The paucity of selective agonists for TWIK-related acid-sensitive K+ 3 (TASK-3) channel, a member of two-pore domain K+ (K2P) channels, has contributed to our limited understanding of its biological functions. By targeting a druggable transmembrane cavity using a structure-based drug design approach, we discovered a biguanide compound, CHET3, as a highly selective allosteric activator for TASK-3–containing K2P channels, including TASK-3 homomers and TASK-3/TASK-1 heteromers. CHET3 displayed potent analgesic effects in vivo in a variety of acute and chronic pain models in rodents that could be abolished pharmacologically or by genetic ablation of TASK-3. We further found that TASK-3–containing channels anatomically define a unique population of small-sized, transient receptor potential cation channel subfamily M member 8 (TRPM8)–, transient receptor potential cation channel subfamily V member 1 (TRPV1)–, or tyrosine hydroxylase (TH)–positive nociceptive sensory neurons and functionally regulate their membrane excitability, supporting CHET3 analgesic effects in thermal hyperalgesia and mechanical allodynia under chronic pain. Overall, our proof-of-concept study reveals TASK-3–containing K2P channels as a druggable target for treating pain.
The voltage-gated KCNQ1 potassium channel is expressed in cardiac tissues, and coassembly of KCNQ1 with an auxiliary KCNE1 subunit mediates a slowly activating current that accelerates the repolarization of action potential in cardiomyocytes. Mutations of KCNQ1 genes that result in reduction or loss of channel activity cause prolongation of repolarization during action potential, thereby causing long QT syndrome (LQTs). Small molecule activators of KCNQ1/KCNE1 are useful both for understanding the mechanism of the complex activity and for developing therapeutics for LQTs. In this study we report that hexachlorophene (HCP), the active component of the topical anti-infective prescription drug pHisoHex, is a KCNQ1/KCNE1 activator. HCP potently increases the current amplitude of KCNQ1/KCNE1 expressed by stabilizing the channel in an open state with an EC50 of 4.61±1.29 μM. Further studies in cardiomyocytes showed that HCP significantly shortens the action potential duration at 1 μM. In addition, HCP is capable of rescuing the loss of function of the LQTs mutants caused by either impaired activation gating or phosphatidylinositol-4,5-bisphosphate (PIP2) binding affinity. Our results indicate HCP is a novel KCNQ1/KCNE1 activator and may be a useful tool compound for the development of LQTs therapeutics.
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 N-(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 STATEMENTTASK-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.
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