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TRAAK, TREK-1, and TREK-2 are mechanosensitive two-pore domain K+ (K2P) channels that contribute to action potential propagation, sensory transduction, and muscle contraction. While structural and functional studies have led to models that explain their mechanosensitivity, we lack a quantitative understanding of channel activation by membrane tension. Here, we define the tension response of mechanosensitive K2Ps using patch-clamp recording and imaging. All are low-threshold mechanosensitive channels (T10%/50% 0.6-2.7 / 4.4-6.4 mN/m) with distinct response profiles. TRAAK is most sensitive, TREK-1 intermediate, and TREK-2 least sensitive. TRAAK and TREK-1 are activated broadly over a range encompassing nearly all physiologically relevant tensions. TREK-2, in contrast, activates over a narrower range like mechanosensitive channels Piezo1, MscS, and MscL. We further show that low-frequency, low-intensity focused ultrasound increases membrane tension to activate TRAAK and MscS. This work provides insight into tension gating of mechanosensitive K2Ps relevant to understanding their physiological roles and potential applications for ultrasonic neuromodulation.

Leak currents, defined as voltage and time independent flows of ions across cell membranes, are central to cellular electrical excitability control. The K2P (KCNK) potassium channel class comprises an ion channel family that produces potassium leak currents that oppose excitation and stabilize the resting membrane potential in cells in the brain, cardiovascular system, immune system, and sensory organs. Due to their widespread tissue distribution, K2Ps contribute to many physiological and pathophysiological processes including anesthesia, pain, arrythmias, ischemia, hypertension, migraine, intraocular pressure regulation, and lung injury responses. Structural studies of six homomeric K2Ps have established the basic architecture of this channel family, revealed key moving parts involved in K2P function, uncovered the importance of asymmetric pinching and dilation motions in the K2P selectivity filter (SF) C-type gate, and defined two K2P structural classes based on the absence or presence of an intracellular gate. Further, a series of structures characterizing K2P:modulator interactions have revealed a striking polysite pharmacology housed within a relatively modestly sized (~70 kDa) channel. Binding sites for small molecules or lipids that control channel function are found at every layer of the channel structure, starting from its extracellular side through the portion that interacts with the membrane bilayer inner leaflet. This framework provides the basis for understanding how gating cues sensed by different channel parts control function and how small molecules and lipids modulate K2P activity. Such knowledge should catalyze development of new K2P modulators to probe function and treat a wide range of disorders.

Ultrasound modulates the electrical activity of excitable cells and offers advantages over other neuromodulatory techniques; for example, it can be noninvasively transmitted through the skull and focused to deep brain regions. However, the fundamental cellular, molecular, and mechanistic bases of ultrasonic neuromodulation are largely unknown. Here, we demonstrate ultrasound activation of the mechanosensitive K+ channel TRAAK with submillisecond kinetics to an extent comparable to canonical mechanical activation. Single-channel recordings reveal a common basis for ultrasonic and mechanical activation with stimulus-graded destabilization of long-duration closures and promotion of full conductance openings. Ultrasonic energy is transduced to TRAAK through the membrane in the absence of other cellular components, likely increasing membrane tension to promote channel opening. We further demonstrate ultrasonic modulation of neuronally expressed TRAAK. These results suggest mechanosensitive channels underlie physiological responses to ultrasound and could serve as sonogenetic actuators for acoustic neuromodulation of genetically targeted cells.

We recently established a role for the stretch-activated two-pore-domain K+ (K2P) channel TREK-1 (K2P2.1) in inflammatory cytokine secretion using models of hyperoxia-, mechanical stretch-, and TNF-α-induced acute lung injury. We have now discovered the expression of large conductance, Ca2+-activated K+ (BK) channels in human pulmonary microvascular endothelial cells and primary human alveolar epithelial cells using semiquantitative real-time PCR, IP and Western blot, and investigated their role in inflammatory cytokine secretion using an LPS-induced acute lung injury model. As expected, LPS induced IL-6 and CCL-2 secretion from pulmonary endothelial and epithelial cells. BK activation with NS1619 decreased LPS-induced CCL-2 but not IL-6 secretion from endothelial cells and had no effect on epithelial cells, although fluorometric assays revealed that BK activation hyperpolarized the plasma membrane potential (Em) of both cell types. Interestingly, BK inhibition (Paxilline) did not alter cytokine secretion or the Em in either cell type. Furthermore, LPS treatment by itself did not affect the Em or intracellular Ca2+ concentrations. Therefore, we propose BK channel activation as a novel targeted approach to counteract LPS-induced CCL-2 secretion from endothelial cells. This protective effect appears to occur via Em hyperpolarization but independent of intracellular Ca2+ concentrations.

