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.

scholarly journals Tuning voltage-gated channel activity and cellular excitability with a sphingomyelinase

2013 ◽  
Vol 142 (4) ◽  
pp. 367-380 ◽  
Author(s):  
David J. Combs ◽  
Hyeon-Gyu Shin ◽  
Yanping Xu ◽  
Yajamana Ramu ◽  
Zhe Lu
Keyword(s):  
Membrane Potential ◽  
Mammalian Cells ◽  
Resting Membrane Potential ◽  
Xenopus Laevis Oocytes ◽  
Channel Activity ◽  
Excitable Cells ◽  
Voltage Sensors ◽  
Voltage Gated ◽  
Activated State ◽  
Gated Channel

Voltage-gated ion channels generate action potentials in excitable cells and help set the resting membrane potential in nonexcitable cells like lymphocytes. It has been difficult to investigate what kinds of phospholipids interact with these membrane proteins in their native environments and what functional impacts such interactions create. This problem might be circumvented if we could modify specific lipid types in situ. Using certain voltage-gated K+ (KV) channels heterologously expressed in Xenopus laevis oocytes as a model, our group has shown previously that sphingomyelinase (SMase) D may serve this purpose. SMase D is known to remove the choline group from sphingomyelin, a phospholipid primarily present in the outer leaflet of plasma membranes. This SMase D action lowers the energy required for voltage sensors of a KV channel to enter the activated state, causing a hyperpolarizing shift of the Q-V and G-V curves and thus activating them at more hyperpolarized potentials. Here, we find that this SMase D effect vanishes after removing most of the voltage-sensor paddle sequence, a finding supporting the notion that SMase D modification of sphingomyelin molecules alters these lipids’ interactions with voltage sensors. Then, using SMase D to probe lipid–channel interactions, we find that SMase D not only similarly stimulates voltage-gated Na+ (NaV) and Ca2+ channels but also markedly slows NaV channel inactivation. However, the latter effect is not observed in tested mammalian cells, an observation highlighting the profound impact of the membrane environment on channel function. Finally, we directly demonstrate that SMase D stimulates both native KV1.3 in nonexcitable human T lymphocytes at their typical resting membrane potential and native NaV channels in excitable cells, such that it shifts the action potential threshold in the hyperpolarized direction. These proof-of-concept studies illustrate that the voltage-gated channel activity in both excitable and nonexcitable cells can be tuned by enzymatically modifying lipid head groups.

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 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.

scholarly journals A Ba2+-Sensitive K+ Current Contributes to the Resting Membrane Potential of Neurons in Rat Suprachiasmatic Nucleus

2002 ◽  
Vol 88 (2) ◽  
pp. 869-878 ◽  
Author(s):  
Marcel de Jeu ◽  
Alwin Geurtsen ◽  
Cyriel Pennartz
Keyword(s):  
Membrane Potential ◽  
Suprachiasmatic Nucleus ◽  
Voltage Dependence ◽  
Brain Slices ◽  
Resting Potential ◽  
Resting Membrane Potential ◽  
Delayed Rectifier ◽  
Patch Clamp Technique ◽  
M Current ◽  
Whole Cell Patch Clamp

A Ba2+-sensitive K+ current was studied in neurons of the suprachiasmatic nucleus (SCN) using the whole cell patch-clamp technique in acutely prepared brain slices. This Ba2+-sensitive K+ current was found in approximately 90% of the SCN neurons and was uniformly distributed across the SCN. Current-clamp studies revealed that Ba2+ (500 μM) reversibly depolarized the membrane potential by 6.7 ± 1.3 mV ( n = 22) and concomitantly Ba2+ induced an increase in the spontaneous firing rate of 0.8 ± 0.2 Hz ( n = 12). The Ba2+-evoked depolarizations did not depend on firing activity or spike dependent synaptic transmission. No significant day/night difference in the hyperpolarizing contribution to the resting membrane potential of the present Ba2+-sensitive current was observed. Voltage-clamp experiments showed that Ba2+ (500 μM) reduced a fast-activating, voltage-dependent K+ current. This current was activated at levels below firing threshold and exhibited outward rectification. The Ba2+-sensitive K+ current was strongly reduced by tetraethylammonium (TEA; 20 and 60 mM) but was insensitive to 4-aminopyridine (4-AP; 5 mM) and quinine (100 μM). A component of Ba2+-sensitive K+ current remaining in the presence of TEA exhibited no clear voltage dependence and is less likely to contribute to the resting membrane potential. The voltage dependence, kinetics and pharmacological properties of the Ba2+- and TEA-sensitive K+ current make it unlikely that this current is a delayed rectifier, Ca2+-activated K+ current, ATP-sensitive K+ current, M-current or K+ inward rectifier. Our data are consistent with the Ba2+- and TEA-sensitive K+ current in SCN neurons being an outward rectifying K+ current of a novel identity or belonging to a known family of K+ channels with related properties. Regardless of its precise molecular identity, the current appears to exert a significant hyperpolarizing effect on the resting potential of SCN neurons.

