Increase In Potassium Current Research Articles

Targeting heterozygous dominant negative variant of KCNA2 using Gapmer ASO for the treatment of drug-resistant epilepsy

A missense mutation c.1220C>G of KCN2A gene was recently identified in an infant with epilepsy. KCNA2 encodes Kv1.2 subunits that form voltage-gated potassium channels (VGKC) via tetrameric assembly. The mutation results in amino acid change P407R at the highly conserved PVP motif. Functional characterization revealed that mutant Kv1.2_P407R subunits formed loss-of-function channels and suppressed both Kv1.2 and Kv1.1 channel activities. Hetero-tetrameric assembly of the Kv1.2_P407R subunits with other neuronal voltage-gated potassium channels of Shaker subfamily could lead to general deficit of repolarising potassium current and potentially underlie the enhanced seizure susceptibility. Indeed, expression of human Kv1.2_P407R in early postnatal rat cortical neurons or genetically engineered hESC-derived neurons disclosed broadening of action potential duration and early afterdepolarization (EAD), associating with reduced potassium current. We hypothesize that Gapmer antisense oligonucleotides (ASO) targeted to c.1220C>G mutation will selectively degrade the mutant mRNA while allowing the remaining WT subunits to form functional channels. As a proof of principle, delivery of Gapmer packaged in lipid nanoparticle into cortical neurons selectively suppressed Kv1.2_P407R over the WT protein expression, reversing the broadening of action potential duration, abrogating the EAD and leading to overall increase in potassium current.

Effects of cisplatin on potassium currents in CT26 cells.

Cisplatin, a platinum-based drug, is an important weapon against many types of cancer. It is well-known that cisplatin induces apoptosis. Potassium channel plays very important role in several signaling pathways. To investigate the possibility that potassium channels also have a role in the cellular response to cisplatin, we examined the effect of cisplatin on the activity of potassium channels on CT26 cell, the colon carcinoma cell line. The cells were cultured in DMEM, supplemented with 10< heat-inactivated fetal bovine serum. At mid-log phase, cultures were harvested, washed twice in phosphate-buffered saline, and resuspended in culture medium before use. Cells were voltage-clamped using the whole-cell patch clamp technique. Membrane current data were collected and amplified. Differences between two groups were assessed by paired t-test and one sample t-test to compare the relative values. One-way ANOVA was used for all experiment with more than two groups. Potassium currents were detected in CT26 cells and the currents were reduced by the application of tetraethylammonium (TEA) chloride, iberiotoxin, a big conductance calcium-activated potassium channel blocker and barium. The potassium currents were enhanced to 192< by the application of cisplatin (0.5 mM). Moreover, the increase of potassium currents by cisplatin was further inhibited by the application of TEA confirming the action of cisplatin on potassium channels. In addition, relative current induced by cisplatin in CT26 cells was bit larger than in normal IEC-6 cells. Potassium currents were detected in CT26 cells and the currents were reduced by the application of tetraethylammonium (TEA) chloride, iberiotoxin, a big conductance calcium-activated potassium channel blocker and barium. The potassium currents were enhanced to 192< by the application of cisplatin (0.5 mM). Moreover, the increase of potassium currents by cisplatin was further inhibited by the application of TEA confirming the action of cisplatin on potassium channels. In addition, relative current induced by cisplatin in CT26 cells was bit larger than in normal IEC-6 cells.

Cholinergic input from the pedunculopontine nucleus to the cerebellum: implications for deep brain stimulation in Parkinson's disease.

