P1601N-glycosylation of TREK-1/hK2P2.1 two-pore-domain (K2P) potassium channels

2019 ◽  
Vol 40 (Supplement_1) ◽  
Author(s):  
F Wiedmann ◽  
D Schlund ◽  
A Ratte ◽  
H A Katus ◽  
M Kraft ◽  
...  
Keyword(s):  
Potassium Channels ◽  
Posttranslational Modifications ◽  
Enzymatic Digestion ◽  
Resting Membrane Potential ◽  
Xenopus Laevis Oocytes ◽  
Channel Function ◽  
Excitable Tissue ◽  
Electrode Voltage ◽  
Channel Surface ◽  
Pore Domain

Abstract Background and purpose Mechanosensitive hTREK-1 (hK2P2.1) two-pore-domain potassium channels give rise to background currents that control resting membrane potential in excitable tissue. Recently TREK-1 currents have been linked to regulation of cardiac rhythm as well as hypertrophy and fibrosis. Even though pharmacological and biophysical characteristics of hTREK-1 channels have been widely studied, less is known about its posttranslational modifications. This study aims to evaluate whether hTREK-1 channels are N-glycosylated and whether glycosylation may affect channel functionality. Experimental approach Following pharmacological inhibition of N glycosylation, enzymatic digestion or mutagenesis, immunoblots of Xenopus laevis oocytes and HEK-233T cell lysates were used to assess electrophoretic mobility. Two-electrode voltage clamp measurements were employed to study channel function. Key results TREK-1 channels subunits undergo N-glycosylation at asparagine residues 110 and 134. The presence of sugar moieties at these two sites increases channel function. Detection of glycosylation-deficient mutant channels in surface fractions and recordings of macroscopic potassium currents mediated by these subunits demonstrate that non-glycosylated hTREK-1 channels subunits are able to reach the cell surface in general, but seemingly with reduced efficiency. Conclusion and implications hTREK-1 are glycoproteins and N glycosylation at positions 110 and 134 is involved in channel surface trafficking. These findings extend our view on regulation of hTREK-1 currents by posttranslational modifications and provide novel insights into how glycosylation deficiency disorders may promote arrhythmogenesis.

scholarly journals N-Glycosylation of TREK-1/hK2P2.1 Two-Pore-Domain Potassium (K2P) Channels

2019 ◽  
Vol 20 (20) ◽  
pp. 5193 ◽  
Author(s):  
Felix Wiedmann ◽  
Daniel Schlund ◽  
Francisco Faustino ◽  
Manuel Kraft ◽  
Antonius Ratte ◽  
...  
Keyword(s):  
Posttranslational Modifications ◽  
Cardiac Rhythm ◽  
293T Cell ◽  
Enzymatic Digestion ◽  
Xenopus Laevis Oocytes ◽  
Channel Function ◽  
Cellular Excitability ◽  
K2p Channels ◽  
Electrode Voltage ◽  
Pore Domain

Mechanosensitive hTREK-1 two-pore-domain potassium (hK2P2.1) channels give rise to background currents that control cellular excitability. Recently, TREK-1 currents have been linked to the regulation of cardiac rhythm as well as to hypertrophy and fibrosis. Even though the pharmacological and biophysical characteristics of hTREK-1 channels have been widely studied, relatively little is known about their posttranslational modifications. This study aimed to evaluate whether hTREK-1 channels are N-glycosylated and whether glycosylation may affect channel functionality. Following pharmacological inhibition of N-glycosylation, enzymatic digestion or mutagenesis, immunoblots of Xenopus laevis oocytes and HEK-293T cell lysates were used to assess electrophoretic mobility. Two-electrode voltage clamp measurements were employed to study channel function. TREK-1 channel subunits undergo N-glycosylation at asparagine residues 110 and 134. The presence of sugar moieties at these two sites increases channel function. Detection of glycosylation-deficient mutant channels in surface fractions and recordings of macroscopic potassium currents mediated by these subunits demonstrated that nonglycosylated hTREK-1 channel subunits are able to reach the cell surface in general but with seemingly reduced efficiency compared to glycosylated subunits. These findings extend our understanding of the regulation of hTREK-1 currents by posttranslational modifications and provide novel insights into how altered ion channel glycosylation may promote arrhythmogenesis.

