scholarly journals Elucidating the Structural Basis of the Intracellular pH Sensing Mechanism of TASK-2 K2P Channels

2020 ◽  
Vol 21 (2) ◽  
pp. 532 ◽  
Author(s):  
Daniel Bustos ◽  
Mauricio Bedoya ◽  
David Ramírez ◽  
Guierdy Concha ◽  
Leandro Zúñiga ◽  
...  
Keyword(s):  
Ion Transport ◽  
Lipid Membrane ◽  
Structural Basis ◽  
Resting Membrane Potential ◽  
Ph Sensing ◽  
Transmembrane Helices ◽  
Energy Profiles ◽  
K2p Channels ◽  
Important Barrier ◽  
Pore Domain

Two-pore domain potassium (K2P) channels maintain the cell’s background conductance by stabilizing the resting membrane potential. They assemble as dimers possessing four transmembrane helices in each subunit. K2P channels were crystallized in “up” and “down” states. The movements of the pore-lining transmembrane TM4 helix produce the aperture or closure of side fenestrations that connect the lipid membrane with the central cavity. When the TM4 helix is in the up-state, the fenestrations are closed, while they are open in the down-state. It is thought that the fenestration states are related to the activity of K2P channels and the opening of the channels preferentially occurs from the up-state. TASK-2, a member of the TALK subfamily of K2P channels, is opened by intracellular alkalization leading the deprotonation of the K245 residue at the end of the TM4 helix. This charge neutralization of K245 could be sensitive or coupled to the fenestration state. Here, we describe the relationship between the states of the intramembrane fenestrations and K245 residue in TASK-2 channel. By using molecular modeling and simulations, we show that the protonated state of K245 (K245+) favors the open fenestration state and, symmetrically, that the open fenestration state favors the protonated state of the lysine residue. We show that the channel can be completely blocked by Prozac, which is known to induce fenestration opening in TREK-2. K245 protonation and fenestration aperture have an additive effect on the conductance of the channel. The opening of the fenestrations with K245+ increases the entrance of lipids into the selectivity filter, blocking the channel. At the same time, the protonation of K245 introduces electrostatic potential energy barriers to ion entrance. We computed the free energy profiles of ion penetration into the channel in different fenestration and K245 protonation states, to show that the effects of the two transformations are summed up, leading to maximum channel blocking. Estimated rates of ion transport are in qualitative agreement with experimental results and support the hypothesis that the most important barrier for ion transport under K245+ and open fenestration conditions is the entrance of the ions into the channel.

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 A “Target Class” Screen to Identify Activators of Two-Pore Domain Potassium (K2P) Channels

2020 ◽  
pp. 247255522097612
Author(s):  
David McCoull ◽  
Emma Ococks ◽  
Jonathan M. Large ◽  
David C. Tickle ◽  
Alistair Mathie ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Point Of View ◽  
Resting Membrane Potential ◽  
Small Molecule Activation ◽  
Target Class ◽  
Index Set ◽  
Diverse Range ◽  
K2p Channels ◽  
Additional Approach ◽  
Pore Domain

Two-pore domain potassium (K2P) channels carry background (or leak) potassium current and play a key role in regulating resting membrane potential and cellular excitability. Accumulating evidence points to a role for K2Ps in human pathophysiologies, most notably in pain and migraine, making them attractive targets for therapeutic intervention. However, there remains a lack of selective pharmacological tools. The aim of this work was to apply a “target class” approach to investigate the K2P superfamily and identify novel activators across all the described subclasses of K2P channels. Target class drug discovery allows for the leveraging of accumulated knowledge and maximizing synergies across a family of targets and serves as an additional approach to standard target-based screening. A common assay platform using baculovirus (BacMam) to transiently express K2P channels in mammalian cells and a thallium flux assay to determine channel activity was developed, allowing the simultaneous screening of multiple targets. Importantly, this system, by allowing precise titration of channel function, allows optimization to facilitate the identification of activators. A representative set of channels (THIK-1, TWIK-1, TREK-2, TASK-3, and TASK-2) were screened against a library of Food and Drug Administration (FDA)-approved compounds and the LifeArc Index Set. Activators were then analyzed in concentration–response format across all channels to assess selectivity. Using the target class approach to investigate the K2P channels has enabled us to determine which of the K2Ps are amenable to small-molecule activation, de-risk multiple channels from a technical point of view, and identify a diverse range of previously undescribed pharmacology.

