2P Channels Research Articles

Expression of mechanogated two-pore domain potassium channels in mouse lungs: special reference to mechanosensory airway receptors

Afferent activities arising from sensory nerve terminals located in lungs and airways are carried almost exclusively by fibres travelling through the vagus nerve. Based on electrophysiological investigations, intrapulmonary airway-related vagal afferent receptors have been classified into three main subtypes, two of which are myelinated and mechanosensitive, i.e., rapidly and slowly adapting receptors. To allow for a full functional identification of the distinct populations of airway receptors, morphological and neurochemical characteristics still need to be determined. Nerve terminals visualised using markers for myelinated vagal afferents seem to be almost uniquely associated with two morphologically well-formed airway receptor end organs, smooth muscle-associated airway receptors (SMARs) and neuroepithelial bodies (NEBs), localised in airway smooth muscle and epithelium, respectively. Due to the lack of a selective marker for SMARs in mice, no further neurochemical coding is available today. NEBs are extensively innervated diffusely spread groups of neuroendocrine cells in the airway epithelium, and are known to receive at least two separate populations of myelinated vagal afferent nerve terminals. So far, however, no evidence has been reported for the expression of channels that may underlie direct sensing and transduction of mechanical stimuli by the receptor terminals in NEBs and SMARs. This study focused on the expression of mechanogated two-pore domain K(+) (K(2P)) channels, TREK-1 and TRAAK, in mouse airways and more particular in the NEB micro-environment and in SMARs by multiple immunostaining. TREK-1 could be detected on smooth muscle cells surrounding intrapulmonary airways and blood vessels, while TRAAK was expressed on myelinated vagal afferents terminating both in SMARs and in the NEB micro-environment. Co-stainings with known markers for subpopulations of myelinated vagal afferents and general neuronal markers revealed that all identified SMARs exhibit TRAAK immunoreactivity, and that at least three subpopulations exist in mouse airways. Also, the intraepithelial terminals of both subpopulations of NEB-associated myelinated vagal sensory nerve fibres were shown to express TRAAK. In conclusion, the present study finally characterised an intrinsically mechanosensitive ion channel, the K(2P) channel TRAAK, on the terminals of identified myelinated vagal nodose airway afferents, organised as SMARs and as components of the innervation of NEBs. These data support the hypothesis that both SMARs and NEBs harbour the morphological counterparts of electrophysiologically identified myelinated vagal airway mechanoreceptors. TRAAK appears to be strongly involved in regulating airway mechanosensing since it was found to be expressed on the terminals of all subpopulations of potential vagal mechanosensors.

TREK-1 isoforms generated by alternative translation initiation display different susceptibility to the antidepressant fluoxetine

Two-pore-domain K + (K 2P) channels are highly expressed in neurons and cardiac myocytes. In this study we investigated the potency of the antidepressant fluoxetine to inhibit brain and cardiac K 2P channels, TREK-1, TASK-1 and THIK-1. Maximal sensitivity was detected for TREK-1, which was inhibited by 77% when expressed in HEK-293 cells and Xenopus oocytes. Alternative translation initiation (ATI) generates two different protein products from a single transcript of TREK-1. Electrophysiological analysis of two polypeptides engineered by mutagenesis (TREK-1[M53I], TREK-1[ΔN52]) revealed reduced current amplitude and K + selectivity of the truncated TREK-1 isoform. The sensitivity of TREK-1[ΔN52] to fluoxetine decreased by 70%, indicating that the first 52 amino acids are essential for TREK-1 sensitivity to this drug.

Carvedilol targets human K2P 3.1 (TASK1) K+ leak channels.

Human K(2P) 3.1 (TASK1) channels represent potential targets for pharmacological management of atrial fibrillation. K(2P) channels control excitability by stabilizing membrane potential and by expediting repolarization. In the heart, inhibition of K(2P) currents by class III antiarrhythmic drugs results in action potential prolongation and suppression of electrical automaticity. Carvedilol exerts antiarrhythmic activity and suppresses atrial fibrillation following cardiac surgery or cardioversion. The objective of this study was to investigate acute effects of carvedilol on human K(2P) 3.1 (hK(2P) 3.1) channels. Two-electrode voltage clamp and whole-cell patch clamp electrophysiology was used to record hK(2P) 3.1 currents from Xenopus oocytes, Chinese hamster ovary (CHO) cells and human pulmonary artery smooth muscle cells (hPASMC). Carvedilol concentration-dependently inhibited hK(2P) 3.1 currents in Xenopus oocytes (IC(50) = 3.8 µM) and in mammalian CHO cells (IC(50) = 0.83 µM). In addition, carvedilol sensitivity of native I(K2P3.1) was demonstrated in hPASMC. Channels were blocked in open and closed states in frequency-dependent fashion, resulting in resting membrane potential depolarization by 7.7 mV. Carvedilol shifted the current-voltage (I-V) relationship by -6.9 mV towards hyperpolarized potentials. Open rectification, characteristic of K(2P) currents, was not affected. The antiarrhythmic drug carvedilol targets hK(2P) 3.1 background channels. We propose that cardiac hK(2P) 3.1 current blockade may suppress electrical automaticity, prolong atrial refractoriness and contribute to the class III antiarrhythmic action in patients treated with the drug.