Local Anesthetic Inhibition of Baseline Potassium Channels with Two Pore Domains in Tandem 

1999 ◽  
Vol 90 (4) ◽  
pp. 1092-1102 ◽  
Author(s):  
Christoph H. Kindler ◽  
Spencer C. Yost ◽  
Andrew T. Gray
Keyword(s):  
Membrane Potential ◽  
Potassium Channels ◽  
Local Anesthetic ◽  
Local Anesthetics ◽  
Resting Membrane Potential ◽  
Electrode Voltage ◽  
Primary Amino ◽  
Anesthetic Action ◽  
Pore Domain ◽  
Pore Domains

Background Recently, a new structural family of potassium channels characterized by two pore domains in tandem within their primary amino acid sequence was identified. These tandem pore domain potassium channels are not gated by voltage and appear to be involved in the control of baseline membrane conductances. The goal of this study was to identify mechanisms of local anesthetic action on these channels. Methods Oocytes of Xenopus laevis were injected with cRNA from five cloned tandem pore domain baseline potassium channels (TASK, TREK-1, TOK1, ORK1, and TWIK-1), and the effects of several local anesthetics on the heterologously expressed channels were assayed using two-electrode voltage-clamp and current-clamp techniques. Results Bupivacaine (1 mM) inhibited all studied tandem pore potassium channels, with TASK inhibited most potently. The potency of inhibition was directly correlated with the octanol: buffer distribution coefficient of the local anesthetic, with the exception of tetracaine, to which TASK is relatively insensitive. The approximate 50% inhibitory concentrations of TASK were 709 microM mepivacaine, 222 microM lidocaine, 51 microM R(+)-ropivacaine, 53 microM S(-)-ropivacaine, 668 microM tetracaine, 41 microM bupivacaine, and 39 microM etidocaine. Local anesthetics (1 mM) significantly depolarized the resting membrane potential of TASK cRNA-injected oocytes compared with saline-injected control oocytes (tetracaine 22+/-6 mV rs. 7+/-1 mV, respectively, and bupivacaine 31+/-7 mV vs. 6+/-4 mV). Conclusions Local anesthetics inhibit tandem pore domain baseline potassium channels, and they could depolarize the resting membrane potential of cells expressing these channels. Whether inhibition of these channels contributes to conduction blockade or to the adverse effects of local anesthetics remains to be determined.

scholarly journals Structures of the human two-pore domain potassium channels TREK-1 and TREK-2

2014 ◽  
Vol 70 (a1) ◽  
pp. C1489-C1489
Author(s):  
Ashley Pike ◽  
Yin Dong ◽  
Alexandra Mackenzie ◽  
Conor McClenaghan ◽  
Shubhashish Mukhopadhyay ◽  
...  
Keyword(s):  
Potassium Channels ◽  
Channel Gating ◽  
Resting Membrane Potential ◽  
Transmembrane Helices ◽  
Ion Conductance ◽  
K2p Channels ◽  
Extracellular Stimuli ◽  
Structural Mechanisms ◽  
Pore Domain ◽  
Pore Lining

TREK-1/2 are members of the mechano-gated subfamily of two-pore (K2P) domain potassium channels leaking K+ out of the cell and contributing to the resting membrane potential. In contrast to the classical tetrameric potassium channels, K2P channels are dimeric with an atypical architecture and the structural mechanisms underlying their channel gating are poorly understood. Here we present the crystal structures of human TREK-1 and TREK-2 at resolutions of 2.7 and 3.4Å which provide insights into the basis of intracellular and extracellular gating in this unique family of ion channels. We have solved the structure of TREK-2 in two distinct conformations differing in the orientation of the pore-lining transmembrane helices. The C-terminal M4 helix is hinged at a conserved glycine residue so that it adopts one of two distinct orientations. The M4 helix is either kinked towards the membrane, packing against the M2 inner helix of the adjacent subunit ("M4 up") or straightens and interacts with the M2/M3 helices from the same subunit ("M4 down"). In the M4 down state, a hydrophobic lateral opening runs perpendicular to the ion conductance pathway between M2 and M4 and links the inner vestibule to the membrane-exposed face of the channel. Transition between the "M4 down" and "M4 up" conformations may play a role in channel activation and gating. Cocrystallisation with a TREK-1/2 channel inhibitor promotes the "M4 down" state. The structure of TREK-1 exhibits an "M4-up" conformation but is unusual in that the selectivity filter is significantly distorted with only two correctly-formed potassium sites. The structure also reveals a divalent ion binding site between the extracellular cap and the pore domain loop. The TREK-1 structure illustrates how changes at an extracellular site can affect the pore structure. The structures will be described in detail along with their implications for channel gating in response to intracellular and extracellular stimuli.