Tandem Pore Domain Potassium Channels

2019 ◽  
Author(s):  
Douglas A. Bayliss
Keyword(s):  
Single Channel ◽  
Resting Membrane Potential ◽  
K2p Channel ◽  
Physiological Processes ◽  
System A ◽  
Wide Range ◽  
K2p Channels ◽  
To Come ◽  
A Site ◽  
Pore Domain

The KCNK gene family encodes two-pore-domain potassium (K2P) channels, which generate the background (“leak”) K+ currents that establish a negative resting membrane potential in cells of the nervous system. A pseudotetrameric K+-selective pore is formed by pairing channel subunits, each with two pore-domains, in homo- or heterodimeric conformations. Unique features apparent from high-resolution K2P channel structures include a domain-swapped extracellular cap domain, a lateral hydrophobic-lined fenestration connecting the lipid bilayer to the channel vestibule, and an antiparallel proximal C-terminal region that links the paired subunits and provides a site for polymodal channel modulation. Individual channels transition between open and closed states, with the channel gate located at the selectivity filter. In general, K2P channels display relatively modest voltage- and time-dependent gating, together with distinct single-channel rectification properties, that conspire to yield characteristic weakly rectifying macroscopic currents over a broad range of membrane potentials (i.e., background K+ currents). Of particular note, K2P channel activity can be regulated by a wide range of physicochemical factors, neuromodulators, and clinically useful drugs; a distinct repertoire of activators and inhibitors for different K2P channel subtypes endows each with unique modulatory potential. Thus, by mediating background currents and serving as targets for multiple modulators, K2P channels are able to dynamically regulate key determinants of cell-intrinsic electroresponsive properties. The roles of specific K2P channels in various physiological processes and pathological conditions are now beginning to come into focus, and this may portend utility for these channels as potential therapeutic targets.

scholarly journals The SPICA Coarse-Grained Force Field for Proteins and Peptides

2021 ◽  
Author(s):  
Shuhei Kawamoto ◽  
Huihui Liu ◽  
Sangjae Seo ◽  
Yusuke Miyazaki ◽  
Mayank Dixit ◽  
...  
Keyword(s):  
Force Field ◽  
Lipid Membranes ◽  
Lipid Membrane ◽  
Coarse Grained ◽  
Transmembrane Helices ◽  
Free Energies ◽  
Transmembrane Peptides ◽  
Energy Profiles ◽  
Protein Model ◽  
Preliminary Version

ABSTRACTA coarse-grained (CG) model for peptides and proteins was developed as an extension of the SPICA (Surface Property fItting Coarse grAined) force field (FF). The model was designed to examine membrane proteins that are fully compatible with the lipid membranes of the SPICA FF. A preliminary version of this protein model was created using thermodynamic properties, including the surface tension and density in the SPICA (formerly called SDK) FF. In this study, we improved the CG protein model to facilitate molecular dynamics (MD) simulation with a reproduction of multiple properties from both experiments and all-atom (AA) simulations. The side chain analogs reproduced the transfer free energy profiles across the lipid membrane and demonstrated reasonable dimerization free energies in water compared to those from AA-MD. A series of peptides/proteins adsorbed or penetrated into the membrane simulated by the CG-MD correctly predicted the penetration depths and tilt angles of peripheral and transmembrane peptides/proteins comparable to those in the orientation of protein in membrane (OPM) database. In addition, the dimerization free energies of several transmembrane helices within a lipid bilayer were comparable to those from experimental estimation. Application studies on a series of membrane protein assemblies, scramblases, and poliovirus capsids demonstrated a good performance of the SPICA FF.

