Activation Of IK Research Articles

A mutation in the cardiac KV7.1 channel possibly disrupts interaction with Yotiao protein

Here, we characterized the p.Arg583His (R583H) Kv7.1 mutation, identified in two unrelated families suffered from LQT syndrome. This mutation is located in the HС-HD linker of the cytoplasmic portion of the Kv7.1 channel. This linker, together with HD helix are responsible for binding the A-kinase anchoring protein 9 (AKAP9), Yotiao. We studied the electrophysiological characteristics of the mutated channel expressed in CHO–K1 along with KCNE1 subunit and Yotiao protein, using the whole-cell patch-clamp technique. We found that R583H mutation, even at the heterozygous state, impedes IKs activation. Molecular modeling showed that HС and HD helixes of the C-terminal part of Kv7.1 channel are swapped along the C-terminus length of the channel and that R583 position is exposed to the outer surface of HC-HD tandem coiled-coil. Interestingly, the adenylate cyclase activator, forskolin had a smaller effect on the mutant channel comparing with the WT protein, suggesting that R583H mutation may disrupt the interaction of the channel with the adaptor protein Yotiao and, therefore, may impair phosphorylation of the KCNQ1 channel.

Evaluating sequential and allosteric activation models in IKs channels with mutated voltage sensors.

The ion-conducting IKs channel complex, important in cardiac repolarization and arrhythmias, comprises tetramers of KCNQ1 α-subunits along with 1-4 KCNE1 accessory subunits and calmodulin regulatory molecules. The E160R mutation in individual KCNQ1 subunits was used to prevent activation of voltage sensors and allow direct determination of transition rate data from complexes opening with a fixed number of 1, 2, or 4 activatable voltage sensors. Markov models were used to test the suitability of sequential versus allosteric models of IKs activation by comparing simulations with experimental steady-state and transient activation kinetics, voltage-sensor fluorescence from channels with two or four activatable domains, and limiting slope currents at negative potentials. Sequential Hodgkin-Huxley-type models approximately describe IKs currents but cannot explain an activation delay in channels with only one activatable subunit or the hyperpolarizing shift in the conductance-voltage relationship with more activatable voltage sensors. Incorporating two voltage sensor activation steps in sequential models and a concerted step in opening via rates derived from fluorescence measurements improves models but does not resolve fundamental differences with experimental data. Limiting slope current data that show the opening of channels at negative potentials and very low open probability are better simulated using allosteric models of activation with one transition per voltage sensor, which implies that movement of all four sensors is not required for IKs conductance. Tiered allosteric models with two activating transitions per voltage sensor can fully account for IKs current and fluorescence activation kinetics in constructs with different numbers of activatable voltage sensors.

KCNQ1 p.D446E Variant as a Risk Allele for Arrhythmogenic Phenotypes: Electrophysiological Characterization Reveals a Complex Phenotype Affecting the Slow Delayed Rectifier Potassium Current (IKs) Voltage Dependence by Causing a Hyperpolarizing Shift and a Lack of Response to Protein Kinase A Activation.

Genetic testing is crucial in inherited arrhythmogenic channelopathies; however, the clinical interpretation of genetic variants remains challenging. Incomplete penetrance, oligogenic, polygenic or multifactorial forms of channelopathies further complicate variant interpretation. We identified the KCNQ1/p.D446E variant in 2/63 patients with long QT syndrome, 30-fold more frequent than in public databases. We thus characterized the biophysical phenotypes of wildtype and mutant IKs co-expressing these alleles with the β-subunit minK in HEK293 cells. KCNQ1 p.446E homozygosity significantly shifted IKs voltage dependence to hyperpolarizing potentials in basal conditions (gain of function) but failed to shift voltage dependence to hyperpolarizing potentials (loss of function) in the presence of 8Br-cAMP, a protein kinase A activator. Basal IKs activation kinetics did not differ among genotypes, but in response to 8Br-cAMP, IKs 446 E/E (homozygous) activation kinetics were slower at the most positive potentials. Protein modeling predicted a slower transition of the 446E Kv7.1 tetrameric channel to the stabilized open state. In conclusion, biophysical and modelling evidence shows that the KCNQ1 p.D446E variant has complex functional consequences including both gain and loss of function, suggesting a contribution to the pathogenesis of arrhythmogenic phenotypes as a functional risk allele.

