Inhibition Of Potassium Currents Research Articles

Toxicity and physiological actions of biflavones on potassium current in insect neuronal cells

Stellera chamaejasme L. is a Chinese traditional herb. It has a long history and many medicinal usages. Biflavones, one of the main active ingredients in S. chamaejasme's roots, possess excellent insecticidal activities both in vivo and in vitro. However, the mechanism of these compounds and its potential molecular targets on insect cell were still not clear. Here the whole cell patch clamp technique was used to investigate whether biflavones affects voltage-gated potassium channels (Kv) on insect neuronal cells (AW1 and WG2). The results confirmed that both the three biflavones: neochamaejasmin A (NCA), neochamaejasmin B (NCB) and isochamaejasmin A (ICM) can significantly inhibit the A-type potassium current (IA) than delayed rectifier potassium current (IK) expressed on insect cells. Moreover, ICM stood out as the strongest inhibition activity on IA with IC50 value of 106.75 μM. Multiple results suggest that the inhibition of potassium current was related to the gating modification of biflavones. ICM produced concentration dependent hyperpolarizing shifts in the voltage dependence of channel steady-state activation and inactivation. Maximal shifts of the ICM-induced V0.5, were −15.1 mV for activation and −6.93 mV for inactivation. ICM also prolonged recovery from inactivation of current. Moreover, the biflavones could inhibited AW1 cell survival in both dose- and time-dependent manners with well correlation of K+ inhibitory activity. Our study showed that biflavones from S. chamaejasme exhibiting significant blocked effects on Kv of AW1 cells and inhibited cell proliferation. These findings may not only show the toxic mechanisms of biflavones on insect cells, but also suggest that Kv channel play an important role in biflavones' mode of action and may be the new targets for designing novel insecticides.

Two KCNQ2 Encephalopathy Variants in the Calmodulin-Binding Helix A Exhibit Dominant-Negative Effects and Altered PIP2 Interaction

Heterozygous missense variants in KCNQ2, which encodes the potassium channel subunit Kv7.2, are among the most common genetic causes of severe neonatal-onset epileptic encephalopathy. Because about 20% of known severe Kv7.2 missense changes lie within the intracellular C-terminal region, improving understanding of the underlying pathogenic mechanisms is important. We analyzed the basis for the severe phenotypes of Kv7.2 A337T and A337G, variants in the C-terminal’s calmodulin (CaM)-binding Helix A. When expressed heterologously in mammalian cells, alone or in combination with wild type Kv7.2 or with wild type Kv7.2 and Kv7.3, both variants strongly suppressed channel currents. A337T channels expressed alone exhibited significantly reduced protein half-life and surface trafficking and co-immunoprecipitated less CaM. For both variants, increasing cellular phosphatidylinositol 4,5-bisphosphate (PIP2) by overexpression of PI(4)P5-kinase restored current densities. For both variants, the fraction of current suppressed by activation of M1 muscarinic receptors with 10 μM oxotremorine methiodide, which depletes PIP2, was less than for controls. During voltage-sensitive phosphatase-induced transient PIP2 depletion and resynthesize, potassium current inhibition and recovery kinetics were both markedly slowed. These results suggest that these variants may reduce currents by a mechanism not previously described: slowing of PIP2 migration between the bulk membrane and binding sites mediating channel electromechanical coupling. A novel Kv7.2/3-selective opener, SF0034, rescued current amplitudes. Our findings show that these two Helix A variants suppress channel current density strongly, consistent with their severe heterozygous phenotypes, implicate impairment of CaM and PIP2 regulation in KCNQ2 encephalopathy pathogenesis, and highlight the potential usefulness of selective Kv7 openers for this distinctive pathogenic mechanism and patient subgroup.

Activation of GABAB receptors results in excitatory modulation of calyx terminals in rat semicircular canal cristae.