scholarly journals Enhancement of TWIK-related Acid-sensitive Potassium Channel 3 (TASK3) Two-pore Domain Potassium Channel Activity by Tumor Necrosis Factor α

2013 ◽  
Vol 289 (3) ◽  
pp. 1388-1401 ◽  
Author(s):  
Mickael-F El Hachmane ◽  
Kathryn A. Rees ◽  
Emma L. Veale ◽  
Vadim V. Sumbayev ◽  
Alistair Mathie
Keyword(s):  
Potassium Channel ◽  
Cell Voltage ◽  
Cell Types ◽  
Resting Membrane Potential ◽  
Channel Activity ◽  
Inflammatory Conditions ◽  
C Terminus ◽  
K2p Channels ◽  
Factor Α ◽  
Pore Domain

TASK3 two-pore domain potassium (K2P) channels are responsible for native leak K channels in many cell types which regulate cell resting membrane potential and excitability. In addition, TASK3 channels contribute to the regulation of cellular potassium homeostasis. Because TASK3 channels are important for cell viability, having putative roles in both neuronal apoptosis and oncogenesis, we sought to determine their behavior under inflammatory conditions by investigating the effect of TNFα on TASK3 channel current. TASK3 channels were expressed in tsA-201 cells, and the current through them was measured using whole cell voltage clamp recordings. We show that THP-1 human myeloid leukemia monocytes, co-cultured with hTASK3-transfected tsA-201 cells, can be activated by the specific Toll-like receptor 7/8 activator, R848, to release TNFα that subsequently enhances hTASK3 current. Both hTASK3 and mTASK3 channel activity is increased by incubation with recombinant TNFα (10 ng/ml for 2–15 h), but other K2P channels (hTASK1, hTASK2, hTREK1, and hTRESK) are unaffected. This enhancement by TNFα is not due to alterations in levels of channel expression at the membrane but rather to an alteration in channel gating. The enhancement by TNFα can be blocked by extracellular acidification but persists for mutated TASK3 (H98A) channels that are no longer acid-sensitive even in an acidic extracellular environment. TNFα action on TASK3 channels is mediated through the intracellular C terminus of the channel. Furthermore, it occurs through the ASK1 pathway and is JNK- and p38-dependent. In combination, TNFα activation and TASK3 channel activity can promote cellular apoptosis.

Two-Pore Domain Potassium Channels as Drug Targets: Anesthesia and Beyond

2021 ◽  
Vol 61 (1) ◽  
pp. 401-420 ◽  
Author(s):  
Alistair Mathie ◽  
Emma L. Veale ◽  
Kevin P. Cunningham ◽  
Robyn G. Holden ◽  
Paul D. Wright
Keyword(s):  
Small Molecule ◽  
Drug Targets ◽  
Cell Activity ◽  
Therapeutic Benefit ◽  
Resting Membrane Potential ◽  
Screening Programs ◽  
K2p Channel ◽  
K2p Channels ◽  
Pharmacological Regulation ◽  
Pore Domain

Two-pore domain potassium (K2P) channels stabilize the resting membrane potential of both excitable and nonexcitable cells and, as such, are important regulators of cell activity. There are many conditions where pharmacological regulation of K2P channel activity would be of therapeutic benefit, including, but not limited to, atrial fibrillation, respiratory depression, pulmonary hypertension, neuropathic pain, migraine, depression, and some forms of cancer. Up until now, few if any selective pharmacological regulators of K2P channels have been available. However, recent publications of solved structures with small-molecule activators and inhibitors bound to TREK-1, TREK-2, and TASK-1 K2P channels have given insight into the pharmacophore requirements for compound binding to these sites. Together with the increasing availability of a number of novel, active, small-molecule compounds from K2P channel screening programs, these advances have opened up the possibility of rational activator and inhibitor design to selectively target K2P channels.