scholarly journals N-glycosylation–dependent regulation of hK2P17.1 currents

2019 ◽  
Vol 30 (12) ◽  
pp. 1425-1436 ◽  
Author(s):  
Felix Wiedmann ◽  
Daniel Schlund ◽  
Niels Voigt ◽  
Antonius Ratte ◽  
Manuel Kraft ◽  
...  
Keyword(s):  
Membrane Trafficking ◽  
Posttranslational Modifications ◽  
Immunoblot Analysis ◽  
Surface Expression ◽  
Genetic Alterations ◽  
Cardiac Conduction ◽  
Resting Membrane Potential ◽  
Xenopus Laevis Oocytes ◽  
Glycosylation Sites ◽  
Expression And Activity

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.

scholarly journals Retigabine holds KV7 channels open and stabilizes the resting potential

2016 ◽  
Vol 147 (3) ◽  
pp. 229-241 ◽  
Author(s):  
Aaron Corbin-Leftwich ◽  
Sayeed M. Mossadeq ◽  
Junghoon Ha ◽  
Iwona Ruchala ◽  
Audrey Han Ngoc Le ◽  
...  
Keyword(s):  
Voltage Dependence ◽  
Resting Potential ◽  
Resting Membrane Potential ◽  
Xenopus Laevis Oocytes ◽  
Channel Activity ◽  
Pore Region ◽  
Threshold Potential ◽  
Kv7 Channels ◽  
Open States ◽  
Pore Domain

The anticonvulsant Retigabine is a KV7 channel agonist used to treat hyperexcitability disorders in humans. Retigabine shifts the voltage dependence for activation of the heteromeric KV7.2/KV7.3 channel to more negative potentials, thus facilitating activation. Although the molecular mechanism underlying Retigabine’s action remains unknown, previous studies have identified the pore region of KV7 channels as the drug’s target. This suggested that the Retigabine-induced shift in voltage dependence likely derives from the stabilization of the pore domain in an open (conducting) conformation. Testing this idea, we show that the heteromeric KV7.2/KV7.3 channel has at least two open states, which we named O1 and O2, with O2 being more stable. The O1 state was reached after short membrane depolarizations, whereas O2 was reached after prolonged depolarization or during steady state at the typical neuronal resting potentials. We also found that activation and deactivation seem to follow distinct pathways, suggesting that the KV7.2/KV7.3 channel activity displays hysteresis. As for the action of Retigabine, we discovered that this agonist discriminates between open states, preferentially acting on the O2 state and further stabilizing it. Based on these findings, we proposed a novel mechanism for the therapeutic effect of Retigabine whereby this drug reduces excitability by enhancing the resting potential open state stability of KV7.2/KV7.3 channels. To address this hypothesis, we used a model for action potential (AP) in Xenopus laevis oocytes and found that the resting membrane potential became more negative as a function of Retigabine concentration, whereas the threshold potential for AP firing remained unaltered.

Volatile Anesthetics Activate the Human Tandem Pore Domain Baseline K+Channel KCNK5

2000 ◽  
Vol 92 (6) ◽  
pp. 1722-1730 ◽  
Author(s):  
Andrew T. Gray ◽  
Byron B. Zhao ◽  
Christoph H. Kindler ◽  
Bruce D. Winegar ◽  
Matthew J. Mazurek ◽  
...  
Keyword(s):  
Potassium Channels ◽  
Volatile Anesthetic ◽  
Volatile Anesthetics ◽  
Functional Expression ◽  
K Channel ◽  
Xenopus Laevis Oocytes ◽  
General Anesthetics ◽  
Neuronal Inhibition ◽  
Pore Domain

Background Previous studies have identified a volatile anesthetic-induced increase in baseline potassium permeability and concomitant neuronal inhibition. The emerging family of tandem pore domain potassium channels seems to function as baseline potassium channels in vivo. Therefore, we studied the effects of clinically used volatile anesthetics on a recently described member of this family. Methods A cDNA clone containing the coding sequence of KCNK5 was isolated from a human brain library. Expression of KCNK5 in the central nervous system was determined by Northern blot analysis and reverse-transcription polymerase chain reaction. Functional expression of the channel was achieved by injection of cRNA into Xenopus laevis oocytes. Results Expression of KCNK5 was detected in cerebral cortex, medulla, and spinal cord. When heterologously expressed in Xenopus oocytes, KCNK5 currents exhibited delayed activation, outward rectification, proton sensitivity, and modulation by protein kinase C. Clinical concentrations of volatile general anesthetics potentiated KCNK5 currents by 8-30%. Conclusion Human KCNK5 is a tandem pore domain potassium channel exhibiting delayed activation and sensitivity to volatile anesthetics and may therefore have a role in suppressing cellular excitability during general anesthesia.