Previous studies have found GABA in vestibular end organs. However, existence of GABA receptors or possible GABAergic effects on vestibular nerve afferents has not been investigated. The current study was conducted to determine whether activation of GABAB receptors affects calyx afferent terminals in the central region of the cristae of semicircular canals. We used patch-clamp recording in postnatal day 13-18 (P13-P18) Sprague-Dawley rats of either sex. Application of GABAB receptor agonist baclofen inhibited voltage-sensitive potassium currents. This effect was blocked by selective GABAB receptor antagonist CGP 35348. Application of antagonists of small (SK)- and large-conductance potassium (BK) channels almost completely blocked the effects of baclofen. The remaining baclofen effect was blocked by cadmium chloride, suggesting that it could be due to inhibition of voltage-gated calcium channels. Furthermore, baclofen had no effect in the absence of calcium in the extracellular fluid. Inhibition of potassium currents by GABAB activation resulted in an excitatory effect on calyx terminal action potential firing. While in the control condition calyces could only fire a single action potential during step depolarizations, in the presence of baclofen they fired continuously during steps and a few even showed repetitive discharge. We also found a decrease in threshold for action potential generation and a decrease in first-spike latency during step depolarization. These results provide the first evidence for the presence of GABAB receptors on calyx terminals, showing that their activation results in an excitatory effect and that GABA inputs could be used to modulate calyx response properties.NEW & NOTEWORTHY Using in vitro whole cell patch-clamp recordings from calyx terminals in the vestibular end organs, we show that activation of GABAB receptors result in an excitatory effect, with decreased spike-frequency adaptation and shortened first-spike latencies. Our results suggest that these effects are mediated through inhibition of calcium-sensitive potassium channels.

Ghrelin Selectively Inhibits CaV3.3 Subtype of Low-Voltage-Gated Calcium Channels.

The mechanisms by which ghrelin controls electrical activity in the hypothalamus are not fully understood. One unexplored target of ghrelin is CaV3, responsible for transient calcium currents (T-currents) that control neuronal firing. We investigated the effect of ghrelin on CaV3 subtypes and how this modulation impacts on neuronal activity. We performed whole-cell patch-clamp recordings in primary mouse hypothalamic cultures to explore the effect of ghrelin on T-currents. We also recorded calcium currents from transiently transfected tsA201 cells to study the sensitivity of each CaV3 subtype to GHSR activation. Finally, we ran a computational model combining the well-known reduction of potassium current by ghrelin with the CaV3 biophysical parameter modifications induced by ghrelin to predict the impact on neuronal electrical behavior. We found that ghrelin inhibits native NiCl2 sensitive current currents in hypothalamic neurons. We determined that CaV3.3 is the only CaV3 subtype sensitive to ghrelin. The modulation of CaV3.3 by ghrelin comprises a reduction in maximum conductance, a shift to hyperpolarized voltages of the I-V and steady-state inactivation curves, and an acceleration of activation and inactivation kinetics. Our model-based prediction indicates that the inhibition of CaV3.3 would attenuate the stimulation of firing originating from the inhibition of potassium currents by ghrelin. In summary, we discovered a new target of ghrelin in neurons: the CaV3.3. This mechanism would imply a negative feed-forward regulation of the neuronal activation exerted by ghrelin. Our work expands the knowledge of the wide range of actions of GHSR, a receptor potentially targeted by therapeutics for several diseases.

Structure-activity relationships for unit C pyridyl analogues of the tuberculosis drug bedaquiline.

The ATP-synthase inhibitor bedaquiline is effective against drug-resistant tuberculosis but is extremely lipophilic (clogP 7.25) with a very long plasma half-life. Additionally, inhibition of potassium current through the cardiac hERG channel by bedaquiline, is associated with prolongation of the QT interval, necessitating cardiovascular monitoring. Analogues were prepared where the naphthalene C-unit was replaced with substituted pyridines to produce compounds with reduced lipophilicity, anticipating a reduction in half-life. While there was a direct correlation between in vitro inhibitory activity against M. tuberculosis (MIC90) and compound lipophilicity, potency only fell off sharply below a clogP of about 4.0, providing a useful lower bound for analogue design. The bulk of the compounds remained potent inhibitors of the hERG potassium channel, with notable exceptions where IC50 values were at least 5-fold higher than that of bedaquiline. Many of the compounds had desirably higher rates of clearance than bedaquiline, but this was associated with lower plasma exposures in mice, and similar or higher MICs resulted in lower AUC/MIC ratios than bedaquiline for most compounds. The two compounds with lower potency against hERG exhibited similar clearance to bedaquiline and excellent efficacy in vivo, suggesting further exploration of C-ring pyridyls is worthwhile.