scholarly journals Nonreciprocal mechanisms in up- and downregulation of spinal motoneuron excitability by modulators of KCNQ/Kv7 channels

2016 ◽  
Vol 116 (5) ◽  
pp. 2114-2124 ◽  
Author(s):  
Joseph Lombardo ◽  
Melissa A. Harrington
Keyword(s):  
Initial Segment ◽  
Input Resistance ◽  
Resting Membrane Potential ◽  
Channel Blockers ◽  
Channel Activity ◽  
Spinal Motoneurons ◽  
M Current ◽  
Kv7 Channels ◽  
Axonal Initial Segment ◽  
The Central Nervous System

KCNQ/Kv7 channels form a slow noninactivating K+ current, also known as the M current. They activate in the subthreshold range of membrane potentials and regulate different aspects of excitability in neurons of the central nervous system. In spinal motoneurons (MNs), KCNQ/Kv7 channels have been identified in the somata, axonal initial segment, and nodes of Ranvier, where they generate a slow, noninactivating, K+ current sensitive to both muscarinic receptor-mediated inhibition and KCNQ/Kv7 channel blockers. In this study, we thoroughly reevaluated the function of up- and downregulation of KCNQ/Kv7 channels in mouse immature spinal MNs. Using electrophysiological techniques together with specific pharmacological modulators of the activity of KCNQ/Kv7 channels, we show that enhancement of the activity of these channels decreases the excitability of spinal MNs in mouse neonates. This action on MNs results from a combination of hyperpolarization of the resting membrane potential, a decrease in the input resistance, and depolarization of the voltage threshold. On the other hand, the effect of inhibition of KCNQ/Kv7 channels suggested that these channels play a limited role in regulating basal excitability. Computer simulations confirmed that pharmacological enhancement of KCNQ/Kv7 channel activity decreases excitability and also suggested that the effects of inhibition of KCNQ/Kv7 channels on the excitability of spinal MNs do not depend on a direct effect in these neurons but likely on spinal cord synaptic partners. These results indicate that KCNQ/Kv7 channels have a fundamental role in the modulation of the excitability of spinal MNs acting both in these neurons and in their local presynaptic partners.

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.

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.

Inward rectification in rat nucleus accumbens neurons

1989 ◽  
Vol 62 (6) ◽  
pp. 1280-1286 ◽  
Author(s):  
N. Uchimura ◽  
E. Cherubini ◽  
R. A. North
Keyword(s):  
Steady State ◽  
Nucleus Accumbens ◽  
Time Course ◽  
Potassium Conductance ◽  
Resting Potential ◽  
Inward Rectification ◽  
Resting Membrane Potential ◽  
Potential Range ◽  
Inward Current ◽  
Rat Nucleus Accumbens

1. Intracellular recordings were made from neurons in slices cut from the rat nucleus accumbens septi. Membrane currents were measured with a single-electrode voltage-clamp amplifier in the potential range -50 to -140 mV. 2. In control conditions (2.5 mM potassium), the resting membrane potential of the neurons was -83.4 +/- 1.1 (SE) mV (n = 157). Steady state membrane conductance was voltage dependent, being 34.8 +/- 1.7 nS (n = 25) at -100 mV and 8.0 +/- 0.7 nS (n = 25) at -60 mV. 3. Barium (1 microM) markedly reduced the inward rectification and caused a small inward current (40.6 +/- 8.7 pA, n = 8) at the resting potential. These effects became larger with higher barium concentrations, and, in 100 microM barium, the current-voltage relation was straight. 4. The block of the inward current by barium (at -130 mV) occurred with an exponential time course; the time constant was approximately 1 s at 1 microM barium and less than 90 ms with 100 microM. Strontium had effects similar to those of barium, but 1000-fold higher concentrations were required. Cesium chloride (2 mM) and rubidium chloride (2 mM) also blocked the inward rectification; their action reached steady state within 50 ms. 5. It is concluded that the nucleus accumbens neurons have a potassium conductance with many features of a typical inward rectifier and that this contributes to the potassium conductance at the resting potential.

