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Active removal of Na⁺ from the cytosol into the vacuole plays a critical role in salinity tissue tolerance, but another, often neglected component of this trait is Na⁺ retention in vacuoles. This retention is based on an efficient control of Na⁺-permeable slow- and fast-vacuolar channels that mediate the back-leak of Na⁺ into cytosol and, if not regulated tightly, could result in a futile cycle. This Tansley insight summarizes our current knowledge of regulation of tonoplast Na⁺-permeable channels and discusses the energy cost of vacuolar Na⁺ sequestration, under different scenarios. We also report on a phylogenetic and bioinformatic analysis of the plant two-pore channel family and the difference in its structure and regulation between halophytes and glycophytes, in the context of salinity tolerance.

TWIK-related acid-sensitive potassium (TASK) channels—members of the two pore domain potassium (K 2P ) channel family—are found in neurons 1 , cardiomyocytes 2 – 4 and vascular smooth muscle cells 5 , where they are involved in the regulation of heart rate 6 , pulmonary artery tone 5 , 7 , sleep/wake cycles 8 and responses to volatile anaesthetics 8 – 11 . K 2P channels regulate the resting membrane potential, providing background K + currents controlled by numerous physiological stimuli 12 – 15 . Unlike other K 2P channels, TASK channels are able to bind inhibitors with high affinity, exceptional selectivity and very slow compound washout rates. As such, these channels are attractive drug targets, and TASK-1 inhibitors are currently in clinical trials for obstructive sleep apnoea and atrial fibrillation 16 . In general, potassium channels have an intramembrane vestibule with a selectivity filter situated above and a gate with four parallel helices located below; however, the K 2P channels studied so far all lack a lower gate. Here we present the X-ray crystal structure of TASK-1, and show that it contains a lower gate—which we designate as an ‘X-gate’—created by interaction of the two crossed C-terminal M4 transmembrane helices at the vestibule entrance. This structure is formed by six residues ( 243 VLRFMT 248 ) that are essential for responses to volatile anaesthetics 10 , neurotransmitters 13 and G-protein-coupled receptors 13 . Mutations within the X-gate and the surrounding regions markedly affect both the channel-open probability and the activation of the channel by anaesthetics. Structures of TASK-1 bound to two high-affinity inhibitors show that both compounds bind below the selectivity filter and are trapped in the vestibule by the X-gate, which explains their exceptionally low washout rates. The presence of the X-gate in TASK channels explains many aspects of their physiological and pharmacological behaviour, which will be beneficial for the future development and optimization of TASK modulators for the treatment of heart, lung and sleep disorders. The X-ray crystal structure of the potassium channel TASK-1 reveals the presence of an X-gate, which traps small-molecule inhibitors in the intramembrane vestibule and explains their low washout rates from the channel.

THIK-1 ( KCNK13 ) is a halothane-inhibited and anionic-lipid-activated two-pore domain (K2P) K + channel implicated in microglial activation and neuroinflammation, and a current target for the treatment of neurodegenerative disorders, for example Alzheimer’s disease and amyothropic lateral sclerosis (ALS). However, compared to other K2P channels, little is known about the structural and functional properties of THIK-1. Here we present a 3.16-Å-resolution cryo-EM structure of human THIK-1 that reveals several distinct features, in particular, a tyrosine in M4 that contributes to a lower ‘Y gate’ that opens upon activation by physiologically relevant G-protein-coupled receptor and lipid signaling pathways. We demonstrate that linoleic acid bound within a modulatory pocket adjacent to the filter influences channel activity, and that halothane inhibition involves a binding site within the inner cavity, both resulting in conformational changes to the Y gate. Finally, the extracellular cap domain contains positively charged residues that line the ion exit pathway and contribute to the distinct biophysical properties of this channel. Overall, our results provide structural insights into THIK-1 function and identify distinct regulatory sites that expand its potential as a drug target for the modulation of microglial function. In this study, Rödström et al. use cryo-EM to reveal a range of structural features in THIK-1 K + channel and demonstrate how these features define many functional properties of the channel, thus expanding its potential as a drug target.

Controlling body temperature is a matter of life or death for most animals, and in mammals the complex thermoregulatory system is comprised of thermoreceptors, thermosensors, and effectors. The activity of thermoreceptors and thermoeffectors has been studied for many years, yet only recently have we begun to obtain a clear picture of the thermosensors and the molecular mechanisms involved in thermosensory reception. An important step in this direction was the discovery of the thermosensitive transient receptor potential (TRP) cationic channels, some of which are activated by increases in temperature and others by a drop in temperature, potentially converting the cells in which they are expressed into heat and cold receptors. More recently, the TWIK-related potassium (TREK) channels were seen to be strongly activated by increases in temperature. Hence, in this review we want to assess the hypothesis that both these groups of channels can collaborate, possibly along with other channels, to generate the wide range of thermal sensations that the nervous system is capable of handling.

TREK-2 (KCNK10/K2P10), a two-pore domain potassium (K2P) channel, is gated by multiple stimuli such as stretch, fatty acids, and pH and by several drugs. However, the mechanisms that control channel gating are unclear. Here we present crystal structures of the human TREK-2 channel (up to 3.4 angstrom resolution) in two conformations and in complex with norfluoxetine, the active metabolite of fluoxetine (Prozac) and a state-dependent blocker of TREK channels. Norfluoxetine binds within intramembrane fenestrations found in only one of these two conformations. Channel activation by arachidonic acid and mechanical stretch involves conversion between these states through movement of the pore-lining helices. These results provide an explanation for TREK channel mechanosensitivity, regulation by diverse stimuli, and possible off-target effects of the serotonin reuptake inhibitor Prozac.