Deep brain stimulation (DBS) is a well established electrophysiological treatment initially applied to treat medication-refractory motor symptoms in Parkinson's disease (PD), and is now being explored for several neurological and psychiatric disorders. The specific physiological mechanisms underlying the effectiveness of DBS are not fully understood, although some hypothesized general mechanisms may be acceptable (Wichmann and DeLong, 2016). Early hypotheses suggested that stimulation of the subthalamic nucleus (STN) in PD produced the same clinical effect as a lesion. In other words, DBS was initially considered to suppress or modulate the abnormal bursting discharge patterns that occur in STN neurons in parkinsonian patients. Several mechanisms have been proposed to explain this effect, invoking what would happen at the site of stimulation and/or in the neuronal circuitry to which the targeted region for stimulation is functionally connected. These mechanisms include depolarization block caused by increase of potassium currents, inactivation of sodium channels, presynaptic depression of excitatory afferents, and stimulation-induced activation of inhibitory afferents. However, evidence in favor of activation of STN neurons was also provided, possibly mediated by stimulation-induced activation of excitatory projections from the motor cortex, or by a direct effect of stimulation directly on STN neurons. Moreover, neuronal activity may be phase locked to the pulse train, with following frequencies dictated by the stimulus interval.

Contribution of BKCa channels of neurons in rostral ventrolateral medulla to CO-mediated central regulation of respiratory rhythm in medullary slices of neonatal rats

We recently described that carbon monoxide (CO) participated in the regulation of rhythmic respiration in medullary slices. The present study was undertaken to further assess whether the large-conductance calcium-activated potassium channels (BKCa channels) are involved in the CO-mediated central regulation of respiratory rhythm in medullary slices. The rhythmic discharge of hypoglossal rootlets of medullary slices of neonatal rats was recorded. We observed that blocking BKCa channels could partially abolish the effects of CO on the rhythmic bursts of hypoglossal rootlets. With whole-cell patch-clamp recording technique, we further observed that CO could reversibly augment potassium current density of the neurons in the rostral ventrolateral medulla. The CO-induced increase in potassium current was entirely blocked by the pretreatment of slices with BKCa channels blocker; whereas blockade of CO generation with zinc protoporphyrin-IX produced an opposite response. Altogether, these data indicate that BKCa channels of the neurons in neonatal rostral ventrolateral medulla could be activated by CO and involved in CO-mediated central regulation of respiratory rhythm in medullary slices.

Ethanol Affects Striatal Interneurons Directly and Projection Neurons Through a Reduction in Cholinergic Tone

The acute effects of ethanol on the neurons of the striatum, a basal ganglia nucleus crucially involved in motor control and action selection, were investigated using whole-cell recordings. An intoxicating concentration of ethanol (50 mM) produced inhibitory effects on striatal large aspiny cholinergic interneurons (LAIs) and low-threshold spike interneurons (LTSIs). These effects persisted in the presence of tetrodotoxin and were because of an increase in potassium currents, including those responsible for medium and slow afterhyperpolarizations. In contrast, fast-spiking interneurons (FSIs) were directly excited by ethanol, which depolarized these neurons through the suppression of potassium currents. Medium spiny neurons (MSNs) became hyperpolarized in the presence of ethanol, but this effect did not persist in the presence of tetrodotoxin and was mimicked and occluded by application of the M1 muscarinic receptor antagonist telenzepine. Ethanol effects on MSNs were also abolished by 100 μM barium. This showed that the hyperpolarizations observed in MSNs were because of decreased tonic activation of M1 muscarinic receptors, resulting in an increase in Kir2 conductances. Evoked GABAergic responses of MSNs were reversibly decreased by ethanol with no change in paired-pulse ratio. Furthermore, ethanol impaired the ability of thalamostriatal inputs to inhibit a subsequent corticostriatal glutamatergic response in MSNs. These results offer the first comprehensive description of the highly cell type-specific effects of ethanol on striatal neurons and provide a cellular basis for the interpretation of ethanol influence on a brain area crucially involved in the motor and decisional impairment caused by this drug.