A generic binding pocket for small molecule IKs activators at the extracellular inter-subunit interface of KCNQ1 and KCNE1 channel complexes

The cardiac IKs ion channel comprises KCNQ1, calmodulin, and KCNE1 in a dodecameric complex which provides a repolarizing current reserve at higher heart rates and protects from arrhythmia syndromes that cause fainting and sudden death. Pharmacological activators of IKs are therefore of interest both scientifically and therapeutically for treatment of IKs loss-of-function disorders. One group of chemical activators are only active in the presence of the accessory KCNE1 subunit and here we investigate this phenomenon using molecular modeling techniques and mutagenesis scanning in mammalian cells. A generalized activator binding pocket is formed extracellularly by KCNE1, the domain-swapped S1 helices of one KCNQ1 subunit and the pore/turret region made up of two other KCNQ1 subunits. A few residues, including K41, A44 and Y46 in KCNE1, W323 in the KCNQ1 pore, and Y148 in the KCNQ1 S1 domain, appear critical for the binding of structurally diverse molecules, but in addition, molecular modeling studies suggest that induced fit by structurally different molecules underlies the generalized nature of the binding pocket. Activation of IKs is enhanced by stabilization of the KCNQ1-S1/KCNE1/pore complex, which ultimately slows deactivation of the current, and promotes outward current summation at higher pulse rates. Our results provide a mechanistic explanation of enhanced IKs currents by these activator compounds and provide a map for future design of more potent therapeutically useful molecules.

Development of IKs activating compounds for treatment of long QT syndrome.

The purpose of this study is to optimize the therapeutic potential of IKs (KCNQ1/KCNE1) channel activating polyunsaturated fatty acids (PUFAs) by using computational and experimental methods to increase their potency and specificity. We are interested in activating the IKs channel because loss of function mutations in this channel can cause long QT syndrome, specifically type 1 (LQT1). LQT1 can lead to certain cardiac arrhythmia and even sudden cardiac death. We hypothesized that using computational methods, such as site identification by ligand competitive saturation (SILCS), combined with physiological experiments will provide sufficient information for us to edit our PUFA structures to enhance their binding affinities and thus IKs activation. We recently developed a new compound using these methods that has increased activation effects and affinity for the IKs channel and we are now working to optimize this further.

Prolonged repolarization in the early phase of ischemia is associated with ventricular fibrillation development in a porcine model.

Background: Repolarization prolongation can be the earliest electrophysiological change in ischemia, but its role in arrhythmogenesis is unclear. The aim of the present study was to evaluate the early ischemic action potential duration (APD) prolongation concerning its causes, expression in ECG and association with early ischemic ventricular fibrillation (phase 1A VF). Methods: Coronary occlusion was induced in 18 anesthetized pigs, and standard 12 lead ECG along with epicardial electrograms were recorded. Local activation time (AT), end of repolarization time (RT), and activation-repolarization interval (ARIc) were determined as dV/dt minimum during QRS-complex, dV/dt maximum during T-wave, and rate-corrected RT-AT differences, respectively. Patch-clamp studies were done in enzymatically isolated porcine cardiomyocytes. IK(ATP) activation and Ito1 inhibition were tested as possible causes of the APD change. Results: During the initial period of ischemia, a total of 11 pigs demonstrated maximal ARIc prolongation >10ms at 1 and/or 2.5min of occlusion (8 and 6 cases at 1 and 2.5min, respectively) followed by typical ischemic ARIc shortening. The maximal ARIc across all leads was associated with VF development (OR 1.024 95% CI 1.003-1.046, p = 0.025) and maximal rate-corrected QT interval (QTc) (B 0.562 95% CI 0.346-0.775, p < 0.001) in logistic and linear regression analyses, respectively. Phase 1A VF incidence was associated with maximal QTc at the 2.5min of occlusion in ROC curve analysis (AUC 0.867, p = 0.028) with optimal cut-off 456ms (sensitivity 1.00, specificity 0.778). The pigs having maximal QTc at 2.5min more and less than 450ms significantly differed in phase 1A VF incidence in Kaplan-Meier analysis (log-rank p = 0.007). In the patch-clamp experiments, 4-aminopyridine did not produce any effects on the APD; however, pinacidil activated IK(ATP) and caused a biphasic change in the APD with initial prolongation and subsequent shortening. Conclusion: The transiently prolonged repolarization during the initial period of acute ischemia was expressed in the prolongation of the maximal QTc interval in the body surface ECG and was associated with phase 1A VF. IK(ATP) activation in the isolated cardiomyocytes reproduced the biphasic repolarization dynamics observed in vivo, which suggests the probable role of IK(ATP) in early ischemic arrhythmogenesis.