Ion channels play critical roles in cellular function by facilitating the flow of ions across the membrane in response to chemical or mechanical stimuli. Ion channels operate in a lipid bilayer, which can modulate or define their function. Recent technical advancements have led to the solution of numerous ion channel structures solubilized in detergent and/or reconstituted into lipid bilayers, thus providing unprecedented insight into the mechanisms underlying ion channel–lipid interactions. Here, we describe how ion channel structures have evolved to respond to both lipid modulators and lipid activators to control the electrical activities of cells, highlighting diverse mechanisms and common themes. Ion channel structures reveal mechanisms of lipid action, including how channel gating is altered by direct binding of signaling lipids and those within the membrane itself, as well as mechanical and architectural effects of membrane lipids.

A two-pore domain K + (K2P) channel, THIK-1, plays important roles in microglia and macrophage. THIK-1 is known to be activated by arachidonic acid and G protein-coupled receptors and inhibited by anesthetics. Steroids, such as cholesterol, estradiol and progesterone, are known to modulate several K + channels and they might be potential modulators of THIK-1. We examined the effects of steroids on THIK-1 and found that estradiol inhibits mouse THIK-1 by approximately 40% (IC 50 = 4.9 ± 1.5 μM). Docking simulations of THIK-1 with estradiol indicated possible docking sites, which were further assessed by introducing an alanine mutation into a residue at or near these locations. The F142A, V269A, and Y273A mutations reduced the inhibitory effect of estradiol. These residues are situated within the upper cavity above the Y gate in THIK-1 (pond), suggesting that the pond conformation is essential for estradiol-mediated inhibition. Conversely, the F145A and F276A mutations, located outside this region, were inhibited by 10 nM estradiol and enhanced inhibition by estradiol, estrone, estriol, and progesterone, likely due to conformational changes that facilitate steroid inhibition. The mouse T237S mutation, which corresponds to the reported human THIK-1 variant, produced effects similar to those seen with the F145A and F276A mutations, but to a lesser degree. In summary, estradiol-mediated THIK-1 inhibition depends on residues located in the pond, which may have physiological or pathological significance for THIK-1 variants which are inhibited by low concentration of estradiol.

The Tandem of pore domain in a Weak Inward Rectifying K + channel 2 (TWIK-2; KCNK6 ) is a member of the Two-Pore Domain K + (K 2P ) channel family, which is associated with pulmonary hypertension, lung injury, and inflammation. Despite its physiological relevance, the structure, regulatory mechanisms, and selective modulators of TWIK-2 remain largely unknown. Here, we present a 3.7 Å single particle cryo-electron microscopy structure of human TWIK-2 and highlight its conserved and distinctive features. Using automated whole-cell patch clamp recordings, we demonstrate that gating in TWIK-2 is voltage-dependent and insensitive to changes in the extracellular pH. We identify key residues that influence TWIK-2 activity by employing site-directed mutagenesis and provide insights into the possible lipid-mediated mechanism of TWIK-2 regulation. Additionally, we demonstrate the application of high-throughput automated whole-cell patch clamp platforms to screen small molecule modulators of TWIK-2. Our work serves as a foundation for designing high-throughput small molecule screening campaigns to identify specific high-affinity TWIK-2 modulators, including promising- anti-inflammatory therapeutics. TWIK-2 is an endolysosomal potassium channel implicated in inflammatory responses. Here, authors present a cryo-EM structure of human TWIK-2 and establish a high-throughput automated patch-clamp electrophysiology assay to investigate modulation of TWIK-2.

TASK2 (also known as KCNK5) channels generate pH-gated leak-type K + currents to control cellular electrical excitability 1 – 3 . TASK2 is involved in the regulation of breathing by chemosensory neurons of the retrotrapezoid nucleus in the brainstem 4 – 6 and pH homeostasis by kidney proximal tubule cells 7 , 8 . These roles depend on channel activation by intracellular and extracellular alkalization 3 , 8 , 9 , but the mechanistic basis for TASK2 gating by pH is unknown. Here we present cryo-electron microscopy structures of Mus musculus TASK2 in lipid nanodiscs in open and closed conformations. We identify two gates, distinct from previously observed K + channel gates, controlled by stimuli on either side of the membrane. Intracellular gating involves lysine protonation on inner helices and the formation of a protein seal between the cytoplasm and the channel. Extracellular gating involves arginine protonation on the channel surface and correlated conformational changes that displace the K + -selectivity filter to render it nonconductive. These results explain how internal and external protons control intracellular and selectivity filter gates to modulate TASK2 activity. The authors report on the structure of the K + channel TASK2 and how this channel opens in response to pH changes on either side of the cell membrane.

In a family with pulmonary arterial hypertension, whole-exome sequencing led to identification of a mutation in the potassium-channel gene KCNK3 . Additional mutations resulting in loss of function of the channel were found in other families and in patients with idiopathic disease. Pulmonary arterial hypertension is a rare disease that is characterized by increased pulmonary-artery pressure in the absence of common causes of pulmonary hypertension, such as chronic heart, lung, or thromboembolic disease. 1 Before the advent of novel therapies, patients with idiopathic or familial pulmonary arterial hypertension had an estimated median survival of 2.8 years, with 1-year, 3-year, and 5-year survival rates of 68%, 48%, and 34%, respectively. 2 However, despite progress in treatment, pulmonary arterial hypertension remains a progressive, fatal disease. The clinical presentation can be nonspecific, and patients often receive a diagnosis late in their clinical course. The cause of pulmonary . . .