Excessive corticosterone induces excitotoxicity of hippocampal neurons and sensitivity of potassium channels via insulin-signaling pathway.

Corticosterone (CORT) is a kind of corticosteroid produced by cortex of adrenal glands. Hypothalamic-pituitary-adrenal (HPA) axis hyperfunction leads to excessive CORT, which is associated with depression. Few studies have investigated the role of CORT in voltage-gated ion channels and its upstream signaling pathway in central nervous system. In this study, we investigated the mechanism of excessive CORT resulting in brain impairment on voltage-gated ion channels, and its upstream signaling effectors in hippocampal CA1 neurons. The action potential (AP) and voltage-gated potassium currents were determined by using whole-cell patch-clamp. Insulin and CORT improved the neuronal excitability. Independent effects existed in transient potassium channel (IA) and delay rectifier potassium channel (IK). The inhibition of potassium currents, IA in our experiment, could increase neuronal excitability. CORT led to the excitotoxicity of hippocampal neurons via phosphatidylinositol 3 kinase (PI3K)-mediated insulin-signaling pathway. Therefore, the stimulation of excessive CORT induces excitotoxicity of hippocampal neurons and sensitivity of potassium channels via PI3K-mediated insulin-signaling pathway, which indicates one possible way of depression treatment.

P512A new antiarrhythmic drug niferidil prolongs action potentials in guinea pig atrial myocardium via inhibition of potassium currents

P512A new antiarrhythmic drug niferidil prolongs action potentials in guinea pig atrial myocardium via inhibition of potassium currents

Ascorbic acid does not modulate potassium currents in cultured human lymphocytes.

Ascorbic acid (AA) is known to modulate lymphocyte function, but the mechanism of action is not clearly understood. As voltage-gated potassium currents play an important role in lymphocyte function, the effect of AA on voltage-gated potassium currents was studied. Peripheral blood mononuclear cells were cultured in the presence of increasing concentrations of AA (0, 0.125, 0.25, 0.5, and 1 mM). Potassium currents in resting lymphocytes were studied by whole cell patch clamp technique using a depolarizing protocol. Lymphocyte function was assessed by measuring interleukin-2 (IL-2) secretion after mitogenic stimulation by ELISA. The mean current density of potassium currents recorded from cells cultured for 48 h in the presence of 0.125 mM AA was not significantly different from that of cells cultured in the absence of AA. There was about 50% inhibition of IL-2 secretion in cell cultures with 0.125 mM AA when compared to controls without AA. At higher concentrations of AA, the IL-2 secretion decreased further. The results of the study indicate that the inhibition of lymphocyte function by AA in vitro may not be due to inhibition of potassium currents in the concentration tested.

Inhibition of potassium currents is involved in antiarrhythmic effect of moderate ethanol on atrial fibrillation

Excessive consumption of alcohol is a well-established risk factor of atrial fibrillation (AF). However, the effects of moderate alcohol drinking remain to be elucidated. This study was designed to determine the effects of moderate ethanol ingestion on atrial fibrillation and the electrophysiological mechanisms. In acetylcholine-induced canine and mouse AF models, the moderate ethanol prevented the generation and persistence of AF through prolonging the latent period of AF and shortening the duration of AF. The action potential duration (APD) was remarkably prolonged under the concentration range of 12.5–50.0mM ethanol in guinea pig atrial myocytes. Ultra-rapid delayed rectified potassium currents (IKv1.5) were markedly inhibited by 12.5–50.0mM ethanol in a concentration-dependent manner. Ethanol with 50.0mM could inhibit rapid delayed rectifier potassium currents (IhERG). Ethanol under 6.25–50.0mM did not affect on inward rectifier potassium currents (IKir2.1). Collectively, the present study provided an evidence that moderate ethanol intake can prolong the APD of atrial myocytes by inhibition of IKv1.5 and IhERG, which contributed to preventing the development and duration of AF.