TASK2 (also known as KCNK5) channels generate pH-gated leak-type K+ currents to control cellular electrical excitability1-3. TASK2 is involved in the regulation of breathing by chemosensory neurons of the retrotrapezoid nucleus in the brainstem4-6 and pH homeostasis by kidney proximal tubule cells7,8. These roles depend on channel activation by intracellular and extracellular alkalization3,8,9, 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 trinuclear ruthenium amine ruthenium red (RuR) inhibits diverse ion channels, including K2P potassium channels, TRPs, the calcium uniporter, CALHMs, ryanodine receptors, and Piezos. Despite this extraordinary array, there is limited information for how RuR engages targets. Here, using X-ray crystallographic and electrophysiological studies of an RuR-sensitive K2P, K2P2.1 (TREK-1) I110D, we show that RuR acts by binding an acidic residue pair comprising the "Keystone inhibitor site" under the K2P CAP domain archway above the channel pore. We further establish that Ru360, a dinuclear ruthenium amine not known to affect K2Ps, inhibits RuR-sensitive K2Ps using the same mechanism. Structural knowledge enabled a generalizable design strategy for creating K2P RuR "super-responders" having nanomolar sensitivity. Together, the data define a "finger in the dam" inhibition mechanism acting at a novel K2P inhibitor binding site. These findings highlight the polysite nature of K2P pharmacology and provide a new framework for K2P inhibitor development.

No targeted therapies exist to counteract Hyperoxia (HO)-induced Acute Lung Injury (HALI). We previously found that HO downregulates alveolar K2P2.1 (TREK-1) K+ channels, which results in worsening lung injury. This decrease in TREK-1 levels leaves a subset of channels amendable to pharmacological intervention. Therefore, we hypothesized that TREK-1 activation protects against HALI. We treated HO-exposed mice and primary alveolar epithelial cells (AECs) with the novel TREK-1 activators ML335 and BL1249, and quantified physiological, histological, and biochemical lung injury markers. We determined the effects of these drugs on epithelial TREK-1 currents, plasma membrane potential (Em), and intracellular Ca2+ (iCa) concentrations using fluorometric assays, and blocked voltage-gated Ca2+ channels (CaV) as a downstream mechanism of cytokine secretion. Once-daily, intra-tracheal injections of HO-exposed mice with ML335 or BL1249 improved lung compliance, histological lung injury scores, broncho-alveolar lavage protein levels and cell counts, and IL-6 and IP-10 concentrations. TREK-1 activation also decreased IL-6, IP-10, and CCL-2 secretion from primary AECs. Mechanistically, ML335 and BL1249 induced TREK-1 currents in AECs, counteracted HO-induced cell depolarization, and lowered iCa2+ concentrations. In addition, CCL-2 secretion was decreased after L-type CaV inhibition. Therefore, Em stabilization with TREK-1 activators may represent a novel approach to counteract HALI.

The TWIK-related K+ channel (TREK-1) is a two-pore-domain potassium channel that produces background leaky potassium currents. TREK-1 has a protective role against ischemia-induced neuronal damage. TREK-1 is also expressed in the heart, but its role in myocardial ischemia-reperfusion (IR)-induced injury has not been examined. In the current study, we used a TREK-1 knockout (KO) mouse model to show that TREK-1 has a critical role in the cardiac I/R-induced injury and during remodeling after myocardial infarction (MI). At baseline, TREK-1 KO mice had similar blood pressure and heart rate as the wild-type (WT) mice. However, the lack of TREK-1 was associated with increased susceptibility to ischemic injury and compromised functional recovery following ex vivo I/R-induced injury. TREK-1 deficiency increased infarct size following permanent coronary artery ligation, resulting in greater systolic dysfunction than the WT counterpart. Electrocardiographic (ECG) analysis revealed QT interval prolongation in TREK-1 KO mice, but normal heart rate (HR). Acutely isolated TREK-1 KO cardiomyocytes exhibited prolonged Ca2+ transient duration associated with action potential duration (APD) prolongation. Our data suggest that TREK-1 has a protective effect against I/R-induced injury and influences the post-MI remodeling processes by regulating membrane potential and maintaining intracellular Ca2+ homeostasis. These data suggest that TREK-1 activation could be an effective strategy to provide cardioprotection against ischemia-induced damage.

K2P potassium channels generate leak currents that stabilize the resting membrane potential of excitable cells. Various K2P channels are implicated in pain, ischemia, depression, migraine, and anesthetic responses, making this family an attractive target for small molecule modulator development efforts. BL-1249, a compound from the fenamate class of nonsteroidal anti-inflammatory drugs is known to activate K2P2.1(TREK-1), the founding member of the thermo- and mechanosensitive TREK subfamily; however, its mechanism of action and effects on other K2P channels are not well-defined. Here, we demonstrate that BL-1249 extracellular application activates all TREK subfamily members but has no effect on other K2P subfamilies. Patch clamp experiments demonstrate that, similar to the diverse range of other chemical and physical TREK subfamily gating cues, BL-1249 stimulates the selectivity filter "C-type" gate that controls K2P function. BL-1249 displays selectivity among the TREK subfamily, activating K2P2.1(TREK-1) and K2P10.1(TREK-2) ∼10-fold more potently than K2P4.1(TRAAK). Investigation of mutants and K2P2.1(TREK-1)/K2P4.1(TRAAK) chimeras highlight the key roles of the C-terminal tail in BL-1249 action and identify the M2/M3 transmembrane helix interface as a key site of BL-1249 selectivity. Synthesis and characterization of a set of BL-1249 analogs demonstrates that both the tetrazole and opposing tetralin moieties are critical for function, whereas the conformational mobility between the two ring systems impacts selectivity. Together, our findings underscore the landscape of modes by which small molecules can affect K2P channels and provide crucial information for the development of better and more selective K2P modulators of the TREK subfamily.