scholarly journals Two-Pore-Domain Potassium (K2P-) Channels: Cardiac Expression Patterns and Disease-Specific Remodelling Processes

Cells ◽  
2021 ◽  
Vol 10 (11) ◽  
pp. 2914
Author(s):  
Felix Wiedmann ◽  
Norbert Frey ◽  
Constanze Schmidt
Keyword(s):  
Action Potential ◽  
Cardiac Fibrosis ◽  
Cardiac Tissue ◽  
Therapeutic Potential ◽  
Expression Patterns ◽  
Cell Types ◽  
Resting Membrane Potential ◽  
K2p Channel ◽  
K2p Channels ◽  
Pore Domain

Two-pore-domain potassium (K2P-) channels conduct outward K+ currents that maintain the resting membrane potential and modulate action potential repolarization. Members of the K2P channel family are widely expressed among different human cell types and organs where they were shown to regulate important physiological processes. Their functional activity is controlled by a broad variety of different stimuli, like pH level, temperature, and mechanical stress but also by the presence of lipids or pharmacological agents. In patients suffering from cardiovascular diseases, alterations in K2P-channel expression and function have been observed, suggesting functional significance and a potential therapeutic role of these ion channels. For example, upregulation of atrial specific K2P3.1 (TASK-1) currents in atrial fibrillation (AF) patients was shown to contribute to atrial action potential duration shortening, a key feature of AF-associated atrial electrical remodelling. Therefore, targeting K2P3.1 (TASK-1) channels might constitute an intriguing strategy for AF treatment. Further, mechanoactive K2P2.1 (TREK-1) currents have been implicated in the development of cardiac hypertrophy, cardiac fibrosis and heart failure. Cardiovascular expression of other K2P channels has been described, functional evidence in cardiac tissue however remains sparse. In the present review, expression, function, and regulation of cardiovascular K2P channels are summarized and compared among different species. Remodelling patterns, observed in disease models are discussed and compared to findings from clinical patients to assess the therapeutic potential of K2P channels.

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.

TASK, TREK & Co.: a mutable potassium channel family for diverse tasks in the brain

e-Neuroforum ◽  
2015 ◽  
Vol 21 (2) ◽  
Author(s):  
P. Ehling ◽  
Stefan Bittner ◽  
Sven G. Meuth ◽  
Thomas Budde
Keyword(s):  
Neurological Diseases ◽  
Outward Current ◽  
Resting Membrane Potential ◽  
K2p Channel ◽  
Membrane Pores ◽  
Open Questions ◽  
K2p Channels ◽  
In The Beginning ◽  
Pore Domain ◽  
The Brain

AbstractDiscovered during the 1990s and in the beginning regarded as passive membrane pores, the family of two-pore domain potassium (K2P)-channels initially received only little attention. Today the view on this channel family comprising 15 ubiquitously expressed members in mammals has greatly changed. K2P-channels carry potassium outward current that counterbalances membrane depolarization and stabilizes the resting membrane potential. Thereby they are important regulators for the excitability and the firing behaviour especially in neurons. The long list of modulating mechanisms underlines the channels’ relevance. K2P-channels in the thalamus contribute to the regulation of the sleep-wake cycle. They also mediate the effect of volatile anaesthetics by supporting the thalamic activity mode that is also typical for sleep. This review summarizes our knowledge about K2P-channel physiology in the brain, provides an idea of the role of these channels in neurological diseases and lists open questions as well as technical challenges in K2P-channel research.

scholarly journals Structural Basis for Gating of the Two-Pore Domain K+ (K2P) Channels TASK-1 and TALK-2

2021 ◽  
Vol 120 (3) ◽  
pp. 289a
Author(s):  
Marcus Schewe ◽  
Elena B. Riel ◽  
Susanne Rinné ◽  
Wojciech Kopec ◽  
Jan Langer ◽  
...  
Keyword(s):  
Structural Basis ◽  
K2p Channels ◽  
Pore Domain
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