Angiotensin II Regulates Adrenal Vascular Tone Through Zona Glomerulosa Cell–Derived EETs and DHETs

Elevated concentrations of aldosterone are associated with several cardiovascular diseases. Angiotensin II (Ang II) increases aldosterone secretion and adrenal blood flow. This concurrent increase in steroidogenesis and adrenal blood flow is not understood. We investigated the role of zona glomerulosa (ZG) cells in the regulation of vascular tone of bovine adrenal cortical arteries by Ang II. ZG cells enhanced endothelium-dependent relaxations to Ang II. The ZG cell-dependent relaxations to Ang II were unchanged by removing the endothelium-dependent response to Ang II. These ZG cell-mediated relaxations were ablated by cytochrome P450 inhibition, epoxyeicosatrienoic acid (EET) antagonism, and potassium channel blockade. Analysis of ZG cell EET production by liquid chromatography/mass spectrometry demonstrated an increase in EETs and dihydroxyeicosatrienoic acids with Ang II stimulation. These EETs and dihydroxyeicosatrienoic acids produced similar concentration-dependent relaxations of adrenal arteries, which were attenuated by EET antagonism. Whole-cell potassium currents of adrenal artery smooth muscle cells were increased by Ang II stimulation in the presence of ZG cells but decreased in the absence of ZG cells. This increase in potassium current was abolished by iberiotoxin. Similarly, 14,15-EET induced concentration-dependent increases in potassium current, which was abolished by iberiotoxin. ZG cell aldosterone release was not directly altered by EETs. These data suggest that Ang II stimulates ZG cells to release EETs and dihydroxyeicosatrienoic acids, resulting in potassium channel activation and relaxation of adrenal arteries. This provides a mechanism by which Ang II concurrently increases adrenal blood flow and steroidogenesis.

The synergetic modulation of the excitability of central gray matter by a neuropeptide: two protocols using excitation waves in chick retina

The isolated chick retina provides an in vitro tissue model, in which two protocols were developed to verify the efficacy of a peptide in the excitability control of the central gray matter. In the first, extra-cellular potassium homeostasis is challenged at long intervals and in the second, a wave is trapped in a ring of tissue causing the system to be under self-sustained challenge. Within the neuropil, the extra-cellular potassium transient observed in the first protocol was affected from the initial rising phase to the final concentration at the end of the five-minute pulse. There was no change in the concomitants of excitation waves elicited by the extra-cellular rise of potassium. However, there was an increase on the elicited waves latency and/or a rise in the threshold potassium concentration for these waves to appear. In the second protocol, the wave concomitants and the propagation velocity were affected by the peptide. The results suggest a synergetic action of the peptide on glial and synaptic membranes: by accelerating the glial Na/KATPase and changing the kinetics of the glial potassium channels, with glia tending to accumulate KCl. At the same time, there is an increase in potassium currents through nerve terminals.

Histamine Enhances Voltage-Gated Potassium Currents of ON Bipolar Cells in Macaque Retina

The goal was to understand the functions of retinopetal axons containing histamine. In prior work, type 3 histamine receptors (HR3) have been localized to the tips of ON bipolar cell dendrites in macaque retinas. Voltage-gated potassium channels have also been localized to bipolar cell dendrites, and the hypothesis tested in the present study was that these are modulated by histamine. Whole-cell recordings of potassium currents were made from bipolar cells in slice preparations of macaque retina. In voltage-clamp mode, the cells were held at -60 mV and stepped to values from -60 to 80 mV. Recordings of the membrane potential were also made in current-clamp mode. Histamine, the HR3 agonist (R) alpha-methylhistamine (RAMH), tetraethyl ammonium (TEA), and 4-aminopyridine (4-AP) were applied in the superfusate. Histamine produced a dose-dependent increase in potassium currents in a subset of bipolar cells. At 5 microM, histamine increased the currents by 15% or more in the ON bipolar cells but not in the OFF bipolar cells. RAMH at 5 microM increased the amplitude of the potassium currents in the ON bipolar cells. In 10 mM TEA, potassium currents were reduced in all the bipolar cells, and there was no effect of histamine. Histamine hyperpolarized the resting membrane potential of the ON bipolar cells by 5 mV. By enhancing potassium currents in the ON bipolar cells, histamine is expected to reduce the amplitude of the light responses and limit their duration. The hyperpolarization of the resting membrane potential would also reduce neurotransmitter release at their output synapses.