Mechanical force sensation is fundamental to a wide breadth of biology from the classic senses of touch, pain, hearing, and balance to less conspicuous sensations of proprioception, blood pressure, and osmolarity and basic aspects of cell growth, differentiation, and development. These diverse and essential systems use force-gated (or mechanosensitive) ion channels that convert mechanical stimuli into cellular electrical signals. TRAAK, TREK1, and TREK2 are K+-selective ion channels of the two-pore domain K+ (K2P) family that are mechanosensitive: they are gated open by increasing membrane tension. TRAAK and TREK channels are thought to play roles in somatosensory and other mechanosensory processes in neuronal and non-neuronal tissues. Here, we present protocols for three assays to study mechanical activation of these channels in cell membranes: (1) cell swelling, (2) cell poking, and (3) patched membrane stretching. Patched membrane stretching is also applicable to the study of mechanosensitive K2P channel activity in a cell-free system and a procedure for proteoliposome reconstitution and patching is also presented. These approaches are also readily applicable to the study of other mechanosensitive ion channels.

Polymodal thermo- and mechanosensitive two-pore domain potassium (K2P) channels of the TREK subfamily generate 'leak' currents that regulate neuronal excitability, respond to lipids, temperature and mechanical stretch, and influence pain, temperature perception and anaesthetic responses. These dimeric voltage-gated ion channel (VGIC) superfamily members have a unique topology comprising two pore-forming regions per subunit. In contrast to other potassium channels, K2P channels use a selectivity filter 'C-type' gate as the principal gating site. Despite recent advances, poor pharmacological profiles of K2P channels limit mechanistic and biological studies. Here we describe a class of small-molecule TREK activators that directly stimulate the C-type gate by acting as molecular wedges that restrict interdomain interface movement behind the selectivity filter. Structures of K2P2.1 (also known as TREK-1) alone and with two selective K2P2.1 (TREK-1) and K2P10.1 (TREK-2) activators-an N-aryl-sulfonamide, ML335, and a thiophene-carboxamide, ML402-define a cryptic binding pocket unlike other ion channel small-molecule binding sites and, together with functional studies, identify a cation-π interaction that controls selectivity. Together, our data reveal a druggable K2P site that stabilizes the C-type gate 'leak mode' and provide direct evidence for K2P selectivity filter gating.

In this issue of EMBO Molecular Medicine, Decher et al (2017) identify a point mutation in the K2P2 (TREK‐1) potassium (K+) channel that changes function in just those ways expected to predispose to right ventricular outflow tract (RVOT) ventricular tachycardia (VT) in the patient they study. Whereas wild‐type channels are selective for K+ and inhibited by β‐adrenergic stimulation, mutant I267T channels pass sodium (Na+) into the cells, even during β‐adrenergic stimulation, and are more active in response to membrane stretch, changes predicted to enhance cardiac myocyte excitability. The report contributes to accumulating evidence for Na+ leak via K2P channels in association with normal development (Thomas et al, 2008), acquired arrhythmia (Ma et al, 2011), and now a missense mutation. Decher et al (2017) both inform and direct us toward interesting opportunities for further investigation.

Short-acting β2-agonist bronchodilators are the most common medications used in treating chronic obstructive pulmonary disease (COPD). Genetic variants determining bronchodilator responsiveness (BDR) in COPD have not been identified. We performed a genome-wide association study (GWAS) of BDR in 5789 current or former smokers with COPD in one African-American and four white populations. BDR was defined as the quantitative spirometric response to inhaled β2-agonists. We combined results in a meta-analysis. In the meta-analysis, single-nucleotide polymorphisms (SNPs) in the genes KCNK1 (P=2.02 × 10(-7)) and KCNJ2 (P=1.79 × 10(-7)) were the top associations with BDR. Among African Americans, SNPs in CDH13 were significantly associated with BDR (P=5.1 × 10(-9)). A nominal association with CDH13 was identified in a gene-based analysis in all subjects. We identified suggestive association with BDR among COPD subjects for variants near two potassium channel genes (KCNK1 and KCNJ2). SNPs in CDH13 were significantly associated with BDR in African Americans.The Pharmacogenomics Journal advance online publication, 27 October 2015; doi:10.1038/tpj.2015.65.