Neurovascular coupling protects neurons against hypoxic injury via inhibition of potassium currents by generation of nitric oxide in direct neuron and endothelium cocultures

This study examined the effect of neuron-endothelial coupling on the survival of neurons after ischemia and the possible mechanism underlying that effect. Whole-cell patch-clamp experiments were performed on cortical neurons cultured alone or directly cocultured with brain microvascular endothelial cells (BMEC). Propidium iodide (PI) and NeuN staining were performed to examine neuronal death following oxygen and glucose deprivation (OGD). We found that the neuronal transient outward potassium currents (IA) decreased in the coculture system, whereas the outward delayed-rectifier potassium currents (IK) did not. Sodium nitroprusside, a NO donor, enhanced BMEC-induced IA inhibition and nitro-l-arginine methylester, a NOS inhibitor, partially prevented this inhibition. Moreover, the neurons directly cocultured with BMEC showed more resistance to OGD-induced injury compared with the neurons cultured alone, and that neuroprotective effect was abolished by treatment with NS5806, an activator of the IA. These results indicate that vascular endothelial cells assist neurons to prevent hypoxic injury via inhibiting neuronal IA by production of NO in the direct neuron-BMEC coculture system. These results further provide direct evidence of functional coupling between neurons and vascular endothelial cells. This study clearly demonstrates that vascular endothelial cells play beneficial roles in the pathophysiological processes of neurons after hypoxic injury, suggesting that the improvement of neurovascular coupling or functional remodeling may become an important therapeutic target for preventing brain injury.

Cytotoxicity mechanism of the RNase Sa cationic mutants involves inhibition of potassium current through Ca2+-activated channels

Cytotoxicity mechanism of the RNase Sa cationic mutants involves inhibition of potassium current through Ca2+-activated channels

Cytotoxicity mechanism of the RNase Sa cationic mutants involves inhibition of potassium current through Ca2+-activated channels

Bacterial ribonucleases (RNases) are considered to be potential anticancer agents. One of most important determinants of RNase cytotoxicity is the net charge of the molecule. In this work a set of mutants of the RNase from Streptomyces aureofaciens (RNase Sa), differing in the net charge of the protein molecules (from -7 to +6) and localization of additional positive charge at the N- or C-terminus of the molecule is used to study inhibition of cell growth. It has been found that the mutants of RNase with increased cationicity most effectively inhibit the growth of HEKhSK4 cells. Additional positive charge at the C-terminus of the molecule also increases the cytotoxic properties of RNases. It has been shown that RNase cytotoxicity correlated with the level of inhibition of the K+-current in cells.

Drug Trapping in hERG Channels does not Require Closure of the Activation Gate

Human ether-a-go-go related gene (hERG) channel inhibitors can be trapped in the channels at rest. The structural peculiarities of hERG blockers that enable trapping or alternatively resting state dissociation are currently unknown. Propafenone (small molecule, MW 341 g/mol) is efficiently trapped in the closed hERG channel pore (1). To investigate whether the size of the blocking molecule plays a role in trapping we synthesized bulky propafenone derivatives containing benzoyl and trimethylphenyl side chains, attached by piperazine linkers, with molecular weights of 500 (Fba212) and 650 g/mol (Fba213) respectively. hERG channels were expressed in Xenopus laevis oocyte and potassium current inhibition was studied using the two - microelectrode voltage clamp technique.It was found: first, both compounds are potent hERG blockers with IC50 3.7μM (Fba212) and 52μM (Fba213). Secondly, channel block by Fba212 and 213 was prevented by mutations Y652A and F656A as previously shown for propafenone. Third, both propafenone derivatives were trapped at rest. To obtain insights into the molecular mechanism of channel block docking experiments with Fba212 and Fba213 in closed and open conformation were performed. Both compounds interact with the propafenone binding site (Y652A and F656A). Fba213 was found to exceed the size of the closed channel cavity of our hERG homology model. We conclude that drug trapping in hERG channels does not necessarily require full closure of the activation gate.1. Witchel HJ, Dempsey CE, Sessions RB, Perry M, Milnes JT, Hancox JC, Mitcheson JS.Mol Pharmacol. 2004 Nov; 66(5):1201-12.