Endothelial IK and SK channel activation decreases pulmonary arterial pressure and vascular remodeling in pulmonary hypertension.

Endothelial cells (ECs) from small pulmonary arteries (PAs) release nitric oxide (NO) and prostacyclin, which lower pulmonary arterial pressure (PAP). In pulmonary hypertension (PH), the levels of endothelium-derived NO and prostacyclin are reduced, contributing to elevated PAP. Small-and intermediate-conductance Ca2+-activated K+ channels (IK and SK)-additional crucial endothelial mediators of vasodilation-are also present in small PAs, but their function has not been investigated in PH. We hypothesized that endothelial IK and SK channels can be targeted to lower PAP in PH. Whole-cell patch-clamp experiments showed functional IK and SK channels in ECs, but not smooth muscle cells, from small PAs. Using a SU5416 plus chronic hypoxia (Su + CH) mouse model of PH, we found that currents through EC IK and SK channels were unchanged compared with those from normal mice. Moreover, IK/SK channel-mediated dilation of small PAs was preserved in Su + CH mice. Consistent with previous reports, endothelial NO levels and NO-mediated dilation were reduced in small PAs from Su + CH mice. Notably, acute treatment with IK/SK channel activators decreased PAP in Su + CH mice but not in normal mice. Further, chronic activation of IK/SK channels decreased PA remodeling and right ventricular hypertrophy, which are pathological hallmarks of PH, in Su + CH mice. Collectively, our data provide the first evidence that, unlike endothelial NO release, IK/SK channel activity is not altered in PH. Our results also demonstrate proof of principle that IK/SK channel activation can be used as a strategy for lowering PAP in PH.

IKCa channels control breast cancer metabolism including AMPK-driven autophagy

Ca2+-activated K+ channels of intermediate conductance (IK) are frequently overexpressed in breast cancer (BC) cells, while IK channel depletion reduces BC cell proliferation and tumorigenesis. This raises the question, of whether and mechanistically how IK activity interferes with the metabolic activity and energy consumption rates, which are fundamental for rapidly growing cells. Using BC cells obtained from MMTV-PyMT tumor-bearing mice, we show that both, glycolysis and mitochondrial ATP-production are reduced in cells derived from IK-deficient breast tumors. Loss of IK altered the sub-/cellular K+- and Ca2+- homeostasis and mitochondrial membrane potential, ultimately resulting in reduced ATP-production and metabolic activity. Consequently, we find that BC cells lacking IK upregulate AMP-activated protein kinase activity to induce autophagy compensating the glycolytic and mitochondrial energy shortage. Our results emphasize that IK by modulating cellular Ca2+- and K+-dynamics contributes to the remodeling of metabolic pathways in cancer. Thus, targeting IK channel might disturb the metabolic activity of BC cells and reduce malignancy.

Evidence for Dual Activation of IK(M) and IK(Ca) Caused by QO-58 (5-(2,6-Dichloro-5-fluoropyridin-3-yl)-3-phenyl-2-(trifluoromethyl)-1H-pyrazolol[1,5-a]pyrimidin-7-one).

