Pore Domain Potassium Channel Research Articles

The TREK-1 potassium channel is a potential pharmacological target for vasorelaxation in pulmonary hypertension.

Pulmonary arterial hypertension (PAH) is a progressive disease in which chronic membrane potential (Em) depolarisation of the pulmonary arterial smooth muscle cells (PASMCs) causes calcium overload, a key pathological alteration. Under resting conditions, the negative Em is mainly set by two pore domain potassium (K2P) channels, of which the TASK-1 has been extensively investigated. Ion channel currents and membrane potential of primary cultured human(h) PASMCs were measured using the voltage- and current clamp methods. Intracellular [Ca2+] was monitored using fluorescent microscopy. Pulmonary BP and vascular tone measurements were also performed ex vivo using a rat PAH model. TREK-1 was the most abundantly expressed K2P in hPASMCs of healthy donors and idiopathic(I) PAH patients. Background K+-current was similar in hPASMCs for both groups and significantly enhanced by the TREK activator ML-335. In donor hPASMCs, siRNA silencing or pharmacological inhibition of TREK-1 caused depolarisation, reminiscent of the electrophysiological phenotype of idiopathic PAH. ML-335 hyperpolarised donor hPASMCs and normalised the Em of IPAH hPASMCs. A close link was found between TREK-1 activity and intracellular Ca2+-signalling using a channel activator, ML-335, and an inhibitor, spadin. In the rat, ML-335 relaxed isolated pre-constricted pulmonary arteries and significantly decreased pulmonary arterial pressure in the isolated perfused lung. These data suggest that TREK-1is a key factor in Em setting and Ca2+ homeostasis of hPASMC, and therefore, essential for maintenance of a low resting pulmonary vascular tone. Thus TREK-1 may represent a new therapeutic target for PAH.

Membrane phospholipids control gating of the mechanosensitive potassium leak channel TREK1

Tandem pore domain (K2P) potassium channels modulate resting membrane potentials and shape cellular excitability. For the mechanosensitive subfamily of K2Ps, the composition of phospholipids within the bilayer strongly influences channel activity. To examine the molecular details of K2P lipid modulation, we solved cryo-EM structures of the TREK1 K2P channel bound to either the anionic lipid phosphatidic acid (PA) or the zwitterionic lipid phosphatidylethanolamine (PE). At the extracellular face of TREK1, a PA lipid inserts its hydrocarbon tail into a pocket behind the selectivity filter, causing a structural rearrangement that recapitulates mutations and pharmacology known to activate TREK1. At the cytoplasmic face, PA and PE lipids compete to modulate the conformation of the TREK1 TM4 gating helix. Our findings demonstrate two distinct pathways by which anionic lipids enhance TREK1 activity and provide a framework for a model that integrates lipid gating with the effects of other mechanosensitive K2P modulators.

Tandem pore domain acid-sensitive K channel 3 (TASK-3) regulates visual sensitivity in healthy and aging retina.

Retinal ganglion cells (RGCs) not only collect but also integrate visual signals and send them from the retina to the brain. The mechanisms underlying the RGC integration of synaptic activity within retinal circuits have not been fully explored. Here, we identified a pronounced expression of tandem pore domain acid-sensitive potassium channel 3 (TASK-3), a two–pore domain potassium channel (K2P), in RGCs. By using a specific antagonist and TASK-3 knockout mice, we found that TASK-3 regulates the intrinsic excitability and the light sensitivity of RGCs by sensing neuronal activity–dependent extracellular acidification. In vivo, the blockade or loss of TASK-3 dampened pupillary light reflex, visual acuity, and contrast sensitivity. Furthermore, overexpressing TASK-3 specifically in RGCs using an adeno-associated virus approach restored the visual function of TASK-3 knockout mice and aged mice where the expression and function of TASK-3 were reduced. Thus, our results provide evidence that implicates a critical role of K2P in visual processing in the retina.

A Direct Interaction between Cyclodextrins and TASK Channels Decreases the Leak Current in Cerebellar Granule Neurons.