Lanthanum Chloride Promoted Proliferation with Enhanced S‐phase Entry and Inhibited Potassium Currents of NIH 3T3 Cells

AbstractThe effects of La3+ on proliferation, cell cycles, apoptosis and ion channels were investigated in mouse embryo fibroblast NIH 3T3 cells and its possible mechanisms were explored. Our data showed that La3+ promoted cell proliferation with increased S‐phase entry and inhibited the outward potassium currents in a concentration‐dependent manner in NIH 3T3 cells. La3+ and Ca2+ had synergistic effect on cell proliferation and cell cycles. It showed that Ca2+ was needed for La3+‐promoted cell cycle progression. Using the whole‐cell voltage‐clamp technique, we found that La3+ blocked the outward potassium current in a concentration‐dependent manner in NIH 3T3 cells. Lanthanum ions can increase intracellular Ca2+ concentration through inhibition of potassium currents, which induce a series of physiological changes and improve proliferation of cells. This may be one of the molecular mechanisms of lanthanum ions induced cell proliferation. The present work provides a new perspective for understanding the biological and toxicological effects of lanthanum.

The Scaffold Protein NHERF2 Determines the Coupling of P2Y1 Nucleotide and mGluR5 Glutamate Receptor to Different Ion Channels in Neurons

Expressed metabotropic group 1 glutamate mGluR5 receptors and nucleotide P2Y1 receptors (P2Y1Rs) show promiscuous ion channel coupling in sympathetic neurons: their stimulation inhibits M-type [Kv7, K(M)] potassium currents and N-type (Ca(V)2.2) calcium currents (Kammermeier and Ikeda, 1999; Brown et al., 2000). These effects are mediated by G(q) and G(i/o) G-proteins, respectively. Via their C-terminal tetrapeptide, these receptors also bind to the PDZ domain of the scaffold protein NHERF2, which enhances their coupling to G(q)-mediated Ca(2+) signaling (Fam et al., 2005; Paquet et al., 2006b). We investigated whether NHERF2 could modulate coupling to neuronal ion channels. We find that coexpression of NHERF2 in sympathetic neurons (by intranuclear cDNA injections) does not affect the extent of M-type potassium current inhibition produced by either receptor but strongly reduced Ca(V)2.2 inhibition by both P2Y1R and mGluR5 activation. NHERF2 expression had no significant effect on Ca(V)2.2 inhibition by norepinephrine (via alpha(2)-adrenoceptors, which do not bind NHERF2), nor on Ca(V)2.2 inhibition produced by an expressed P2Y1R lacking the NHERF2-binding DTSL motif. Thus, NHERF2 selectively restricts downstream coupling of mGluR5 and P2Y1Rs in neurons to G(q)-mediated responses such as M-current inhibition. Differential distribution of NHERF2 in neurons may therefore determine coupling of mGluR5 receptors and P2Y1 receptors to calcium channels.

The Mechanism for Prejunctional Enhancement of Neuromuscular Transmission by Ethanol in the Mouse

Ethanol has been shown to have both presynaptic and postsynaptic effects on synaptic transmission. However, the mechanisms by which ethanol affects evoked neurotransmitter release have not been studied at the mouse neuromuscular junction, a synapse at which binomial analysis of neurotransmitter release and measurements of prejunctional ionic currents can be made. Ethanol (400 mM) increased neurotransmitter release independently of both the cAMP and phorbol ester/Munc13 signaling pathways. Binomial analysis of neurotransmitter release revealed that ethanol increases the average probability of secretion without an effect on the immediately available store of the neurotransmitter. Application of ethanol also resulted in an inhibition of potassium currents in the motor nerve endings. These results suggest that the potentiating effects of ethanol on neurotransmitter release at the skeletal neuromuscular junction are mediated by an inhibition of the delayed rectifier potassium current, thus increasing both calcium entry into the nerve ending and the probability of neurotransmitter release. Identifying the mechanism through which ethanol enhances neurotransmitter release at the neuromuscular junction may be useful in determining the processes underlying the enhancement of neurotransmitter release at other synapses.