scholarly journals A unique lower X-gate in TASK channels traps inhibitors within the vestibule

2019 ◽  
Author(s):  
Karin E. J. Rödström ◽  
Aytuğ K. Kiper ◽  
Wei Zhang ◽  
Susanne Rinné ◽  
Ashley C. W. Pike ◽  
...  
Keyword(s):  
Potassium Channels ◽  
Selectivity Filter ◽  
Resting Membrane Potential ◽  
Transmembrane Helices ◽  
Volatile Anaesthetics ◽  
Open Probability ◽  
High Affinity ◽  
Obstructive Sleep ◽  
Task Channels ◽  
Pore Domain

TASK channels are unusual members of the two-pore domain potassium (K2P) channel family, with unique and unexplained physiological and pharmacological characteristics. TASKs are found in neurons1,2, cardiomyocytes3–5 and vascular smooth muscle cells6 where they are involved in regulation of heart rate7, pulmonary artery tone6,8, sleep/wake cycles9 and responses to volatile anaesthetics9–12. K2P channels regulate the resting membrane potential, providing background K+ currents controlled by numerous physiological stimuli13,14. Unlike other K2P channels, TASK channels have the capacity to bind inhibitors with high affinity, exceptional selectivity and very slow compound washout rates. These characteristics make the TASK channels some of the the most easily druggable potassium channels, and indeed TASK-1 inhibitors are currently in clinical trials for obstructive sleep apnea (OSA) and atrial fibrillation (Afib)15 (The DOCTOS and SANDMAN Trials). Generally, potassium channels have an intramembrane vestibule with a selectivity filter above and a gate with four parallel helices below. However, K2P channels studied to date all lack a lower gate. Here we present the structure of TASK-1, revealing a unique lower gate created by interaction of the two crossed C-terminal M4 transmembrane helices at the vestibule entrance, which we designate as an ‟X-gate”. This structure is formed by six residues (V243LRFMT248) that are essential for responses to volatile anaesthetics11, neuro-transmitters16 and G-protein coupled receptors16. Interestingly, mutations within the X-gate and surrounding regions drastically affect both open probability and activation by anaesthetics. Structures of TASK-1 with two novel, high-affinity blockers, shows both inhibitors bound below the selectivity filter, trapped in the vestibule by the X-gate, thus explaining their exceptionally low wash-out rates. Thus, the presence of the X-gate in TASK channels explains many aspects of their unusual physiological and pharmacological behaviour, which is invaluable for future development and optimization of TASK modulators for treatment of heart, lung and sleep disorders.

scholarly journals Combining endocannabinoids with retigabine for enhanced M-channel effect and improved KV7 subtype selectivity

2020 ◽  
Vol 152 (8) ◽  
Author(s):  
Johan E. Larsson ◽  
Urban Karlsson ◽  
Xiongyu Wu ◽  
Sara I. Liin
Keyword(s):  
Adverse Effects ◽  
Xenopus Oocytes ◽  
Site Directed Mutagenesis ◽  
Resting Membrane Potential ◽  
Xenopus Laevis Oocytes ◽  
Kv7 Channels ◽  
Subtype Selectivity ◽  
Channel Effect ◽  
Low Concentrations ◽  
Electrode Voltage

Retigabine is unique among anticonvulsant drugs by targeting the neuronal M-channel, which is composed of KV7.2/KV7.3 and contributes to the negative neuronal resting membrane potential. Unfortunately, retigabine causes adverse effects, which limits its clinical use. Adverse effects may be reduced by developing M-channel activators with improved KV7 subtype selectivity. The aim of this study was to evaluate the prospect of endocannabinoids as M-channel activators, either in isolation or combined with retigabine. Human KV7 channels were expressed in Xenopus laevis oocytes. The effect of extracellular application of compounds with different properties was studied using two-electrode voltage clamp electrophysiology. Site-directed mutagenesis was used to construct channels with mutated residues to aid in the mechanistic understanding of these effects. We find that arachidonoyl-L-serine (ARA-S), a weak endocannabinoid, potently activates the human M-channel expressed in Xenopus oocytes. Importantly, we show that ARA-S activates the M-channel via a different mechanism and displays a different KV7 subtype selectivity compared with retigabine. We demonstrate that coapplication of ARA-S and retigabine at low concentrations retains the effect on the M-channel while limiting effects on other KV7 subtypes. Our findings suggest that improved KV7 subtype selectivity of M-channel activators can be achieved through strategically combining compounds with different subtype selectivity.