Volume regulation of murine T lymphocytes relies on voltage-dependent and two-pore domain potassium channels

A variety of ion channels are supposed to orchestrate the homoeostatic volume regulation in T lymphocytes. However, the relative contribution of different potassium channels to the osmotic volume regulation and in particular to the regulatory volume decrease (RVD) in T cells is far from clear. This study explores a putative role of the newly identified K 2P channels (TASK1, TASK2, TASK3 and TRESK) along with the voltage-gated potassium channel K V1.3 and the calcium-activated potassium channel K Ca3.1 in the RVD of murine T lymphocytes, using genetic and pharmacological approaches. K 2P channel knockouts exerted profound effects on the osmotic properties of murine T lymphocytes, as revealed by reduced water and RVD-related solute permeabilities. Moreover, both genetic and pharmacological data proved a key role of K V1.3 and TASK2 channels in the RVD of murine T cells exposed to hypotonic saline. Our experiments demonstrate a leading role of potassium channels in the osmoregulation of T lymphocytes under different conditions. In summary, the present study sheds new light on the complex and partially redundant network of potassium channels involved in the basic physiological process of the cellular volume homeostasis and extends the repertoire of potassium channels by the family of K 2P channels.

Protons, Heat, and Mechanical Force Act Through a Common Gate to Control K 2P Channel Function

Members of the K2P potassium channel family are key regulators of cell excitability and are implicated in pain, anesthetic responses, thermosensation, neuroprotection, and mood. Unlike other potassium channel families, K2P channels are gated by remarkably diverse stimuli that include chemical, thermal, and mechanical modalities. It has remained unclear whether the various gating inputs act through separate or common channel elements. Here, we show that protons, heat, and pressure affect the activity of the prototypical, polymodal K2P channel, K2P2.1 (KCNK2/TREK-1), at a common molecular gate that comprises extracellular elements of the pore-forming segments and part of the putative fourth transmembrane helix, M4, that is proximal to the extracellular end. We further demonstrate that the functional importance of the M4 gating element is conserved across the K2P family, regardless of whether the gating stimuli are inhibitory or activating. Taken together, our results highlight a unique gating mechanism shared by K2P channels and suggest that the diverse sensory properties of K2P channels are achieved by coupling different molecular sensors to a conserved core gating apparatus comprising the selectivity filter and M4.

Gating of a pH-Sensitive K2P Potassium Channel by an Electrostatic Effect of Basic Sensor Residues on the Selectivity Filter

K+ channels share common selectivity characteristics but exhibit a wide diversity in how they are gated open. Leak K2P K+ channels TASK-2, TALK-1 and TALK-2 are gated open by extracellular alkalinization. The mechanism for this alkalinization-dependent gating has been proposed to be the neutralization of the side chain of a single arginine (lysine in TALK-2) residue near the pore of TASK-2, which occurs with the unusual pKa of 8.0. We now corroborate this hypothesis by transplanting the TASK-2 extracellular pH (pHo) sensor in the background of a pHo-insensitive TASK-3 channel, which leads to the restitution of pHo-gating. Using a concatenated channel approach, we also demonstrate that for TASK-2 to open, pHo sensors must be neutralized in each of the two subunits forming these dimeric channels with no apparent cross-talk between the sensors. These results are consistent with adaptive biasing force analysis of K+ permeation using a model selectivity filter in wild-type and mutated channels. The underlying free-energy profiles confirm that either a doubly or a singly charged pHo sensor is sufficient to abolish ion flow. Atomic detail of the associated mechanism reveals that, rather than a collapse of the pore, as proposed for other K2P channels gated at the selectivity filter, an increased height of the energetic barriers for ion translocation accounts for channel blockade at acid pHo. Our data, therefore, strongly suggest that a cycle of protonation/deprotonation of pHo-sensing arginine 224 side chain gates the TASK-2 channel by electrostatically tuning the conformational stability of its selectivity filter.