Repetitive Firing Triggers Clustering of Kv2.1 Potassium Channels in Aplysia Neurons

The Kv2.1 gene encodes a highly conserved delayed rectifier potassium channel that is widely expressed in neurons of the central nervous system. In the bag cell neurons of Aplysia, Kv2.1 channels contribute to the repolarization of action potentials during a prolonged afterdischarge that triggers a series of reproductive behaviors. Partial inactivation of Aplysia Kv2.1 during repetitive firing produces frequency-dependent broadening of action potentials during the afterdischarge. We have now found that, as in mammalian neurons, Kv2.1 channels in bag cell neurons are localized to ring-like clusters in the plasma membrane of the soma and proximal dendrites. Either elevation of cyclic AMP levels or direct electrical stimulation of afterdischarge rapidly enhanced formation of these clusters on the somata of these neurons. In contrast, injection of a 13-amino acid peptide corresponding to a region in the C terminus that is required for clustering of Kv2.1 channels produced disassociation of the clusters, resulting in a more uniform distribution over the somata. Voltage clamp recordings demonstrated that peptide-induced dissociation of the Kv2.1 clusters is associated with an increase in the amplitude of delayed rectifier current and a shift of activation toward more negative potentials. In current clamp recording, injection of the unclustering peptide reduced the width of action potentials and reduced frequency-dependent broadening of action potentials. Our results suggest that rapid redistribution of Kv2.1 channels occurs during physiological changes in neuronal excitability.

Human ether-a-go-go-related Gene 1 Channels Are Physically Linked to β1 Integrins and Modulate Adhesion-dependent Signaling

Adhesive receptors of the integrin family are primarily involved in cell-extracellular matrix adhesion. Additionally, integrins trigger multiple signaling pathways that are involved in cell migration, proliferation, survival, and differentiation. We previously demonstrated that the activation of integrins containing the beta(1) subunit leads to a selective increase in potassium currents carried by the human ether-a-go-go-related gene (hERG) channels in neuroblastoma and leukemia cells; this current activation modulates adhesion-dependent differentiation in these cells. We hypothesized that the cross-talk between integrins and hERG channels could be traced back to the assembly of a macromolecular signaling complex comprising the two proteins. We tested this hypothesis in both SH-SY5Y neuroblastoma cells and in human embryonic kidney 293 cells stably transfected with hERG1 and, therefore, expressing only the full-length hERG1 protein on the plasma membrane. The beta(1) integrin and hERG1 coprecipitate in these cells and colocalize in both intracellular and surface membrane compartments. The two proteins also coprecipitate with caveolin-1, suggesting the localization of the complex in lipid rafts/caveolae. hERG1-transfected cells undergo an activation of hERG currents after beta(1) integrin-mediated adhesion to fibronectin; concomitant with this activation, the focal adhesion kinase associates with the hERG1 protein and becomes tyrosine phosphorylated. Using hERG1-specific inhibitors, we show that the tyrosine phosphorylation of focal adhesion kinase is strictly dependent on hERG channel activity. Similarly, the activity of the small GTPase Rac1 turned out to be dependent on hERG currents. On the whole, these data indicate that the hERG1 protein associates with beta(1) integrins and modulates adhesion receptor signaling.