Blood pressure (BP) is a complex trait that is the consequence of an interaction between genetic and environmental determinants. Previous studies have demonstrated increased BP in mice with global deletion of TASK-1 channels contemporaneous with diverse dysregulation of aldosterone production. In humans, genome-wide association studies in ≈100 000 individuals of European, East Asian, and South Asian ancestry identified a single nucleotide polymorphism (SNP) in KCNK3 (the gene encoding TASK-1) associated with mean arterial pressure. The current study was motivated by the hypotheses that (1) association of KCNK3 SNPs with BP and related traits extends to blacks and Hispanics, and (2) KCNK3 SNPs exhibit associations with plasma renin activity and aldosterone levels. We examined baseline BP measurements for 7840 participants from the Multi-Ethnic Study of Atherosclerosis (MESA), and aldosterone levels and plasma renin activity in a subset of 1653 MESA participants. We identified statistically significant association of the previously reported KCNK3 SNP (rs1275988) with mean arterial pressure in MESA blacks (P=0.024) and a nearby SNP (rs13394970) in MESA Hispanics (P=0.031). We discovered additional KCNK3 SNP associations with systolic BP, mean arterial pressure, and hypertension. We also identified statistically significant association of KCNK3 rs2586886 with plasma aldosterone level in MESA and demonstrated that global deletion of TASK-1 channels in mice produces a mild-hyperaldosteronism, not associated with a decrease in renin. Our results suggest that genetic variation in the KCNK3 gene may contribute to BP variation and less severe hypertensive disorders in which aldosterone may be one of several causative factors.

Because a rank-ordered recruitment of motor units occurs during isometric contraction of jaw-closing muscles, jaw-closing motoneurons (MNs) may be recruited in a manner dependent on their soma sizes or input resistances (IRs). In the dorsolateral part of the trigeminal motor nucleus (dl-TMN) in rats, MNs abundantly express TWIK (two-pore domain weak inwardly rectifying K channel)-related acid-sensitive-K(+) channel (TASK)-1 and TASK3 channels, which determine the IR and resting membrane potential. Here we examined how TASK channels are involved in IR-dependent activation/recruitment of MNs in the rat dl-TMN by using multiple methods. The real-time PCR study revealed that single large MNs (>35 μm) expressed TASK1 and TASK3 mRNAs more abundantly compared with single small MNs (15-20 μm). The immunohistochemistry revealed that TASK1 and TASK3 channels were complementarily distributed in somata and dendrites of MNs, respectively. The density of TASK1 channels seemed to increase with a decrease in soma diameter while there were inverse relationships between the soma size of MNs and IR, resting membrane potential, or spike threshold. Dual whole-cell recordings obtained from smaller and larger MNs revealed that the recruitment of MNs depends on their IRs in response to repetitive stimulation of the presumed Ia afferents. 8-Bromoguanosine-cGMP decreased IRs in small MNs, while it hardly changed those in large MNs, and subsequently decreased the difference in spike-onset latency between the smaller and larger MNs, causing a synchronous activation of MNs. These results suggest that TASK channels play critical roles in rank-ordered recruitment of MNs in the dl-TMN.

Twik-related K(+) channel 1 (TREK1), TREK2, and Twik-related arachidonic-acid stimulated K(+) channel (TRAAK) form the TREK subfamily of two-pore-domain K(+) (K2P) channels. Despite sharing up to 78% sequence homology and overlapping expression profiles in the nervous system, these channels show major differences in their regulation by physiological stimuli. For instance, TREK1 is inhibited by external acidification, whereas TREK2 is activated. Here, we investigated the ability of the members of the TREK subfamily to assemble to form functional heteromeric channels with novel properties. Using single-molecule pull-down (SiMPull) from HEK cell lysate and subunit counting in the plasma membrane of living cells, we show that TREK1, TREK2, and TRAAK readily coassemble. TREK1 and TREK2 can each heterodimerize with TRAAK, but do so less efficiently than with each other. We functionally characterized the heterodimers and found that all combinations form outwardly rectifying potassium-selective channels but with variable voltage sensitivity and pH regulation. TREK1-TREK2 heterodimers show low levels of activity at physiological external pH but, unlike their corresponding homodimers, are activated by both acidic and alkaline conditions. Modeling based on recent crystal structures, along with mutational analysis, suggests that each subunit within a TREK1-TREK2 channel is regulated independently via titratable His. Finally, TREK1/TRAAK heterodimers differ in function from TRAAK homodimers in two critical ways: they are activated by both intracellular acidification and alkalinization and are regulated by the enzyme phospholipase D2. Thus, heterodimerization provides a means for diversifying functionality through an expansion of the channel types within the K2P channels.

The endolymphatic sac (ES) is a cystic organ that is a part of the inner ear and is connected to the cochlea and vestibule. The ES is thought to be involved in inner ear ion homeostasis and fluid volume regulation for the maintenance of hearing and balance function. Many ion channels, transporters, and exchangers have been identified in the ES luminal epithelium, mainly in animal studies, but there has been no functional study investigating ion transport using human ES tissue. We designed the first functional experiments on electrogenic transport in human ES and investigated the contribution of K(+) channels in the electrogenic transport, which has been rarely identified, even in animal studies, using electrophysiological/pharmacological and molecular biological methods. As a result, we identified functional and molecular evidence for the essential participation of K(+) channels in the electrogenic transport of human ES epithelium. The identified K(+) channels involved in the electrogenic transport were KCNN2, KCNJ14, KCNK2, and KCNK6, and the K(+) transports via those channels are thought to play an important role in the maintenance of the unique ionic milieu of the inner ear fluid.