Simple SummaryCyclodextrins are cyclic oligosaccharides used to deplete cholesterol from cellular membranes. The effects of methyl-β-cyclodextrin (MβCD) on cellular functions originate principally from reductions in cholesterol levels. In this study, using immunocytochemistry, heterologous expression of K2P channels, and cholesterol-depleting maneuvers, we provide evidence of expression in cultured rat cerebellar granule neurons (CGNs) of TWIK-1 (K2P1), TASK-1 (K2P3), TASK-3 (K2P9), and TRESK (K2P18) channels and their association with lipid rafts using the specific lipids raft markers. In addition, we show a direct blocking with MβCD of TASK-1 and TASK-3 channels as well as for the covalently concatenated heterodimer TASK-1/TASK-3.Two pore domain potassium channels (K2P) are strongly expressed in the nervous system (CNS), where they play a central role in excitability. These channels give rise to background K+ currents, also known as IKSO (standing-outward potassium current). We detected the expression in primary cultured cerebellar granule neurons (CGNs) of TWIK-1 (K2P1), TASK-1 (K2P3), TASK-3 (K2P9), and TRESK (K2P18) channels by immunocytochemistry and their association with lipid rafts using the specific lipids raft markers flotillin-2 and caveolin-1. At the functional level, methyl-β-cyclodextrin (MβCD, 5 mM) reduced IKSO currents by ~40% in CGN cells. To dissect out this effect, we heterologously expressed the human TWIK-1, TASK-1, TASK-3, and TRESK channels in HEK-293 cells. MβCD directly blocked TASK-1 and TASK-3 channels and the covalently concatenated heterodimer TASK-1/TASK-3 currents. Conversely, MβCD did not affect TWIK-1- and TRESK-mediated K+ currents. On the other hand, the cholesterol-depleting agent filipin III did not affect TASK-1/TASK-3 channels. Together, the results suggest that neuronal background K+ channels are associated to lipid raft environments whilst the functional activity is independent of the cholesterol membrane organization.

Inhibition of Potassium Channels Affects the Ability of Pig Spermatozoa to Elicit Capacitation and Trigger the Acrosome Exocytosis Induced by Progesterone.

During capacitation, sperm undergo a myriad of changes, including remodeling of plasma membrane, modification of sperm motility and kinematic parameters, membrane hyperpolarization, increase in intracellular calcium levels, and tyrosine phosphorylation of certain sperm proteins. While potassium channels have been reported to be crucial for capacitation of mouse and human sperm, their role in pigs has not been investigated. With this purpose, sperm samples from 15 boars were incubated in capacitation medium for 300 min with quinine, a general blocker of potassium channels (including voltage-gated potassium channels, calcium-activated potassium channels, and tandem pore domain potassium channels), and paxilline (PAX), a specific inhibitor of calcium-activated potassium channels. In all samples, acrosome exocytosis was induced after 240 min of incubation with progesterone. Plasma membrane and acrosome integrity, membrane lipid disorder, intracellular calcium levels, mitochondrial membrane potential, and total and progressive sperm motility were evaluated after 0, 120, and 240 min of incubation, and after 5, 30, and 60 min of progesterone addition. Although blocking potassium channels with quinine and PAX prevented sperm to elicit in vitro capacitation by impairing motility and mitochondrial function, as well as reducing intracellular calcium levels, the extent of that inhibition was larger with quinine than with PAX. Therefore, while our data support that calcium-activated potassium channels are essential for sperm capacitation in pigs, they also suggest that other potassium channels, such as the voltage-gated, tandem pore domain, and mitochondrial ATP-regulated ones, are involved in that process. Thus, further research is needed to elucidate the specific functions of these channels and the mechanisms underlying its regulation during sperm capacitation.