SAP97 increases Kv1.5 currents through an indirect N-terminal mechanism

The functional interaction of the voltage-gated potassium channel hKv1.5 with the PDZ domain containing protein SAP97 has been investigated. In marked contrast with the known dependence of SAP97-induced Kv1 potassium current down-regulation on the channel C-termini, SAP97 increased hKv1.5 current through an indirect interaction with the Kv1.5 N-terminus. Deletion of the Kv1.5 N-terminus eliminated the SAP97-mediated increase in potassium currents whereas deletion of the channel’s C-terminal PDZ binding motif had no effect. In contrast with other Kv1–SAP97 interactions, no physical interaction could be detected in vivo or in vitro between the two proteins. The proteins did not co-localize in cardiac myocytes nor did they co-immunoprecipitate from transfected HEK cells. Yeast two-hybrid experiments also failed to detect any interaction between the two proteins, but in one experiment of six, Kv1.5 co-immunoprecipitated very inefficiently with SAP97 from rat ventricular myocytes. Thus, we conclude that the influence of SAP97 on Kv1.5 potassium current levels is dependent upon a novel regulatory mechanism.

Tyrosine Phosphorylation of the κ-Opioid Receptor Regulates Agonist Efficacy

To explore the role of highly conserved tyrosine residues in the putative cytoplasmic domains of the seven-transmembrane G protein-coupled opioid receptors, we expressed the rat kappa-opioid receptor (KOR) in Xenopus oocytes and then activated the intrinsic insulin receptor tyrosine kinase. KOR activation by the agonist produced a strong increase in potassium current through coexpressed G protein-gated inwardly rectifying potassium channels (K(IR)3). Brief pretreatment with insulin caused a 60% potentiation of the KOR-activated response. The insulin-induced increase in kappa-opioid response was blocked by the tyrosine kinase inhibitor genistein. In contrast, insulin had no effect on the basal activity of K(IR)3, suggesting that KOR is the target of the tyrosine kinase cascade. Mutation of tyrosine residues to phenylalanines in either the first or second intracellular loop of KOR to produce KOR(Y87F) and KOR(Y157F) had no effect on either the potency or maximal effect of. However, neither KOR(Y87F)- nor KOR(Y157F)-mediated responses were potentiated by insulin treatment. Insulin pretreatment shifted the dose-response curve for activation of KOR by increasing the maximal response without changing the EC(50) value for. These results suggest that insulin increases the efficacy of KOR activation by phosphorylating two tyrosine residues in the first and second intracellular loops of the receptor. Thus, tyrosine phosphorylation may provide an important mechanism for modulation of G protein-coupled receptor signaling.

Niflumic acid-induced increase in potassium currents in frog motor nerve terminals: effects on transmitter release

The actions of the nonsteroidal antiinflammatory drug niflumic acid were studied on frog neuromuscular preparations by conventional electrophysiological techniques. Niflumic acid reduced the amplitude and increased the latency of endplate potentials in a concentration-dependent manner. Neuromuscular junctions pretreated with niflumic acid (0.05–0.5 mM) showed much less depression than control when they were stimulated with trains of impulses. Inhibition of acetylcholine release was reverted by raising the extracellular Ca 2+ concentration but not by simply washing out the preparations with niflumic acid-free solutions. Pretreatment with indomethacin (0.1 mM), another nonsteroidal antiinflamatory drug, did not affect the niflumic acid-induced inhibition of evoked responses. Niflumic acid (0.1 mM) did not change the amplitude of miniature endplate potentials and had a dual action on the frequency of miniatures: it decreased their frequency at 0.1 mM whereas it produced an enormous increase in the rate of spontaneous discharge at 0.5 mM. Niflumic acid (0.1–1 mM) reversibly increased the amplitude and affected the kinetics of presynaptic voltage-activated K + current and Ca 2+-activated K + current in a concentration-dependent manner. Niflumic acid (0.1–1 mM) irreversibly decreased the amplitude and reversibly affected the kinetics of the nodal Na + current. Indomethacin (0.1 mM) had no effect on presynaptic currents. In conclusion, niflumic acid reduces acetylcholine release by increasing presynaptic K + currents. This may shorten the depolarizing phase of the presynaptic action potential and may reduce the entry of Ca 2+ with each impulse.