Brown adipose tissue (BAT) acts in mammals as a natural defense system against hypothermia, and its activation to a state of increased energy expenditure is believed to protect against the development of obesity. Even though the existence of BAT in adult humans has been widely appreciated, its cellular origin and molecular identity remain elusive largely because of high cellular heterogeneity within various adipose tissue depots. To understand the nature of adult human brown adipocytes at single cell resolution, we isolated clonally derived adipocytes from stromal vascular fractions of adult human BAT from two individuals and globally analyzed their molecular signatures. We used RNA sequencing followed by unbiased genome-wide expression analyses and found that a population of uncoupling protein 1 (UCP1)-positive human adipocytes possessed molecular signatures resembling those of a recruitable form of thermogenic adipocytes (that is, beige adipocytes). In addition, we identified molecular markers that were highly enriched in UCP1-positive human adipocytes, a set that included potassium channel K3 (KCNK3) and mitochondrial tumor suppressor 1 (MTUS1). Further, we functionally characterized these two markers using a loss-of-function approach and found that KCNK3 and MTUS1 were required for beige adipocyte differentiation and thermogenic function. The results of this study present new opportunities for human BAT research, such as facilitating cell-based disease modeling and unbiased screens for thermogenic regulators.

BackgroundTREK-1 deficient alveolar epithelial cells (AECs) secrete less IL-6, more MCP-1, and contain less F-actin. Whether these alterations in cytokine secretion and F-actin content are related remains unknown. We now hypothesized that cytokine secretion from TREK-1-deficient AECs was regulated by cytoskeletal rearrangements.MethodsWe determined F-actin and α-tubulin contents of control, TREK-1-deficient and TREK-1-overexpressing human A549 cells by confocal microscopy and western blotting, and measured IL-6 and MCP-1 levels using real-time PCR and ELISA.ResultsCytochalasin D decreased the F-actin content of control cells. Jasplakinolide increased the F-actin content of TREK-1 deficient cells, similar to the effect of TREK-1 overexpression in control cells. Treatment of control and TREK-1 deficient cells with TNF-α, a strong stimulus for IL-6 and MCP-1 secretion, had no effect on F-actin structures. The combination of TNF-α+cytochalasin D or TNF-α+jasplakinolide had no additional effect on the F-actin content or architecture when compared to cytochalasin D or jasplakinolide alone. Although TREK-1 deficient AECs contained less F-actin at baseline, quantified biochemically, they contained more α-tubulin. Exposure to nocodazole disrupted α-tubulin filaments in control and TREK-1 deficient cells, but left the overall amount of α-tubulin unchanged. Although TNF-α had no effect on the F-actin or α-tubulin contents, it increased IL-6 and MCP-1 production and secretion from control and TREK-1 deficient cells. IL-6 and MCP-1 secretions from control and TREK-1 deficient cells after TNF-α+jasplakinolide or TNF-α+nocodazole treatment was similar to the effect of TNF-α alone. Interestingly, cytochalasin D decreased TNF-α-induced IL-6 but not MCP-1 secretion from control but not TREK-1 deficient cells.ConclusionAlthough cytochalasin D, jasplakinolide and nocodazole altered the F-actin and α-tubulin structures of control and TREK-1 deficient AEC, the changes in cytokine secretion from TREK-1 deficient cells cannot be explained by cytoskeletal rearrangements in these cells.

Age at menarche is a marker of timing of puberty in females. It varies widely between individuals, is a heritable trait and is associated with risks for obesity, type 2 diabetes, cardiovascular disease, breast cancer and all-cause mortality. Studies of rare human disorders of puberty and animal models point to a complex hypothalamic-pituitary-hormonal regulation, but the mechanisms that determine pubertal timing and underlie its links to disease risk remain unclear. Here, using genome-wide and custom-genotyping arrays in up to 182,416 women of European descent from 57 studies, we found robust evidence (P

Membrane lipids serve as second messengers and docking sites for proteins and play central roles in cell signaling. A major question about lipid signaling is whether diffusible lipids can selectively target specific proteins. One family of lipid-regulated membrane proteins is the TWIK-related K channel (TREK) subfamily of K2P channels: TREK1, TREK2, and TWIK-related arachdonic acid stimulated K(+) channel (TRAAK). We investigated the regulation of TREK channels by phosphatidic acid (PA), which is generated by phospholipase D (PLD) via hydrolysis of phosphatidylcholine. Even though all three of the channels are sensitive to PA, we found that only TREK1 and TREK2 are potentiated by PLD2 and that none of these channels is modulated by PLD1, indicating surprising selectivity. We found that PLD2, but not PLD1, directly binds to the C terminus of TREK1 and TREK2, but not to TRAAK. The results have led to a model for selective lipid regulation by localization of phospholipid enzymes to specific effector proteins. Finally, we show that regulation of TREK channels by PLD2 occurs natively in hippocampal neurons.