The Phosphodiesterase Inhibitor IBMX Blocks the Potassium Channel THIK-1 from the Extracellular Side

The two-pore domain potassium channel (K2P-channel) THIK-1 has several predicted protein kinase A (PKA) phosphorylation sites. In trying to elucidate whether THIK-1 is regulated via PKA, we expressed THIK-1 channels in a mammalian cell line (CHO cells) and used the phosphodiesterase inhibitor 3-isobutyl-1-methyl-xanthine (IBMX) as a pharmacological tool to induce activation of PKA. Using the whole-cell patch-clamp recording, we found that THIK-1 currents were inhibited by application of IBMX with an IC50 of 120 µM. Surprisingly, intracellular application of IBMX or of the second messenger cAMP via the patch pipette had no effect on THIK-1 currents. In contrast, extracellular application of IBMX produced a rapid and reversible inhibition of THIK-1. In patch-clamp experiments with outside-out patches, THIK-1 currents were also inhibited by extracellular application of IBMX. Expression of THIK-1 channels in Xenopus oocytes was used to compare wild-type channels with mutated channels. Mutation of the putative PKA phosphorylation sites did not change the inhibitory effect of IBMX on THIK-1 currents. Mutational analysis of all residues of the (extracellular) helical cap of THIK-1 showed that mutation of the arginine residue at position 92, which is in the linker between cap helix 2 and pore helix 1, markedly reduced the inhibitory effect of IBMX. This flexible linker region, which is unique for each K2P-channel subtype, may be a possible target of channel-specific blockers. SIGNIFICANCE STATEMENT: The potassium channel THIK-1 is strongly expressed in the central nervous system. We studied the effect of 3-isobutyl-1-methyl-xanthine (IBMX) on THIK-1 currents. IBMX inhibits breakdown of cAMP and thus activates protein kinase A (PKA). Surprisingly, THIK-1 current was inhibited when IBMX was applied from the extracellular side of the membrane, but not from the intracellular side. Our results suggest that IBMX binds directly to the channel and that the inhibition of THIK-1 current was not related to activation of PKA.

A lower X-gate in TASK channels traps inhibitors within the vestibule.

TWIK-related acid-sensitive potassium (TASK) channels-members of the two pore domain potassium (K2P) channel family-are found in neurons1, cardiomyocytes2-4 and vascular smooth muscle cells5, where they are involved in the regulation of heart rate6, pulmonary artery tone5,7, sleep/wake cycles8 and responses to volatile anaesthetics8-11. K2P channels regulate the resting membrane potential, providing background K+ currents controlled by numerous physiological stimuli12-15. Unlike other K2P 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 fibrillation16. 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 K2P 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 (243VLRFMT248) that are essential for responses to volatile anaesthetics10, neurotransmitters13 and G-protein-coupled receptors13. 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.

Heterodimerization of TALK Subunits

The potassium channels form a large and diverse family of ions channels. The 15 members that constitute the Two Pore domain Potassium channels (K2P) subfamily contribute to resting membrane potential and are involved in physiological functions such as depression, migraine, pain or neuronal protection. These K2P channels have been classified into six subfamilies. Most of these channels respond to a diverse regulatory inputs that include extracellular and intracellular pH, temperature, membrane stretch and noxious chemicals. Their membrane topology is distinguishable from the others K+ channels by the presence of two pore and 4 transmembrane domains per subunit. As a consequence of this singular topology, two subunits of K2P channels assemble to constitute a functional channel. Recently, we have shown that different subunits can organize as dimers among the same subfamily members. These heterodimers present different properties and regulations compared to the ones of each homomers. This heteromultimerization represent an alternative to the regulation of relative expression levels. Here we report the heterodimerization between members of TALK subfamily (TWIK-Related AlkalinepH-ActivatedK+channel): TALK-1, TALK-2 and TASK-2. Although, these channels belong to the same subfamily, they have different sensitivity to extracellular pH variations and differential subcellular distribution. TALK-2 is localized at the plasma membrane, whereas TALK-1 channel is mostly in the ER membrane and has been recently implicated in the control of intracellular Ca2+ homeostasis. TASK-2, for its part, is equally distributed to the membrane and in the ER. Using PLA in HEK293 and dominant negative assays in oocytes, we show that TALK-1, TALK-2 and TASK-2 subunits assemble and form active heterodimerics channels with dinstinctive electrophysiological and trafficking properties. Such results emphasize the complexity of this K2P channel family and the need to take into account the co-distribution of each subfamily members within the tissue of interest.

