A Small-Molecule Compound Selectively Activates K2P Channel TASK-3 by Acting at Two Distant Clusters of Residues

Abstract

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.

Polynuclear Ruthenium Amines Inhibit K2P Channels via a “Finger in the Dam” Mechanism

“…A range of physical and chemical signals control K2P function (Enyedi and Czirjak, 2010;Feliciangeli et al, 2014;Renigunta et al, 2015) and various K2P subtypes have emerging roles in a multitude of physiological responses and pathological conditions such as action potential propagation in myelinated axons (Brohawn et al, 2019;Kanda et al, 2019), anesthetic responses (Heurteaux et al, 2004;Lazarenko et al, 2010), microglial surveillance (Madry et al, 2018), sleep duration (Yoshida et al, 2018), pain (Alloui et al, 2006;Devilliers et al, 2013;Vivier et al, 2017), arrythmia (Decher et al, 2017), ischemia (Heurteaux et al, 2004;Laigle et al, 2012;Wu et al, 2013), cardiac fibrosis (Abraham et al, 2018), depression (Heurteaux et al, 2006), migraine (Royal et al, 2019), intraocular pressure regulation (Yarishkin et al, 2018), and pulmonary hypertension (Lambert et al, 2018). Although there have been recent advances in identifying new K2P modulators (Bagriantsev et al, 2013;Lolicato et al, 2017;Pope et al, 2018;Su et al, 2016;Tian et al, 2019;Vivier et al, 2017;Wright et al, 2019) and in defining key structural aspects of K2P channel pharmacology (Dong et al, 2015;Lolicato et al, 2017;Schewe et al, 2019), as is the case with many ion channel classes, pharmacological agents targeting K2Ps remain poorly developed and limit the ability to probe K2P mechanism and biological functions (Sterbuleac, 2019).…”

Silencing KCC2 in mouse dorsal hippocampus compromises spatial and contextual memory

“…We hypothesized a similar effect may also contribute to increase neuronal firing and contamination of SWRs with bursts of action potentials upon KCC2 knockdown. We therefore tested whether increasing Task-3 function, using the selective activator terbinafine (Tian et al , 2019; Wright et al , 2017), might prevent neuronal hyperexcitability and thereby rescue or occlude memory upon KCC2 silencing. In KCC2-knockdown mice, we implanted an intracerebroventricular (icv) canula and a silicon probe to record neuronal activity in the right dorsal hippocampus while injecting terbinafine in the left lateral ventricle (Figure 4A).…”

Small-Molecule Modulators of Mitochondrial Channels as Chemotherapeutic Agents

“…The thiotriazole ML308, developed by the same group, worked with an IC 50 of ~ 0.4 µM, and a >50-fold selectivity for TASK-3 over TASK-1 [269]. Two small-molecule activators have also been identified: NPBA [270] and terbinafine and analogs [271]. In patch-clamp experiments, NPBA increased TASK-3 current with an EC 50 of 6.7 µM (but the current was increased up to 6-fold at 10 µM).…”