Immunocytochemical localization of TASK-3 (K(2P)9.1) channels in monoaminergic and cholinergic neurons.

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(2P) channels. Here we describe the generation and characterization of a polyclonal monospecific antibody against rat TASK-3, a major brain K(2P) 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.

TREK‐1 K+ Channels in the Cardiovascular System: Their Significance and Potential as a Therapeutic Target

Potassium (K(+) ) channels are important in cardiovascular disease both as drug targets and as a cause of underlying pathology. Voltage-dependent K(+) (K(V) ) channels are inhibited by the class III antiarrhythmic agents. Certain vasodilators work by opening K(+) channels in vascular smooth muscle cells (VSMCs), and K(+) channel activation may also be a route to improving endothelial function. The two-pore domain K(+) (K(2P) ) channels form a group of 15 known channels with an expanding list of functions in the cardiovascular system. One of these K(2P) channels, TREK-1, is the focus of this review. TREK-1 channel activity is tightly regulated by intracellular and extracellular pH, membrane stretch, polyunsaturated fatty acids (PUFAs), temperature, and receptor-coupled second messenger systems. TREK-1 channels are also activated by volatile anesthetics and some neuroprotectant agents, and they are inhibited by selective serotonin reuptake inhibitors (SSRIs) as well as amide local anesthetics. Some of the clinical cardiovascular effects and side effects of these drugs may be through their actions on TREK-1 channels. It has recently been suggested that TREK-1 channels have a role in mechano-electrical coupling in the heart. They also seem important in the vascular responses to PUFAs, and this may underlie some of the beneficial cardiovascular effects of the essential dietary fatty acids. Development of selective TREK-1 openers and inhibitors may provide promising routes for intervention in cardiovascular diseases.

A MICROSCOPIC CLUSTER MODEL STUDY OF 3He + p SCATTERINGS

3 He + p scattering phase shifts for the S- and P-waves are studied in a microscopic cluster model in order to investigate the role of the d + 2p channel in the low-energy phase shifts. In the present cluster model, the description of the 3 He wave function is extended from a simple (0s)3 model to a three-body model and two different nucleon-nucleon interactions, the Minnesota and AV8' potentials, are employed. The present extended cluster model shows that the d + 2p channel is indispensable to reproduce the resonant phase shifts in the AV8' potential while it plays a minor role in the MN potential. On the contrary, the role of this channel in the S-wave non-resonant phase shifts is negligible in both potentials.

The Membrane-Bound State of K2P Potassium Channels

Potassium channel subunits composed of two-pore domains arranged in tandem (K(2P)) are of paramount importance for neural function. A variety of stimuli, such as membrane depolarization and tension, acidification, and anesthetic action, activate K(2P) channels. Most of the channel sensitivity is attributed to its intracellular C-terminal moiety, which works as a sensor domain required for proper integration of the electrical, chemical, and mechanical signals into channel activity. Herein, the structure of K(2P) in a membrane environment has been studied using molecular dynamics (MD). Two distinct fully atomistic models for the most studied K(2P) channel, namely, the TWIK-related (TREK)-1 channel have been built. These constructs were then inserted into a fully hydrated zwitterionic lipid bilayer, and each relaxed by means of MD simulations spanning approximately 0.3 micros. Both simulated TREK-1 structures converged to a final conformation characterized by a closed pore and a C-terminal domain adsorbed onto the lipid bilayer surface. The C-terminus, which is physically linked to the pore and energetically coupled to the bilayer, is poised to gate the channel in response to membrane stimulation. The present study indicates the nature of the direct coupling between the C-terminal domain and the membrane, which is a key structural feature underlying K(2P) channel function.

Enhancement of TREK1 channel surface expression by protein–protein interaction with β-COP

TREK1 belongs to a family of two-pore-domain K + (K 2P) channels and produce background currents that regulate cell excitability. In the present study, we identified a vesicle transport protein, β-COP, as an interacting partner by yeast two-hybrid screening of a human brain cDNA library with N-terminal region of TREK1 (TREK1-N) as bait. Several in vitro and in vivo binding assays confirmed the protein–protein interaction between TREK1 and β-COP. We also found that β-COP was associated with TREK1 in native condition at the PC3 cells. When RFP-β-COP was co-transfected with GFP-TREK1 into COS-7 cells, both proteins were found localized to the plasma membrane. In addition, the channel activity and surface expression of GFP-TREK1 increased dramatically by co-transfection with RFP-β-COP. Surface expression of the TREK1 channel was also clearly reduced with the addition of β-COP-specific shRNA. Collectively, these data suggest that β-COP plays a critical role in the forward transport of TREK1 channel to the plasma membrane.