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.

N-glycosylation-dependent regulation of hK2P17.1 currents.

Two pore-domain potassium (K2P) channels mediate potassium background currents that stabilize the resting membrane potential and facilitate action potential repolarization. In the human heart, hK2P17.1 channels are predominantly expressed in the atria and Purkinje cells. Reduced atrial hK2P17.1 protein levels were described in patients with atrial fibrillation or heart failure. Genetic alterations in hK2P17.1 were associated with cardiac conduction disorders. Little is known about posttranslational modifications of hK2P17.1. Here, we characterized glycosylation of hK2P17.1 and investigated how glycosylation alters its surface expression and activity. Wild-type hK2P17.1 channels and channels lacking specific glycosylation sites were expressed in Xenopus laevis oocytes, HEK-293T cells, and HeLa cells. N-glycosylation was disrupted using N-glycosidase F and tunicamycin. hK2P17.1 expression and activity were assessed using immunoblot analysis and a two-electrode voltage clamp technique. Channel subunits of hK2P17.1 harbor two functional N-glycosylation sites at positions N65 and N94. In hemi-glycosylated hK2P17.1 channels, functionality and membrane trafficking remain preserved. Disruption of both N-glycosylation sites results in loss of hK2P17.1 currents, presumably caused by impaired surface expression. This study confirms diglycosylation of hK2P17.1 channel subunits and its pivotal role in cell-surface targeting. Our findings underline the functional relevance of N-glycosylation in biogenesis and membrane trafficking of ion channels.

Exploring the Hydrophobic Barrier of Human K2P Channel TWIK1 with Steered MD Simulations

We used steered molecular dynamics simulations to explore the hydrophobic barrier within the two pore domain potassium channel TWIK-1. This hydrophobic barrier is formed by Leucine 146 and Leucine 261 that lie below the selectivity filter on the cytoplasmic side of the protein. Experimental studies have shown that mutations of Leucine 146 and 261 allow more water to permeate to the selectivity filter and increase ion conduction (Nature Commun., 5, 4377, 2014). We use a collective variable (CV) that mimics tension by applying forces on lipids in the vicinity of the channel based on their proximity to explore the role of membrane interactions in modulating the hydrophobic barrier. Our group has studied the mechanosensitive channel of large conductance using a similar approach. By applying a harmonic potential to this CV, we observed different solvent accessibility to the selectivity filter. We found that TWIK-1 interacts strongly with lipids on the cytoplasmic side of the membrane through hydrogen bonding, which is higher compared to the periplasmic side. Positive amino acid residues Lysine (176, 275, 278) and Arginine (155, 162, 163, 171) alone contribute to one third of the hydrogen bond made by cytoplasmic side with protein. Furthermore, we explored the effects of mutating the hydrophobic L146 and L261 residues to Asparagine on solvent accessibility to the channel's selectivity filter and compare to the steered CV model.

The Involvement of Sortilin/NTSR3 in Depression as the Progenitor of Spadin and Its Role in the Membrane Expression of TREK-1.

The molecular identification of sortilin, also called neurotensin receptor-3, from three different biochemical approaches already predicted the involvement of the protein in numerous biological and cellular functions. The first important observation was that sortilin is synthesized as a precursor that is converted to a mature protein after cleavage by the protein convertase furin in late Golgi compartments. This maturation leads to the formation of a 44 amino acid peptide, the propeptide (PE). The release of this peptide when matured sortilin reached the plasma membrane remained to be demonstrated. Sortilin has been also shown to be shedded by matrix metalloproteases releasing a large extracellular fragment identified as soluble sortilin. Therefore, sortilin has been shown to interact with several proteins and receptors confirming its role in the sorting of cellular components to the plasma membrane and/or to the lysosomal pathway. Interestingly, sortilin physically interacts with the two pore domain potassium channel TREK-1 and the PE as well as its synthetic analog spadin is able to block the activation of TREK-1 highlighting their role in the depression pathology. The present review describes the advance of research that led to these results and how both the soluble form of sortilin and the sortilin-derived PE have been detected in human serum and whose levels are affected in patients with major depressive disorder (MDD). The use of spadin as an antidepressant and the further role of soluble sortilin and of sortilin-derived PE as potential biomarkers during depression statement and/or remission of the pathology are considered and discussed in this review.