Carbon monoxide stimulates a potassium-selective current in rabbit corneal epithelial cells.

The effects of CO on ion currents in freshly dispersed rabbit corneal epithelial cells were assessed using the perforated patch whole cell voltage-clamp technique. Bath perfusion with 1% CO resulted in a 84 +/- 18% (mean +/- SE, n = 14) increase in potassium current (IK) and a membrane hyperpolarization from -42 +/- 4 to -51 +/- 4 mV. The CO-stimulated current reversed at -64 +/- 7 mV [reverse potential (EK) = -87 mV]. The stimulated current was blocked by 1 mM quinidine or 1 mM diltiazem, agents that inhibit IK in rabbit corneal epithelial cells. Single potassium-channel currents measured in the cell-attached configuration showed that exogenous CO increased the steady-state open probability from 0.003 to 0.156 at a holding potential of -40 mV. CO did not affect open probability in excised patches. The single-channel conductance measured from -40 to +40 mV was unaffected. Intracellular guanosine 3',5'-cyclic monophosphate (cGMP) concentration measured with radioimmunoassay techniques was found to increase from 0.41 +/- 0.24 to 0.55 +/- 0.27 pmol/10(6) cells after the addition of 1% CO (P < 0.05). The data show that bath perfusion with exogenous CO activates IK and hyperpolarizes the resting membrane potential; the data also suggest that CO modulates intracellular cGMP concentration.

Differential G protein-mediated coupling of D 2 dopamine receptors to K + and Ca 2+ currents in rat anterior pituitary cells

In anterior pituitary cells, dopamine, acting on D 2 dopamine receptors, concomitantly reduces calcium currents and increases potassium currents. These dopamine effects require the presence of intracellular GTP and are blocked by pretreatment of the cells with pertussis toxin, suggesting that one or more G protein is involved. To identify the G proteins involved in coupling D 2 receptors to these currents, we performed patch-clamp recordings in the whole-cell configuration using pipettes containing affinity-purified polyclonal antibodies raised against either G oα, G 3α, or G i1,2α. Dialysis with G oα antiserum significantly reduced the inhibition of calcium currents induced by dopamine, while increase of potassium currents was markedly attenuated only by G i3α antiserum. We therefore conclude that in pituitary cells, two different G proteins are involved in the signal transduction mechanism that links D 2 receptor activation to a specific modulation of the four types of ionic channels studied here.

Free radicals mediate actions of oxyhemoglobin on cerebrovascular smooth muscle cells.

Single smooth muscle cells were isolated from the basilar artery of the rat by enzymatic dispersion. The membrane properties of the cells were assessed using the patch-electrode voltage-clamp technique, and cell viability was monitored using fluorescein diacetate uptake. Exposure of the cells to oxyhemoglobin (5 microM) resulted in 1) contraction, 2) the appearance of membrane blebs, 3) an increase in the outward potassium currents, 4) a decrease in the membrane resistance, and 5) cell death. In contrast, no effect of oxyhemoglobin on cultured murine neuroblastoma cells was observed. Methemoglobin (100 microM) had no effects on the smooth muscle cells. Catalase (300 units/ml) or dimethyl sulfoxide (0.5%) protected against the effects of oxyhemoglobin; superoxide dismutase (100-1,000 units/ml) provided only partial protection. Exposure of the cells to superoxide anions generated by xanthine (1 mM) plus xanthine oxidase (10 units/l) or to hydrogen peroxide (500 microM) caused an increase in the outward potassium currents without affecting membrane resistance. Generation of hydroxyl radicals by metal ions plus hydrogen peroxide caused the same effects as oxyhemoglobin, that is, an increase in the potassium currents, followed by a decrease in the membrane resistance and cell death. In conclusion, it appears that oxyhemoglobin exerts its effects on vascular smooth muscle cells by the generation of free radicals, chiefly hydroxyl radicals.