Mechanosensitive ion channels underlie neuronal responses to physical forces in the sensation of touch, hearing, and other mechanical stimuli. The fundamental basis of force transduction in eukaryotic mechanosensitive ion channels is unknown. Are mechanical forces transmitted directly from membrane to channel as in prokaryotic mechanosensors or are they mediated through macromolecular tethers attached to the channel? Here we show in cells that the K(+) channel TRAAK (K2P4.1) is responsive to mechanical forces similar to the ion channel Piezo1 and that mechanical activation of TRAAK can electrically counter Piezo1 activation. We then show that the biophysical origins of force transduction in TRAAK and TREK1 (K2P2.1) two-pore domain K(+) (K2P) channels come from the lipid membrane, not from attached tethers. These findings extend the "force-from-lipid" principle established for prokaryotic mechanosensitive channels MscL and MscS to these eukaryotic mechanosensitive K(+) channels.

Acute Respiratory Distress Syndrome remains challenging partially because the underlying mechanisms are poorly understood. While inflammation and loss of barrier function are associated with disease progression, our understanding of the biophysical mechanisms associated with ventilator-associated lung injury is incomplete. In this line of thinking, we recently showed that changes in the F-actin content and deformability of AECs lead to cell detachment with mechanical stretch. Elsewhere, we discovered that cytokine secretion and proliferation were regulated in part by the stretch-activated 2-pore domain K(+) (K2P) channel TREK-1 in alveolar epithelial cells (AECs). As such, the aim of the current study was to determine whether TREK-1 regulated the mechanobiology of AECs through cytoskeletal remodeling and cell detachment. Using a TREK-1-deficient human AEC line (A549), we examined the cytoskeleton by confocal microscopy and quantified differences in the F-actin content. We used nano-indentation with an atomic force microscope to measure the deformability of cells and detachment assays to quantify the level of injury in our monolayers. We found a decrease in F-actin and an increase in deformability in TREK-1 deficient cells compared to control cells. Although total vinculin and focal adhesion kinase (FAK) levels remained unchanged, focal adhesions appeared to be less prominent and phosphorylation of FAK at the Tyr(925) residue was greater in TREK-1 deficient cells. TREK-1 deficient cells have less F-actin and are more deformable making them more resistant to stretch-induced injury.

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.

The standing outward K(+) current (IKso) governs the response of cerebellar granule neurons to natural and medicinal stimuli including volatile anesthetics. We showed that SUMOylation silenced half of IKso at the surface of cerebellar granule neurons because the underlying channels were heterodimeric assemblies of K2P1, a subunit subject to SUMOylation, and the TASK (two-P domain, acid-sensitive K(+)) channel subunits K2P3 or K2P9. The heterodimeric channels comprised the acid-sensitive portion of IKso and mediated its response to halothane. We anticipate that SUMOylation also influences sensation and homeostatic mechanisms in mammals through TASK channels formed with K2P1.

K(2P)2.1 (TREK-1) is a polymodal two-pore domain leak potassium channel that responds to external pH, GPCR-mediated phosphorylation signals, and temperature through the action of distinct sensors within the channel. How the various intracellular and extracellular sensory elements control channel function remains unresolved. Here, we show that the K(2P)2.1 (TREK-1) intracellular C-terminal tail (Ct), a major sensory element of the channel, perceives metabolic and thermal commands and relays them to the extracellular C-type gate through transmembrane helix M4 and pore helix 1. By decoupling Ct from the pore-forming core, we further demonstrate that Ct is the primary heat-sensing element of the channel, whereas, in contrast, the pore domain lacks robust temperature sensitivity. Together, our findings outline a mechanism for signal transduction within K(2P)2.1 (TREK-1) in which there is a clear crosstalk between the C-type gate and intracellular Ct domain. In addition, our findings support the general notion of the existence of modular temperature-sensing domains in temperature-sensitive ion channels. This marked distinction between gating and sensory elements suggests a general design principle that may underlie the function of a variety of temperature-sensitive channels.

Small ubiquitin modifier 1 (SUMO1) is shown to regulate K2P1 background channels in the plasma membrane (PM) of live mammalian cells. Confocal microscopy reveals native SUMO1, SAE1, and Ubc9 (the enzymes that activate and conjugate SUMO1) at PM where SUMO1 and expressed human K2P1 are demonstrated to colocalize. Silent K2P1 channels in excised PM patches are activated by SUMO isopeptidase (SENP1) and resilenced by SUMO1. K2P1-Lys274 is crucial: when mutated to Gln, Arg, Glu, Asp, Cys, or Ala, the channels are constitutively active and insensitive to SUMO1 and SENP1. Tandem mass spectrometry confirms conjugation of SUMO1 to the epsilon-amino group of Lys274 in vitro. FRET microscopy shows that assembly of K2P1 and SUMO1 requires Lys274. Single-particle TIRF microscopy shows that wild-type channels in PM have two K2P1 subunits and assemble with two SUMO1 monomers. Although channels engineered with one Lys274 site carry just one SUMO1 they are activated and silenced by SENP1 and SUMO1 like wild-type channels.