Characterization and regulation of wild-type and mutant TASK-1 two pore domain potassium channels indicated in pulmonary arterial hypertension.

Key points The TASK‐1 channel gene (KCNK3) has been identified as a possible disease‐causing gene in heritable pulmonary arterial hypertension (PAH).In the present study, we show that novel mutated TASK‐1 channels, seen in PAH patients, have a substantially reduced current compared to wild‐type TASK‐1 channels.These mutated TASK‐1 channels are located at the plasma membrane to the same degree as wild‐type TASK‐1 channels.ONO‐RS‐082 and alkaline pH 8.4 both activate TASK‐1 channels but do not recover current through mutant TASK‐1 channels.We show that the guanylate cyclase activator, riociguat, a novel treatment for PAH, enhances current through TASK‐1 channels but does not recover current through mutant TASK‐1 channels. Pulmonary arterial hypertension (PAH) affects ∼15–50 people per million. KCNK3, the gene that encodes the two pore domain potassium channel TASK‐1 (K2P3.1), has been identified as a possible disease‐causing gene in heritable PAH. Recently, two new mutations have been identified in KCNK3 in PAH patients: G106R and L214R. The present study aimed to characterize the functional properties and regulation of wild‐type (WT) and mutated TASK‐1 channels and determine how these might contribute to PAH and its treatment. Currents through WT and mutated human TASK‐1 channels transiently expressed in tsA201 cells were measured using whole‐cell patch clamp electrophysiology. Localization of fluorescence‐tagged channels was visualized using confocal microscopy and quantified with in‐cell and on‐cell westerns. G106R or L214R mutated channels were located at the plasma membrane to the same degree as WT channels; however, their current was markedly reduced compared to WT TASK‐1 channels. Functional current through these mutated channels could not be restored using activators of WT TASK‐1 channels (pH 8.4, ONO‐RS‐082). The guanylate cyclase activator, riociguat, enhanced current through WT TASK‐1 channels; however, similar to the other activators investigated, riociguat did not have any effect on current through mutated TASK‐1 channels. Thus, novel mutations in TASK‐1 seen in PAH substantially alter the functional properties of these channels. Current through these channels could not be restored by activators of TASK‐1 channels. Riociguat enhancement of current through TASK‐1 channels could contribute to its therapeutic benefit in the treatment of PAH.

Identification and characterization of two zebrafish Twik related potassium channels, Kcnk2a and Kcnk2b

KCNK2 is a 2 pore domain potassium channel involved in maintaining cellular membrane resting potentials. Although KCNK2 is regarded as a mechanosensitive ion channel, it can also be gated chemically. Previous research indicates that KCNK2 expression is particularly enriched in neuronal and cardiac tissues. In this respect, KCNK2 plays an important role in neuroprotection and has also been linked to cardiac arrhythmias. KCNK2 has subsequently become an attractive pharmacologic target for developing preventative/curative strategies for neuro/cardio pathophysiological conditions. Zebrafish represent an important in vivo model for rapidly analysing pharmacological compounds. We therefore sought to identify and characterise zebrafish kcnk2 to allow this model system to be incorporated into therapeutic research. Our data indicates that zebrafish possess two kcnk2 orthologs, kcnk2a and kcnk2b. Electrophysiological analysis of both zebrafish Kcnk2 orthologs shows that, like their human counterparts, they are activated by different physiological stimuli such as mechanical stretch, polyunsaturated fatty acids and intracellular acidification. Furthermore, both zebrafish Kcnk2 channels are inhibited by the human KCNK2 inhibitory peptide spadin. Taken together, our results demonstrate that both Kcnk2a and Kcnk2b share similar biophysiological and pharmacological properties to human KCNK2 and indicate that the zebrafish will be a useful model for developing KCNK2 targeting strategies.