Electrical and Mechanical Properties of the Red and White Muscles in the Silver Carp

1972 ◽  
Vol 57 (2) ◽  
pp. 551-567
Author(s):  
T. YAMAMOTO
Keyword(s):  
Mechanical Properties ◽  
White Muscle ◽  
Silver Carp ◽  
Membrane Resistance ◽  
Resting Potential ◽  
Resting Membrane Potential ◽  
Mechanical Responses ◽  
Red Muscle ◽  
Red And White Muscles ◽  
Specific Membrane

1. Electrical and mechanical properties of the red muscle (M. levator pinnae pectoralis) and white muscle (M. levator pinnae lateralis abdominis) in the silver carp (Carassius auratus Linné) were investigated by using caffeine and thymol. 2. A complete tetanus could be produced in the red muscle. But in the white muscle no tetanus was produced and there was a gradual decrease in tension during continuous stimulation, even at a frequency of 1 c/s or less. 3. Caffeine (0.5-1 mM) and thymol (0.25-0.5 mM) potentiated the twitch tension in both muscles without an increase in the resting tension; they produced a contracture in both muscles when the concentration was increased further. 4. The falling phase of the active state of contraction was nearly the same in both muscles and was prolonged by caffeine (0.5 mmM) and by thymol (0.25 mM). 5. The resting membrane potential of the red muscle was scarcely affected by caffeine (0.5-5 mM), whereas in the white muscles a depolarization of 10 mV was observed with caffeine of more than 2 mM. The resting potential of both muscles was little changed by o.25 mm thymol. However, at a concentration of more than 0.5mM thymol depolarized the membrane in both muscles to the same extent. 6. In caffeine (2-3 mM) solution the mean specific membrane resistance was reduced from 8.8 kΩ cm2 to 6.0 kΩ cm2 in the red muscle, and from 5.0 kΩ cm2 to 2.7 kΩ cm2 in the white muscle. In thymol (0.5-1 mM) solution it was reduced from 11.2 kΩcm2 to 6.5 kΩ cm2 in the red muscle, and from 5.4kΩ cm2 to 3.1 kΩ) cm2 in the white muscle. The specific membrane capacitance, calculated from the time constant and the membrane resistance, remained more or less the same in both muscles after a treatment with these agents. 7. Electrical properties of the muscles and the effects of caffeine and thymol on mechanical responses suggest that there are no fundamental differences between red and white muscles except for the excitation-contraction coupling. A lack of summation of twitch, a successive decline of twitch, and inability to produce potassium contracture in the white muscle may be due to the fact that the Ca-releasing mechanism is easily inactivated by depolarization of the membrane.

Inhibition of TASK-1 potassium channel by phospholipase C

2001 ◽  
Vol 281 (2) ◽  
pp. C700-C708 ◽  
Author(s):  
Gábor Czirják ◽  
Gábor L. Petheő ◽  
András Spät ◽  
Péter Enyedi
Keyword(s):  
Phospholipase C ◽  
Lysophosphatidic Acid ◽  
Protein S ◽  
Receptor Activation ◽  
Xenopus Laevis Oocytes ◽  
Ang Ii ◽  
Inositol 1,4,5 Trisphosphate ◽  
Glomerulosa Cells ◽  
Pore Domain ◽  
Stimulation Of

The two-pore-domain K+ channel, TASK-1, was recently shown to be a target of receptor-mediated regulation in neurons and in adrenal glomerulosa cells. Here, we demonstrate that TASK-1 expressed in Xenopus laevis oocytes is inhibited by different Ca2+-mobilizing agonists. Lysophosphatidic acid, via its endogenous receptor, and ANG II and carbachol, via their heterologously expressed ANG II type 1a and M1 muscarinic receptors, respectively, inhibit TASK-1. This effect can be mimicked by guanosine 5′- O-(3-thiotriphosphate), indicating the involvement of GTP-binding protein(s). The phospholipase C inhibitor U-73122 reduced the receptor-mediated inhibition of TASK-1. Downstream signals of phospholipase C action (inositol 1,4,5-trisphosphate, cytoplasmic Ca2+ concentration, and diacylglycerol) do not mediate the inhibition. Unlike the Gq-coupled receptors, stimulation of the Gi-activating M2 muscarinic receptor coexpressed with TASK-1 results in an only minimal decrease of the TASK-1 current. However, additional coexpression of phospholipase C-β2 (which is responsive also to Giβγ-subunits) renders M2 receptor activation effective. This indicates the significance of phospholipase C activity in the receptor-mediated inhibition of TASK-1.

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