K(2P) channels mediate potassium background currents essential to central nervous system function, controlling excitability by stabilizing membrane potential below firing threshold and expediting repolarization. Here, we show that alternative translation initiation (ATI) regulates function of K(2P)2.1 (TREK-1) via an unexpected strategy. Full-length K(2P)2.1 and an isoform lacking the first 56 residues of the intracellular N terminus (K(2P)2.1Delta1-56) are produced differentially in a regional and developmental manner in the rat central nervous system, the latter passing sodium under physiological conditions leading to membrane depolarization. Control of ion selectivity via ATI is proposed to be a natural, epigenetic mechanism for spatial and temporal regulation of neuronal excitability.

Surface expression of the K(2P)3.1 two-pore domain potassium channel is regulated by phosphorylation-dependent binding of 14-3-3, leading to suppression of coatomer coat protein I (COPI)-mediated retention in endoplasmic reticulum (ER). Here, we investigate the nature of the macromolecular regulatory complexes that mediate forward and retrograde transport. We demonstrate that (i) the channel employs two separate but interacting COPI binding sites on the N- and C-termini; (ii) disrupting COPI binding to either site interferes with the ER retention; (iii) p11 and 14-3-3 do not interact on their own; (iv) p11 binding to the C-terminal retention motif is dependent on 14-3-3; and (v) p11 is coexpressed in only a subset of tissues with K(2P)3.1, while 14-3-3 expression is ubiquitous. We conclude that K(2P)3.1 forward transport requires 14-3-3 suppression of COPI binding, whereas p11 serves a modulatory role.

Surface expression of the K(2P)3.1 two-pore domain potassium channel is regulated by phosphorylation-dependent binding of 14-3-3, leading to suppression of coatomer coat protein I (COPI)-mediated retention in endoplasmic reticulum (ER). Here, we investigate the nature of the macromolecular regulatory complexes that mediate forward and retrograde transport. We demonstrate that (i) the channel employs two separate but interacting COPI binding sites on the N- and C-termini; (ii) disrupting COPI binding to either site interferes with the ER retention; (iii) p11 and 14-3-3 do not interact on their own; (iv) p11 binding to the C-terminal retention motif is dependent on 14-3-3; and (v) p11 is coexpressed in only a subset of tissues with K(2P)3.1, while 14-3-3 expression is ubiquitous. We conclude that K(2P)3.1 forward transport requires 14-3-3 suppression of COPI binding, whereas p11 serves a modulatory role.

Reversible, covalent modification with small ubiquitin-related modifier proteins (SUMOs) is known to mediate nuclear import/export and activity of transcription factors. Here, the SUMO pathway is shown to operate at the plasma membrane to control ion channel function. SUMO-conjugating enzyme is seen to be resident in plasma membrane, to assemble with K2P1, and to modify K2P1 lysine 274. K2P1 had not previously shown function despite mRNA expression in heart, brain, and kidney and sequence features like other two-P loop K+ leak (K2P) pores that control activity of excitable cells. Removal of the peptide adduct by SUMO protease reveals K2P1 to be a K+-selective, pH-sensitive, openly rectifying channel regulated by reversible peptide linkage.

BackgroundThe 2P domain potassium (K2P) channels are a recently discovered ion channel superfamily. Structurally, K2P channels are distinguished by the presence of two pore forming loops within one channel subunit. Functionally, they are characterized by their ability to pass potassium across the physiologic voltage range. Thus, K2P channels are also called open rectifier, background, or leak potassium channels. Patch clamp studies of renal tubules have described several open rectifier potassium channels that have as yet eluded molecular identification. We sought to determine the segment-specific expression of transcripts for the 14 known K2P channel genes in human nephron to identify potential correlates of native leak channels.MethodsHuman kidney samples were obtained from surgical cases and specific nephron segments were dissected. RNA was extracted and used as template for the generation of cDNA libraries. Real-time polymerase chain reaction (PCR) (TaqMan) was used to analyze gene expression.ResultsWe found significant (P < 0.05) expression of K2P10 in glomerulus, K2P5 in proximal tubule and K2P1 in cortical thick ascending limb of Henle's loop (cTAL) and in distal nephron segments. In addition, we repeatedly detected message for several other K2P channels with less abundance, including K2P3 and K2P6 in glomerulus, K2P10 in proximal tubule, K2P5 in thick ascending limb of Henle's loop, and K2P3, K2P5, and K2P13 in distal nephron segments.ConclusionK2P channels are expressed in specific segments of human kidney. These results provide a step toward assigning K2P channels to previously described native renal leaks.

Proteins with dibasic retention motifs are subject to retrograde transport to endoplasmic reticulum (ER) by COPI-coated vesicles. As forward transport requires escape from ER retention, general release mechanisms have been expected. Here, KCNK3 potassium channels are shown to bear two cytoplasmic trafficking motifs: an N-terminal dibasic site that binds beta-COP to hold channels in ER and a C-terminal "release" site that binds the ubiquitous intracellular regulator 14-3-3beta on a nonclassical motif in a phosphorylation-dependent fashion to suppress beta-COP binding and allow forward transport. The strategy appears to be common. The major histocompatibility antigen class II-associated invariant chain Iip35 exhibits dibasic retention, carries a release motif, and shows mutually exclusive binding of beta-COP and 14-3-3beta on adjacent N-terminal sites. Other retained proteins are demonstrated to carry functional 14-3-3beta release motifs.

