wake control, cognition, and epilepsy. However, there is still a lack of selective pharmacological tools for TASK-3, which limits further research on channel function. In this work, using a high-throughput screen, we discovered that -(2-((4-nitro-2-(trifluoromethyl)phenyl)amino)ethyl)benzamide (NPBA) showed excellent potency and selectivity as a novel TASK-3 activator. The molecular determinants of NPBA activation were then investigated by combining chimera and mutagenesis analysis. Two distant clusters of residues located at the extracellular end of the second transmembrane domain (A105 and A108) and the intracellular end of the third transmembrane domain (E157) were found to be critical for NPBA activation. We then compared the essentials of the actions of NPBA with inhalation anesthetics that nonselectively activate TASK-3 and found that they may activate TASK-3 channels through different mechanisms. Finally, we transplanted the three residues A105, A108, and E157 into the TASK-1 channel, which resists NPBA activation, and the constructed mutant TASK-1(G105A, V108A, A157E) showed dramatically increased activation by NPBA, confirming the importance of these two distant clusters of residues. SIGNIFICANCE STATEMENT: TASK-3 channels conduct potassium and are involved in various physiological and pathological processes. However, the lack of selective modulators has hindered efforts to increase our understanding of the physiological roles of TASK-3 channels. By using a high-throughput screen, we identified NPBA as a potent and selective TASK-3 activator, and we show that NPBA is a more potent activator than terbinafine, the only reported TASK-3 selective activator to date. We also show here that NPBA has outstanding selectivity for TAS-3 channels. These characteristics make NPBA a promising pharmacological probe for research focused on defining TASK-3 channel function(s). In addition, we identified two distant clusters of residues as determinants of NPBA activation providing new molecular clues for the understanding of the gating mechanism of K2P channels.
TASK-3 通道是 K2P 家族的成员，对于维持静息膜电位很重要。先前的研究表明，TASK-3 通道参与了许多生理和病理过程，包括睡眠/觉醒控制、认知和癫痫。然而，目前仍然缺乏对 TASK-3 的选择性药理学工具，这限制了对通道功能的进一步研究。在这项工作中，我们使用高通量筛选发现，-(2-((4-硝基-2-(三氟甲基)苯基)氨基)乙基)苯甲酰胺（NPBA）作为一种新型 TASK-3 激活剂具有优异的效力和选择性。然后通过组合嵌合体和突变分析研究了 NPBA 激活的分子决定因素。发现位于第二跨膜域（A105 和 A108）的细胞外端和第三跨膜域（E157）的细胞内端的两个遥远的残基簇对于 NPBA 的激活至关重要。然后，我们将 NPBA 的作用与非选择性激活 TASK-3 的吸入麻醉剂进行了比较，发现它们可能通过不同的机制激活 TASK-3 通道。最后，我们将三个残基 A105、A108 和 E157 移植到对 NPBA 激活有抵抗力的 TASK-1 通道中，构建的突变体 TASK-1（G105A、V108A、A157E）对 NPBA 的激活显著增加，证实了这两个遥远的残基簇的重要性。
TASK-3 通道传导钾离子，并参与各种生理和病理过程。然而，缺乏选择性调节剂阻碍了我们对 TASK-3 通道生理作用的理解。通过使用高通量筛选，我们鉴定出 NPBA 是一种有效的 TASK-3 激活剂，并且我们发现 NPBA 比迄今为止报道的唯一的 TASK-3 选择性激活剂特比萘芬更有效。我们还在这里表明，NPBA 对 TAS-3 通道具有出色的选择性。这些特性使 NPBA 成为研究 TASK-3 通道功能的有前途的药理学探针。此外，我们鉴定出两个遥远的残基簇作为 NPBA 激活的决定因素，为理解 K2P 通道的门控机制提供了新的分子线索。