QO-58 (5-(2,6-dichloro-5-fluoropyridin-3-yl)-3-phenyl-2-(trifluoromethyl)-1H-pyrazolol[1,5-a]pyrimidin-7-one) has been regarded to be an activator of KV7 channels with analgesic properties. However, whether and how the presence of this compound can result in any modifications of other types of membrane ion channels in native cells are not thoroughly investigated. In this study, we investigated its perturbations on M-type K+ current (IK(M)), Ca2+-activated K+ current (IK(Ca)), large-conductance Ca2+-activated K+ (BKCa) channels, and erg-mediated K+ current (IK(erg)) identified from pituitary tumor (GH3) cells. Addition of QO-58 can increase the amplitude of IK(M) and IK(Ca) in a concentration-dependent fashion, with effective EC50 of 3.1 and 4.2 μM, respectively. This compound could shift the activation curve of IK(M) toward a leftward direction with being void of changes in the gating charge. The strength in voltage-dependent hysteresis (Vhys) of IK(M) evoked by upright triangular ramp pulse (Vramp) was enhanced by adding QO-58. The probabilities of M-type K+ (KM) channels that will be open increased upon the exposure to QO-58, although no modification in single-channel conductance was seen. Furthermore, GH3-cell exposure to QO-58 effectively increased the amplitude of IK(Ca) as well as enhanced the activity of BKCa channels. Under inside-out configuration, QO-58, applied at the cytosolic leaflet of the channel, activated BKCa-channel activity, and its increase could be attenuated by further addition of verruculogen, but not by linopirdine (10 μM). The application of QO-58 could lead to a leftward shift in the activation curve of BKCa channels with neither change in the gating charge nor in single-channel conductance. Moreover, cell exposure of QO-58 (10 μM) resulted in a minor suppression of IK(erg) amplitude in response to membrane hyperpolarization. The docking results also revealed that there are possible interactions of the QO-58 molecule with the KCNQ or KCa1.1 channel. Overall, dual activation of IK(M) and IK(Ca) caused by the presence of QO-58 eventually may have high impacts on the functional activity (e.g., anti-nociceptive effect) residing in electrically excitable cells. Care must be exercised when interpreting data generated with QO-58 as it is not entirely KCNQ/KV7 selective.

Optimization of novel lead compounds for treatment of long QT syndrome

The purpose of this study is to optimize the therapeutic potential of IKs (KCNQ1/KCNE1) channel activating Poly Unsaturated Fatty Acids (PUFAs) by using computational and experimental methods to increase their potency and specificity. We hypothesize that the computational method, Site Identification by Ligand Competitive Saturation (SILCS), will further elucidate the chemical environment and potential compound interactions in the known PUFA binding sites on the IKs channel allowing us to edit our PUFA structures to enhance their binding affinities and thus IKs activation.

Carbon monoxide activation of delayed rectifier potassium currents of human cardiac fibroblasts through diverse pathways.

To identify the effect and mechanism of carbon monoxide (CO) on delayed rectifier K+ currents (IK) of human cardiac fibroblasts (HCFs), we used the whole-cell mode patch-clamp technique. Application of CO delivered by carbon monoxide-releasing molecule-3 (CORM3) increased the amplitude of outward K+ currents, and diphenyl phosphine oxide-1 (a specific IK blocker) inhibited the currents. CORM3-induced augmentation was blocked by pretreatment with nitric oxide synthase blockers (L-NG-monomethyl arginine citrate and L-NG-nitro arginine methyl ester). Pretreatment with KT5823 (a protein kinas G blocker), 1H-[1,-2,-4] oxadiazolo-[4,-3-a] quinoxalin-1-on (ODQ, a soluble guanylate cyclase blocker), KT5720 (a protein kinase A blocker), and SQ22536 (an adenylate cyclase blocker) blocked the CORM3 stimulating effect on IK. In addition, pretreatment with SB239063 (a p38 mitogen-activated protein kinase [MAPK] blocker) and PD98059 (a p44/42 MAPK blocker) also blocked the CORM3’s effect on the currents. When testing the involvement of S-nitrosylation, pretreatment of N-ethylmaleimide (a thiol-alkylating reagent) blocked CO-induced IK activation and DL-dithiothreitol (a reducing agent) reversed this effect. Pretreatment with 5,10,15,20-tetrakis(1-methylpyridinium-4-yl)-21H,23H porphyrin manganese (III) pentachloride and manganese (III) tetrakis (4-benzoic acid) porphyrin chloride (superoxide dismutase mimetics), diphenyleneiodonium chloride (an NADPH oxidase blocker), or allopurinol (a xanthine oxidase blocker) also inhibited CO-induced IK activation. These results suggest that CO enhances IK in HCFs through the nitric oxide, phosphorylation by protein kinase G, protein kinase A, and MAPK, S-nitrosylation and reduction/oxidation (redox) signaling pathways.