HSV-1 promotes Ca2+-mediated APP phosphorylation and Aβ accumulation in rat cortical neurons

Epidemiological and experimental findings suggest that chronic infection with Herpes simplex virus type 1 (HSV-1) may be a risk factor for Alzheimer's disease (AD), but the molecular mechanisms underlying this association have not been fully identified. We investigated the effects of HSV-1 on excitability and intracellular calcium signaling in rat cortical neurons and the impact of these effects on amyloid precursor protein (APP) processing and the production of amyloid-β peptide (Aβ). Membrane depolarization triggering firing rate increases was observed shortly after neurons were challenged with HSV-1 and was still evident 12 hours postinfection. These effects depended on persistent sodium current activation and potassium current inhibition. The virally induced hyperexcitability triggered intracellular Ca(2+) signals that significantly increased intraneuronal Ca(2+) levels. It also enhanced activity- and Ca(2+)-dependent APP phosphorylation and intracellular accumulation of Aβ42. These findings indicate that HSV-1 causes functional changes in cortical neurons that promote APP processing and Aβ production, and they are compatible with the co-factorial role for HSV-1 in the pathogenesis of AD suggested by previous findings.

Muscarinic Acetylcholine Receptors (mAChRs) in the Nervous System: Some Functions and Mechanisms

This article summarizes some of the effects of stimulating muscarinic receptors on nerve cell activity as observed by recording from single nerve cells and cholinergic synapses in the peripheral and central nervous sytems. It addresses the nature of the muscarinic receptor(s) involved and the ion channels and subcellular mechanisms responsible for the effects. The article concentrates on three effects: postsynaptic excitation, postsynaptic inhibition, and presynaptic (auto) inhibition. Postsynaptic excitation results primarily from the inhibition of potassium currents by M(1)/M(3)/M(5) receptors, consequent upon activation of phospholipase C by the G protein Gq. Postsynaptic inhibition results from M2-activation of inward rectifier potassium channels, consequent upon activation of Gi. Presynaptic inhibition results from M(2) or M(4) inhibition of voltage-gated calcium channels, consequent upon activation of Go. The segregation receptors, G proteins and ion channels, and the corelease of acetylcholine and glutamate from cholinergic fibres in the brain are also discussed.

Blockade of hERG K+ channel by antimalarial drug, primaquine

Lengthening of the Q-T interval and proarrhythmia are adverse effects associated with antimalarial agents. Also, lengthening of the Q-T interval is a definite outcome when patients are administered with an overdose of primaquine. Inhibition of potassium current I (Kr) and resultant QT prolongation is suggested as the reason behind drug-induced arrhythmias. The present study investigated the molecular mechanisms of voltage-dependent inhibition of human Ether-a-go-go Related Gene (hERG) delayed rectifier K(+) channels expressed in HEK-293 cells by primaquine. Primaquine inhibited hERG current in a concentration-dependent manner with the half-maximal inhibitory concentration (IC(50)) of 21.5 microM. The voltage-dependent inhibition of hERG current resulted in the activation curve to be shifted to a negative voltage after primaquine exposure in a dose-dependent manner. Blockade of hERG by primaquine was also found to be time-dependent, occurring rather rapidly. Blockade of wild-type hERG channel by primaquine was similar to those of both the S6 residue hERG mutants (F656A and Y652A) and the pore region mutants (T623A). In conclusion, these results indicate that primaquine preferentially inhibits the hERG potassium channel, but blockade of hERG channel by primaquine may not be related to the S6 residue or the pore region, but may be induced through other pathways such as binding other region or effect by drug binding receptor which indicates a need for further exploration.

Inhibition of potassium currents as a pharmacologic target for investigation in chronic lymphocytic leukemia

Chronic lymphocytic leukemia (CLL) represents 22-30% of all leukemia cases, thus being the most commonly diagnosed form of adult leukemia in the Western world. On a cellular level, the disease progresses due to the prolonged survival of B-cell CLL cells arrested in the G₀ stage of the cell cycle. The current standard treatment for CLL is a combination regimen containing purine analogues and monoclonal antibodies. Although response rates to such regimens in previously untreated patients are high, patients with CLL invariably experience relapse and often acquire high-risk chromosomal abnormalities. Therefore, the search for novel avenues in CLL treatment is warranted. In this manuscript, we will describe theoretical premises and some preliminary data making the case for inhibitors of the potassium currents as possible proapoptotic agents that warrant investigation as a potential pharmacologic target in CLL.