Potassium leak channels are essential to neurophysiological function. Leaks suppress excitability through maintenance of resting membrane potential below the threshold for action potential firing. Conversely, voltage-dependent potassium channels permit excitation because they do not interfere with rise to threshold, and they actively promote recovery and rapid re-firing. Previously attributed to distinct transport pathways, we demonstrate here that phosphorylation of single, native hippocampal and cloned KCNK2 potassium channels produces reversible interconversion between leak and voltage-dependent phenotypes. The findings reveal a pathway for dynamic regulation of excitability.

With a bang, a new family of potassium channels has exploded into view. Although KCNK channels were discovered only five years ago, they already outnumber other channel types. KCNK channels are easy to identify because of their unique structure--they possess two preforming domains in each subunit. The new channels function in a most remarkable fashion: they are highly regulated, potassium-selective leak channels. Although leak currents are fundamental to the function of nerves and muscles, the molecular basis for this type of conductance had been a mystery. Here we review the discovery of KCNK channels, what has been learned about them and what lies ahead. Even though two-P-domain channels are widespread and essential, they were hidden from sight in plain view--our most basic questions remain to be answered.

A 2 P domain potassium channel expressed in eye, lung, and stomach, Kcnk8, has recently been identified. To initiate further biochemical and genetic studies of this channel, we assembled the murine Kcnk8 cDNA sequence, characterized the genomic structure of the Kcnk8 gene, determined its chromosomal localization, and analyzed its activity in a Xenopus laevis oocyte expression system. The composite cDNA has an open reading frame of 1029 bp and encodes a protein of 343 amino acids with a predicted molecular mass of 36 kDa. Structure analyses predict 2 P domains and four potential transmembrane helices with a potential single EF-hand motif and four potential SH3-binding motifs in the COOH-terminus. Cloning of the Kcnk8 chromosomal gene revealed that it is composed of three exons distributed over 4 kb of genomic DNA. Genome database searching revealed that one of the intron/exon boundaries identified in Kcnk8 is present in other mammalian 2 P domain potassium channels genes and many C. elegans 2P domain potassium channel genes, revealing evolutionary conservation of gene structure. Using fluorescence in situ hybridization, the murine Kcnk8 gene was mapped to chromosome 19, 2B, the locus of the murine dancer phenotype, and syntenic to 11q11-11q13, the location of the human homologue. No significant currents were generated in a Xenopus laevis oocyte expression system using the composite Kcnk8 cDNA sequence, suggesting, like many potassium channels, additional channel subunits, modulator substances, or cellular chaperones are required for channel function.

Potassium leak conductances were recently revealed to exist as independent molecular entities. Here, the genomic structure, cardiac localization, and biophysical properties of a murine example are considered. Kcnk3 subunits have two pore-forming P domains and unique functional attributes. At steady state, Kcnk3 channels behave like open, potassium-selective, transmembrane holes that are inhibited by physiological levels of proton. With voltage steps, Kcnk3 channels open and close in two phases, one appears to be immediate and one is time-dependent (tau = approximately 5 ms). Both proton block and gating are potassium-sensitive; this produces an anomalous increase in outward flux as external potassium levels rise because of decreased proton block. Single Kcnk3 channels open across the physiological voltage range; hence they are "leak" conductances; however, they open only briefly and rarely even after exposure to agents that activate other potassium channels.

Monoaminergic and cholinergic systems are important regulators of cortical and subcortical systems, and a variety of vegetative functions are controlled by the respective neurotransmitters. Neuronal excitability and transmitter release of these neurons are strongly regulated by their potassium conductances carried by Kir and K channels. Here we describe the generation and characterization of a polyclonal monospecific antibody against rat TASK-3, a major brain K channel. After removal of cross-reactivities and affinity purification the antibody was characterized by ELISA, immunocytochemistry of TASK-3 transfected cells, and Western blots indicating that the antibody only detects TASK-3 protein, but not its paralogs TASK-1 and TASK-5. Western blot analysis of brain membrane fractions showed a single band around 45 kD, close to the predicted molecular weight of the TASK-3 protein. In addition, specific immunolabeling using the anti-TASK-3 antibody in Western blot analysis and immunocytochemistry was blocked in a concentration dependent manner by its cognate antigen only. Immunocytochemical analysis of rat brain revealed strong expression of TASK-3 channels in serotoninergic neurons of the dorsal and median raphe, noradrenergic neurons of the locus coeruleus, histaminergic neurons of the tuberomammillary nucleus and in the cholinergic neurons of the basal nucleus of Meynert. Immunofluorescence double-labeling experiments with appropriate marker enzymes confirmed the expression of TASK-3 in cholinergic, serotoninergic, and noradrenergic neurons. In the dopaminergic system strong TASK-3 expression was found in the ventral tegmental area, whereas TASK-3 immunoreactivity in the substantia nigra compacta was only weak. All immunocytochemical results were supported by in situ hybridization using TASK-3 specific riboprobes. [ABSTRACT FROM AUTHOR]