Docosahexaenoic acid normalizes QT interval in long QT type 2 transgenic rabbit models in a genotype-specific fashion.

Long QT syndrome (LQTS) is a cardiac channelopathy predisposing to ventricular arrhythmias and sudden cardiac death. Since current therapies often fail to prevent arrhythmic events in certain LQTS subtypes, new therapeutic strategies are needed. Docosahexaenoic acid (DHA) is a polyunsaturated fatty acid, which enhances the repolarizing IKs current. We investigated the effects of DHA in wild type (WT) and transgenic long QT Type 1 (LQT1; loss of IKs), LQT2 (loss of IKr), LQT5 (reduction of IKs), and LQT2-5 (loss of IKr and reduction of IKs) rabbits. In vivo ECGs were recorded at baseline and after 10 µM/kg DHA to assess changes in heart-rate corrected QT (QTc) and short-term variability of QT (STVQT). Ex vivo monophasic action potentials were recorded in Langendorff-perfused rabbit hearts, and action potential duration (APD75) and triangulation were assessed. Docosahexaenoic acid significantly shortened QTc in vivo only in WT and LQT2 rabbits, in which both α- and β-subunits of IKs-conducting channels are functionally intact. In LQT2, this led to a normalization of QTc and of its short-term variability. Docosahexaenoic acid had no effect on QTc in LQT1, LQT5, and LQT2-5. Similarly, ex vivo, DHA shortened APD75 in WT and normalized it in LQT2, and additionally decreased AP triangulation in LQT2. Docosahexaenoic acid exerts a genotype-specific beneficial shortening/normalizing effect on QTc and APD75 and reduces pro-arrhythmia markers STVQT and AP triangulation through activation of IKs in LQT2 rabbits but has no effects if either α- or β-subunits to IKs are functionally impaired. Docosahexaenoic acid could represent a new genotype-specific therapy in LQT2.

Midazolam's Effects on Delayed-Rectifier K+ Current and Intermediate-Conductance Ca2+-Activated K+ Channel in Jurkat T-lymphocytes.

Midazolam (MDZ) could affect lymphocyte immune functions. However, the influence of MDZ on cell’s K+ currents has never been investigated. Thus, in the present study, the effects of MDZ on Jurkat T lymphocytes were studied using the patch-clamp technique. Results showed that MDZ suppressed the amplitude of delayed-rectifier K+ current (IK(DR)) in concentration-, time-, and state-dependent manners. The IC50 for MDZ-mediated reduction of IK(DR) density was 5.87 μM. Increasing MDZ concentration raised the rate of current-density inactivation and its inhibitory action on IK(DR) density was estimated with a dissociation constant of 5.14 μM. In addition, the inactivation curve of IK(DR) associated with MDZ was shifted to a hyperpolarized potential with no change on the slope factor. MDZ-induced inhibition of IK(DR) was not reversed by flumazenil. In addition, the activity of intermediate-conductance Ca2+-activated K+ (IKCa) channels was suppressed by MDZ. Furthermore, inhibition by MDZ on both IK(DR) and IKCa-channel activity appeared to be independent from GABAA receptors and affected immune-regulating cytokine expression in LPS/PMA-treated human T lymphocytes. In conclusion, MDZ suppressed current density of IK(DR) in concentration-, time-, and state-dependent manners in Jurkat T-lymphocytes and affected immune-regulating cytokine expression in LPS/PMA-treated human T lymphocytes.