Patch clamp data driven stochastic modeling and simulation of hTREK1 potassium ion channel gating

Chemical reactions involving small number of molecules are noisy and are simulated using stochastic simulation algorithms, which are valid when the reaction environments are well mixed. However, biological media are crowded reaction environments where alternative simulation methods have to be used. Single molecule experiments have revealed non-markovian nature of biological reactions due to a phenomenon called dynamic disorder which makes the rate constants random. This happens when there are additional slow scale conformational transitions, giving the molecule a memory of its previous states. Traditionally, ion channel gating is modeled as Markovian transitions between fixed states. hTREK1 channel, a two pore domain potassium channel, has been shown to have long term memory in its kinetics under the influence of trichloroethanol, an anaesthetic. Here we show that lactate, a metabolite that increases in concentration during ischemia, is also able to induce memory in the channel. We have provided a simple diffusion model for its gating that assumes ion channel diffusion through a continuum of states on its potential energy landscape, derived from the steady state probability distribution of ionic current recorded from patch clamp experiments. A stochastic differential equation (SDE) driven by Gaussian white noise is proposed to model this motion in an asymmetric double well potential which can reproduce the amplitude histogram very well. Such cases where ligands like lactate are added, the SDE is modified by introducing an auxiliary variable to run on coloured noise such that increasing the noise correlation with ligand concentration improves the fits to experimental data. These methods unlike the Markovian models are true to the physical picture of gating and are able to efficiently reproduce the whole raw data trace over a few seconds’ interval.

Investigation of the structure activity relationship of flufenamic acid derivatives at the human TRESK channel K2P18.1

TRESK (Twik RElated Spinal cord K+ channel) is a member of the Twin Pore Domain potassium channel (K2P) family responsible for regulating neuronal excitability in dorsal root ganglion (DRG) and trigeminal (TG) neurons, peripheral neurons involved in pain transmission. As channel opening causes an outward K+ current responsible for cell hyperpolarisation, TRESK represents a potentially interesting target for pain treatment. However, as no crystal structure exists for this protein, the mechanisms involved in the opening action of its ligands are still poorly understood, making the development of new potent and selective openers challenging. In this work we present a structure activity relationship (SAR) of the known TRESK opener flufenamic acid (FFA) and some derivatives, investigating the functional effects of chemical modifications to build a TRESK homology model to support the biological results. A plausible binding mode is proposed, providing the first predictive hypothesis of a human TRESK opener binding site.

Complementary and synergistic therapeutic effects of compounds found in Kampo medicine: analysis of daikenchuto.

Herbal medicines have been used in Japan for more than 1500 years and traditional Japanese medicines (Kampo medicines) are now fully integrated into the modern healthcare system. In total, 148 Kampo formulae are officially approved as prescription drugs and covered by the national health insurance system in Japan. However, despite their long track record of clinical use, the multi-targeted, multi-component properties of Kampo medicines, which are fundamentally different from Western medicines, have made it difficult to create a suitable framework for conducting well-designed, large-scale clinical trials. In turn, this has led to misconceptions among western trained physicians concerning the paucity of scientific evidence for the beneficial effects of Kampo medicines. Fortunately, there has been a recent surge in scientifically robust data from basic and clinical studies for some of the Kampo medicines, e.g., daikenchuto (TU-100). Numerous basic and clinical studies on TU-100, including placebo-controlled double-blind studies for various gastrointestinal disorders, and absorption, distribution, metabolism and excretion (ADME) studies, have been conducted or are in the process of being conducted in both Japan and the USA. Clinical studies suggest that TU-100 is beneficial for postoperative complications, especially ileus and abdominal bloating. ADME and basic studies indicate that the effect of TU-100 is a composite of numerous actions mediated by multiple compounds supplied via multiple routes. In addition to known mechanisms of action via enteric/sensory nerve stimulation, novel mechanisms via the TRPA1 channel and two pore domain potassium channels have recently been elucidated. TU-100 compounds target these channels with and without absorption, both before and after metabolic activation by enteric flora, with different timings and possibly with synergism.