Two-pore potassium ion channels are inhibited by both G q/11- and G i-coupled P2Y receptors

Two-pore potassium (K 2P) ion channels and P2Y receptors modulate the activity of neurones and are targets for the treatment of neuronal disorders. Here we have characterised their interaction. In cells coexpressing the Gα i-coupled hP2Y 12 receptor, ADP and ATP significantly inhibited hK 2P2.1 currents. This was abolished by pertussis toxin (PTX), the hP2Y 12 antagonist AR-C69931MX, the hP2Y 1 antagonist MRS2179 and by mutating potential PKA/PKC phosphorylation sites in the channel C terminal. In cells coexpressing the Gα q/11-coupled hP2Y 1 receptor, ADP and ATP also inhibited hK 2P2.1 currents, which were abolished by MRS2179, but unaffected by AR-C69931MX and PTX. When both receptors were coexpressed with K 2P2.1 channels, ADP-induced inhibition was antagonised by AR-C69913MX and MRS2179, but not PTX. Thus, both Gα q/11- and Gα i-coupled P2Y receptors inhibit K 2P channels and the action of hP2Y 12 receptors appears to involve co-activation of endogenous hP2Y 1 receptors. This represents a novel mechanism by which P2Y receptors may modulate neuronal activity.

Molecular mechanisms underlying membrane-potential-mediated regulation of neuronal K 2P2.1 channels

The activity of background K 2P channels adjusts the resting membrane potential to enable plasticity of excitable cells. Here we have studied the regulation of neuronal K 2P2.1 (KCNK2, TREK-1) channel activity by resting membrane potential. When heterologously expressed, K 2P2.1 currents gradually increased at hyperpolarizing potentials and declined at depolarizing potentials, with a midpoint potential of − 60 mV. As K 2P channels are not equipped with an integral voltage sensor, we sought extrinsic cellular components that could convert changes in the membrane electrical field to cellular activity that would indirectly modify K 2P2.1 currents. We propose that membrane depolarization activated the Gq protein-coupled receptor pathway, in the apparent absence of ligand, resulting in phosphatidylinositol-4,5-bisphosphate (PIP 2) depletion through the action of phospholipase C. Our results suggest a novel mechanism in which an indirect pathway confers membrane potential regulation onto channels that are not intrinsically voltage sensitive to enhance regulation of neuronal excitability levels.

The generalized oscillator strengths for Na: monopole, dipole and quadrupole transitions

The generalized oscillator strengths (GOSs) of Na from the ground state [2p63s]32S excited to [2p6(n + 1)s](n + 1)2S, [2p6np]n2P, [2p6nd]n2D(3 ⩽ n ⩽ ∞) and adjacent continuum states are systematically investigated by the R-matrix method within the first Born approximation. The calculated energy levels of Na+ and Na are in good agreement with the experimental values. The energy dependence of the GOS extrema for the monopole, dipole and quadrupole transitions is investigated. The GOS density surfaces of 2S, 2P and 2D channels are calculated and their accuracies are tested rigorously by the available experiments as well as by the comparisons between the calculations on the different bases and the sum rule. With the surfaces tested, the high-energy electron impact excitation cross sections of the infinite Rydberg states near the threshold can be readily obtained by interpolations. In addition, the GOSDs for the resonance transitions 2p63s → 2p53s2 and 2p63s → 2p53s3p are investigated.

Regional expression of the anesthetic-activated potassium channel TRESK in the rat nervous system

The two-pore-domain potassium (K 2P) channels contribute to background (leak) potassium currents maintaining the resting membrane potential to play an important role in regulating neuronal excitability. As such they may contribute to nociception and the mechanism of action of volatile anesthetics. In the present study, we examined the protein expression pattern of the K 2P channel TRESK in the rat central nervous system (CNS) and peripheral nervous system (PNS) by immunohistochemistry. The regional distribution expression pattern of TRESK has both similarities and significant differences from that of other K 2P channels expressed in the CNS. TRESK expression is broadly found in the brain, spinal cord and dorsal root ganglia (DRG). TRESK expression is highest in important CNS structures, such as specific cortical layers, periaqueductal gray (PAG), granule cell layer of the cerebellum, and dorsal horn of the spinal cord. TRESK expression is also high in small and medium sized DRG neurons. These results provide an anatomic basis for identifying functional roles of TRESK in the rat nervous system.

