Pharmacologically reversible, loss of function mutations in the TM2 and TM4 inner pore helices of TREK-1 K2P channels.

A better understanding of the gating of TREK two pore domain potassium (K2P) channels and their activation by compounds such as the negatively charged activator, flufenamic acid (FFA) is critical in the search for more potent and selective activators of these channels. Currents through wild-type and mutated human K2P channels expressed in tsA201 cells were measured using whole-cell patch-clamp recordings in the presence and absence of FFA. Mutation of the TM2.6 residue of TREK-1 to a phenylalanine (G171F) and a similar mutation of TM4.6 (A286F) substantially reduced current through TREK-1 channels. In complementary experiments, replacing the natural F residues at the equivalent position in TRESK channels, significantly enhanced current. Known, gain of function mutations of TREK-1 (G137I, Y284A) recovered current through these mutated channels. This reduction in current could be also be reversed pharmacologically, by FFA. However, an appropriate length MTS (MethaneThioSulfonate) cross-linking reagent (MTS14) restricted the activation of TREK-1_A286C channels by repeated application of FFA. This suggests that the cross-linker stabilises the channel in a conformation which blunts FFA activation. Pharmacologically reversible mutations of TREK channels will help to clarify the importance of these channels in pathophysiological conditions such as pain and depression.

Molecular cloning of two kcnk3 genes from the Northern snakehead (Channa argus) for quantification of their transcriptions in response to fasting and refeeding.

Potassium channel subfamily K member 3 (KCNK3) has been reported to play important roles in membrane potential conduction, pulmonary hypertension and thermogenesis regulation in mammals. However, its roles remain largely unknown and scarce reports were seen in fish. In the present study, we for the first time identified two kcnk3 genes (kcnk3a and kcnk3b) from the carnivorous Northern snakehead (Channa argus) by molecular cloning and a genomic survey. Subsequently, their transcription changes in response to different feeding status were investigated. Full-length coding sequences of the kcnk3a and kcnk3b genes are 1203 and 1176 bp, encoding 400 and 391 amino acids, respectively. Multiple alignments, 3D-structure prediction and phylogenetic analysis further suggested that these kcnk3 genes may be highly conserved in vertebrates. Tissue distribution analysis by real-time PCR demonstrated that both the snakehead kcnk3s were widely transcribed in majority of the examined tissues but with different distribution patterns. In a short-term (24-h) fasting experiment, we observed that brain kcnk3a and kcnk3b genes showed totally opposite transcription patterns. In a long-term (2-week) fasting and refeeding experiment, we also observed differential change patterns for the brain kcnk3 genes. In summary, our findings suggest that the two kcnk3 genes are close while present different transcription responses to fasting and refeeding. They therefore can be potentially selected as novel target genes for improvement of production and quality of this economically important fish.

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

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 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.

A high-throughput functional screen identifies small molecule regulators of temperature- and mechano-sensitive K2P channels.

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.

Identification and functional characterization of zebrafish K(2P)10.1 (TREK2) two-pore-domain K(+) channels.

Two-pore-domain potassium (K(2P)) channels mediate K(+) background currents that stabilize the resting membrane potential and contribute to repolarization of action potentials in excitable cells. The functional significance of K(2P) currents in cardiac electrophysiology remains poorly understood. Danio rerio (zebrafish) may be utilized to elucidate the role of cardiac K(2P) channels in vivo. The aim of this work was to identify and functionally characterize a zebrafish otholog of the human K(2P)10.1 channel. K(2P)10.1 orthologs in the D. rerio genome were identified by database analysis, and the full zK(2P)10.1 coding sequence was amplified from zebrafish cDNA. Human and zebrafish K(2P)10.1 proteins share 61% identity. High degrees of conservation were observed in protein domains relevant for structural integrity and regulation. K(2P)10.1 channels were heterologously expressed in Xenopus oocytes, and currents were recorded using two-electrode voltage clamp electrophysiology. Human and zebrafish channels mediated K(+) selective background currents leading to membrane hyperpolarization. Arachidonic acid, an activator of hK(2P)10.1, induced robust activation of zK(2P)10.1. Activity of both channels was reduced by protein kinase C. Similar to its human counterpart, zK(2P)10.1 was inhibited by the antiarrhythmic drug amiodarone. In summary, zebrafish harbor K(2P)10.1 two-pore-domain K(+) channels that exhibit structural and functional properties largely similar to human K(2P)10.1. We conclude that the zebrafish represents a valid model to study K(2P)10.1 function in vivo.

A specific two-pore domain potassium channel blocker defines the structure of the TASK-1 open pore.

Two-pore domain potassium (K(2P)) channels play a key role in setting the membrane potential of excitable cells. Despite their role as putative targets for drugs and general anesthetics, little is known about the structure and the drug binding site of K(2P) channels. We describe A1899 as a potent and highly selective blocker of the K(2P) channel TASK-1. As A1899 acts as an open-channel blocker and binds to residues forming the wall of the central cavity, the drug was used to further our understanding of the channel pore. Using alanine mutagenesis screens, we have identified residues in both pore loops, the M2 and M4 segments, and the halothane response element to form the drug binding site of TASK-1. Our experimental data were used to validate a K(2P) open-pore homology model of TASK-1, providing structural insights for future rational design of drugs targeting K(2P) channels.

Two-pore domain k(+) channels and their role in chemoreception.

A number of tandem P-domain K(+)- channels (K(2)P) generate background K(+)-currents similar to those found in enteroreceptors that sense a diverse range of physiological stimuli including blood pH, carbon dioxide, oxygen, potassium and glucose. This review presents an overview of the properties of both cloned K(2)P tandem-P-domain K-channels and the endogenous chemosensitive background K-currents found in central chemoreceptors, peripheral chemoreceptors, the adrenal gland and the hypothalamus. Although the identity of many of these endogenous channels has yet to be confirmed they show striking similarities to a number of K(2)P channels especially those of the TASK subgroup. Moreover these channels seem often (albeit not exclusively) to be involved in pH and nutrient/metabolic sensing.

Deafness associated changes in expression of two-pore domain potassium channels in the rat cochlear nucleus.

Two-pore domain potassium channels (K(2PD)+) play an important role in setting resting membrane potential by regulating background leakage of potassium ions, which in turn controls neuronal excitability. To determine whether these channels contribute to activity-dependent plasticity following deafness, we used quantitative real-time PCR to examine the expression of 10 K(2PD)+ subunits in the rat cochlear nucleus at 3 days, 3 weeks and 3 months after bilateral cochlear ablation. There was a large sustained decrease in the expression of TASK-5, a subunit that is predominantly expressed in auditory brain stem neurons, and in the TASK-1 subunit which is highly expressed in several types of cochlear nucleus neurons. TWIK-1 and THIK-2 also showed significant decreases in expression that were maintained across all time points. TWIK-2, TREK-1 and TREK-2 showed no significant change in expression at 3 days but showed large decreases at 3 weeks and 3 months following deafness. TRAAK and TASK-3 subunits showed significant decreases at 3 days and 3 weeks following deafness, but these differences were no longer significant at 3 months. Dramatic changes in expression of K(2PD)+ subunits suggest these channels may play a role in deafness-associated changes in the excitability of cochlear nucleus neurons.