Neuropeptides as novel regulators of human atrial TASK-1 currents

2020 ◽  
Vol 41 (Supplement_2) ◽  
Author(s):  
F Wiedmann ◽  
J Nietfeld ◽  
M Kraft ◽  
A Ratte ◽  
S Benda ◽  
...  
Keyword(s):  
Atrial Fibrillation ◽  
Molecular Mechanisms ◽  
Receptor Activation ◽  
Xenopus Laevis Oocytes ◽  
Specific Expression ◽  
Atrial Cardiomyocytes ◽  
Kinase C ◽  
Electrode Voltage ◽  
Independent Mechanism ◽  
Pore Domain

Abstract Background/Introduction The neurokinin-III receptor (NK3R) was recently shown to regulate action potential duration (APD) in atrial cardiomyocytes by inhibition of a background potassium current. In the human heart, TASK-1 (hK2P3.1) two-pore-domain potassium channels display atrial-specific expression. Because of their phospholipase C (PLC)-dependent regulation, TASK-1 channels are a promising candidate to mediate APD prolongation via the Gq-coupled neurokinin-III receptor. Purpose To investigate whether TASK-1 channels mediate neurokinin-III receptor activation induced APD prolongation and to dissect the underlying molecular mechanisms. Methods Patch clamp measurements were performed in atrial cardiomyocytes isolated from patients with atrial fibrillation. Xenopus laevis oocytes heterologously expressing hTASK-1 and hNK3R were subjected to two-electrode voltage-clamp recordings. Results In Xenopus oocytes heterologously overexpressing hNK3R and hTASK-1 administration of substance P or neurokinin B resulted in TASK-1 current inhibition. This could be reproduced by application of the high affinity neurokinin-III receptor agonist senktide. Moreover, preincubation with the neurokinin-III receptor antagonist osanetant blunted the effect of senktide. Pharmacological experiments and mutagenesis studies could show a protein kinase C (PKC)-independent mechanism of TASK-1 current inhibition: upon NK3R activation TASK-1 channels are blocked via Gq-mediated PLC activation, in a DAG-dependent fashion. Finally, effects of senktide on atrial background currents could be reproduced in human atrial cardiomyocytes isolated from patients with atrial fibrillation. Conclusion Neurokinin-III receptor stimulation suppresses background potassium currents in isolated human atrial cardiomyocytes from patients with atrial fibrillation. Heterologously expressed human TASK-1 channels are inhibited by neurokinin-III receptor activation in a PLC and DAG dependent fashion, suggesting neuropeptides as novel regulators of human atrial TASK-1 currents. Central Illustration Funding Acknowledgement Type of funding source: None

scholarly journals Calcineurin as a Nociceptor Modulator

2009 ◽  
Vol 4;12 (4;7) ◽  
pp. E309-E318
Author(s):  
Howard Smith
Keyword(s):  
Spinal Cord ◽  
Potassium Channels ◽  
Chemokine Receptors ◽  
Calcineurin Inhibitors ◽  
Pain Syndrome ◽  
Resting Membrane Potential ◽  
Activated T Cells ◽  
Key Words Pain ◽  
Weakly Inward ◽  
Pore Domain

Calcineurin may be involved in affecting nociceptive processes in multiple circumstances. It is conceivable that interfering with calcineurin’s normal role in contributing to glial resting membrane potential, via its effects on the ion channel (TRESK) [tandem-pore-domain weakly inward rectifying potassium channels (TWIK)-related spinal cord potassium channels] may facilitate nociception. Another aspect of calcineurin function may be its role in the pronociceptive signaling of nuclear factor of activated T-cells (NFAT). NFAT activation via mediators (e.g. Substance P, brain-derived neurotrophic factor, nerve growth factor, bradykinin) appears to be dependent on calcineurin function. This calcineurin-regulated NFAT signaling may subsequently lead to transcription of pronociceptive genes as well as upregulation of pronociceptive chemokine receptors in the dorsal root ganglion. In fact, multiple articles have described the clinical use of calcineurin-inhibitors leading to pain, a phenomenon referred to as calcineurin inhibitor-induced pain syndrome (CIPS). Thus, it appears that calcineurin functions may encompass actions which promote or dampen nociceptive processes. A greater understanding of the physiology of calcineurin, especially as it relates to modulating nociception may lead to the development of novel analgesic targets in attempts to optimally alleviate patient discomfort. Key words: Pain, neuropathic, calcineurin, NFAT, TRESK-[Tandem-pore-domain weakly inward rectifying potassium channels (TWIK)-related spinal cord potassium channels], CIPS (calcineurin-induced pain syndrome)

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