Understanding the Dynamics of K2P Channels in Complex Lipid Bilayers

Ion channels are proteins that reside in phospholipid bilayers and conduct ions through their trans-membrane pore. Studying the dynamics of interaction between the channel, phospholipids, water and ions is fundamental to understanding ion channel function. We are using a combination of molecular dynamics simulations and functional tools to gain insight into K2P (Two Pore-domain Potassium Channel) function. By initially observing wetting and dewetting transitions in the inner pore of TWIK-1 (K2P1) channels during MD simulations and by subsequently combining in silico and in vitro mutagenesis and electrophysiology, we have found a hydrophobic barrier deep within the inner pore of the TWIK-1 channel [1]. Our study suggests that this barrier contributes to the very low level of functional currents observed for TWIK-1 channels. We have also reviewed the computational, structural and functional evidence for hydrophobic gating in several ion channel families and propose that understanding the dynamic behavior of water and ions within the pore represents an increasingly important element in understanding the relationship between ion channel structure and function [2]. We are now examining the interaction between K2P channels and phospholipids in more detail. Using MD simulations, we find hot spots for K2P channel and lipid interactions. These findings suggest that lipids can play modulatory roles in K2P channel function.[1] Aryal P, Abd-Wahab F, Bucci G, Sansom MSP & Tucker SJ. A hydrophobic barrier deep within the inner pore of the TWIK-1 K2P potassium channel. Nature Communications 5:4377 (2014) [http://dx.doi.org/10.1038/ncomms5377][2] Aryal P, Sansom MSP and Tucker SJ. Hydrophobic Gating in Ion Channels. Journal of Molecular Biology (2014) [http://dx.doi.org/10.1016/j.jmb.2014.07.030]

KCNK10, a tandem pore domain potassium channel, is a regulator of mitotic clonal expansion during the early stage of adipocyte differentiation.

KCNK10, a member of tandem pore domain potassium channel family, gives rise to leak K+ currents. It plays important roles in stabilizing the negative resting membrane potential and in counterbalancing depolarization. We previously demonstrated that kcnk10 expression is quickly elevated during the early stage of adipogenesis of 3T3-L1 cells and that reduction of kcnk10 expression inhibits adipocyte differentiation. However, the molecular mechanism of KCNK10 in adipocyte differentiation remains unclear. Here we revealed that kcnk10 is induced by 3-isobutyl-1-methylxanthine, a cyclic nucleotide phosphodiesterase inhibitor and a potent inducer of adipogenesis, during the early stage of adipocyte differentiation. We also demonstrated that KCNK10 functions as a positive regulator of mitotic clonal expansion (MCE), a necessary process for terminal differentiation. The reduction of kcnk10 expression repressed the expression levels of CCAAT/enhancer-binding protein β (C/EBPβ) and C/EBPδ as well as the phosphorylation level of Akt during the early phase of adipogenesis. In addition, knockdown of kcnk10 expression suppressed insulin-induced Akt phosphorylation. These results indicate that KCNK10 contributes to the regulation of MCE through the control of C/EBPβ and C/EBPδ expression and insulin signaling.

Molecular modeling of a tandem two pore domain potassium channel reveals a putative binding site for general anesthetics.

Anesthetics are thought to mediate a portion of their activity via binding to and modulation of potassium channels. In particular, tandem pore potassium channels (K2P) are transmembrane ion channels whose current is modulated by the presence of general anesthetics and whose genetic absence has been shown to confer a level of anesthetic resistance. While the exact molecular structure of all K2P forms remains unknown, significant progress has been made toward understanding their structure and interactions with anesthetics via the methods of molecular modeling, coupled with the recently released higher resolution structures of homologous potassium channels to act as templates. Such models reveal the convergence of amino acid regions that are known to modulate anesthetic activity onto a common three- dimensional cavity that forms a putative anesthetic binding site. The model successfully predicts additional important residues that are also involved in the putative binding site as validated by the results of suggested experimental mutations. Such a model can now be used to further predict other amino acid residues that may be intimately involved in the target-based structure–activity relationships that are necessary for anesthetic binding.