Gating the pore of potassium leak channels

A key feature of potassium channel function is the ability to switch between conducting and non-conducting states by undergoing conformational changes in response to cellular or extracellular signals. Such switching is facilitated by the mechanical coupling of gating domain movements to pore opening and closing. Two-pore domain potassium channels (K(2P)) conduct leak or background potassium-selective currents that are mostly time- and voltage-independent. These channels play a significant role in setting the cell resting membrane potential and, therefore modulate cell responsiveness and excitability. Thus, K(2P) channels are key players in numerous physiological processes and were recently shown to also be involved in human pathologies. It is well established that K(2P) channel conductance, open probability and cell surface expression are significantly modulated by various physical and chemical stimuli. However, in understanding how such signals are translated into conformational changes that open or close the channels gate, there remain more open questions than answers. A growing line of evidence suggests that the outer pore area assumes a critical role in gating K(2P) channels, in a manner reminiscent of C-type inactivation of voltage-gated potassium channels. In some K(2P) channels, this gating mechanism is facilitated in response to external pH levels. Recently, it was suggested that K(2P) channels also possess a lower activation gate that is positively coupled to the outer pore gate. The purpose of this review is to present an up-to-date summary of research describing the conformational changes and gating events that take place at the K(2P) channel ion-conducting pathway during the channel regulation.

Inverse coupling in leak and voltage-activated K+ channel gates underlies distinct roles in electrical signaling

Voltage-activated (Kv) and leak (K(2P)) K(+) channels have key, yet distinct, roles in electrical signaling in the nervous system. Here we examine how differences in the operation of the activation and slow inactivation pore gates of Kv and K(2P) channels underlie their unique roles in electrical signaling. We report that (i) leak K(+) channels possess a lower activation gate, (ii) the activation gate is an important determinant controlling the conformational stability of the K(+) channel pore, (iii) the lower activation and upper slow inactivation gates of leak channels cross-talk and (iv) unlike Kv channels, where the two gates are negatively coupled, these two gates are positively coupled in K(2P) channels. Our results demonstrate how basic thermodynamic properties of the K(+) channel pore, particularly conformational stability and coupling between gates, underlie the specialized roles of Kv and K(2P) channel families in electrical signaling.

Thrilling Moment of an Inhibitory Channel

Long thought to be potassium channels with calming influence quietly working away in the background, in this issue of Neuron, Thomas et al. demonstrate that K(2P)2.1 (TREK-1) channels have two endogenous isoforms due to a mechanism called alternative translation initiation (ATI). Hence, they can actually be excitable.

Alternative Translation Initiation in Rat Brain Yields K2P2.1 Potassium Channels Permeable to Sodium

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.

Knockout of the gene encoding the K 2P channel KCNK7 does not alter volatile anesthetic sensitivity

The molecular site of action for volatile anesthetics remains unknown despite many years of study. Members of the K 2P potassium channel family, whose currents are potentiated by volatile anesthetics have emerged as possible anesthetic targets. In fact, a mouse model in which the gene for TREK-1 (KCNK2) has been inactivated shows resistance to volatile anesthetics. In this study we tested whether inactivation of another member of this ion channel family, KCNK7, in a knockout mouse displayed altered sensitivity to the anesthetizing effect of volatile anesthetics. KCNK7 knockout mice were produced by standard gene inactivation methods. Heterozygous breeding pairs produced animals that were homozygous, heterozygous or wild-type for the inactivated gene. Knockout animals were tested for movement in response to noxious stimulus (tail clamp) under varying concentrations of isoflurane, halothane, and desflurane to define the minimum alveolar concentration (MAC) preventing movement. Mice homozygous for inactivated KCNK7 were viable and indistinguishable in weight, general development and behavior from heterozygotes or wild-type littermates. Knockout mice (KCNK7−/−) displayed no difference in MAC for the three volatile anesthetics compared to heterozygous (+/−) or wild-type (+/+) littermates. Because inactivation of KCNK7 does not alter MAC, KCNK7 may play only a minor role in normal CNS function or may have had its function compensated for by other inhibitory mechanisms. Additional studies with transgenic animals will help define the overall role of the K 2P channels in normal neurophysiology and in volatile anesthetic mechanisms.