Effects of modulation on sodium and potassium channel currents by extremely low frequency electromagnetic fields stimulation on hippocampal CA1 pyramidal cells

ABSTRACT To investigate the effects of extremely low-frequency electromagnetic fields (ELF-EMFs) stimulation on sodium channel currents (INa), transient outward potassium channel currents (IA) and delayed rectifier potassium channel currents (IK) on hippocampal CA1 pyramidal neurons of young Sprague–Dawley rats. CA1 pyramidal neurons of rat hippocampal slices were subjected to ELF-EMFs stimulation with different frequencies (15 and 50 Hz), intensities (0.5, 1 and 2 mT) and durations (10, 20 and 30 min). The INa, IA and IK of neurons were recorded by a whole-cell patch-clamp method. ELF-EMFs stimulation enhanced INa densities, and depressed IA and IK densities. In detail, INa was more sensitive to the variation of intensities and frequencies of ELF-EMFs, whereas IA and IK were mainly affected by the variation of the duration of ELF-EMFs. ELF-EMFs stimulation altered activation and deactivation properties of INa, IA and IK. ELF-EMFs stimulation plays a role as a regulator rather than an inducer for ion channels. It might change the transition probability of ion channel opening or closing, and might also change the structure and function of the ion channel which need to be proved by the further technical method.

Effectiveness in the inhibition of dapagliflozin and canagliflozin on M-type K+ current and α-methylglucoside-induced current in pituitary tumor (GH3) and pheochromocytoma PC12 cells

Dapagliflozin (DAPA) or canagliflozin (CANA), Na+-dependent glucose co-transporter type 2 (SGLT2) inhibitors, were used for treatment of type II diabetes mellitus. Addition of DAPA or CANA suppressed M-type K+ current (IK(M)) in pituitary tumor (GH3) and pheochromocytoma PC12 cells. The IC50 value for DAPA- or CANA-mediated inhibition of IK(M) in GH3 cells was 0.11 or 0.42 μM, respectively. The presence of DAPA (0.1 μM) shifted the steady-state activation of IK(M) to less depolarized potential without changing the gating charge of the current. During high-frequency depolarizing pulses, IK(M) magnitude was reduced by DAPA; however, DAPA-induced block of IK(M) remained effective. The amplitude of neither erg-mediated K+ current nor hyperpolarization-activated cation current in GH3 cells was modified in the presence of 1 μM DAPA. Alternatively, addition of DAPA, CANA, phlorizin or chlorotoxin effectively suppressed α-methylglucoside-(αMG-) induced current (IαMG) in GH3 cells, albeit inability of tefluthrin (activator of INa) to suppress this current. DAPA shifted the charge-voltage relation of presteady-state IαMG in a rightward and downward direction with no change in the gating charge of the IαMG. Under current-clamp recordings, subsequent additions of DAPA, but still in the continued presence of αMG, increased the firing rate of spontaneous action potentials stimulated by αMG. Our results suggested that activity of SGLT was expressed functionally in GH3 and PC12 cells. Therefore, inhibitory actions of DAPA or CANA on the amplitude and gating of IK(M) might provide a yet unidentified mechanism through which the SGLT1 or SGLT2 activity were attenuated in unclamped cells occurring in vivo.

Multiple Dynamical Mechanisms of Phase-2 Early Afterdepolarizations in a Human Ventricular Myocyte Model: Involvement of Spontaneous SR Ca 2+ Release.

Early afterdepolarization (EAD) is known to cause lethal ventricular arrhythmias in long QT syndrome (LQTS). In this study, dynamical mechanisms of EAD formation in human ventricular myocytes (HVMs) were investigated using the mathematical model developed by ten Tusscher and Panfilov (Am J Physiol Heart Circ Physiol 291, 2006). We explored how the rapid (IKr) and slow (IKs) components of delayed-rectifier K+ channel currents, L-type Ca2+ channel current (ICaL), Na+/Ca2+ exchanger current (INCX), and intracellular Ca2+ handling via the sarcoplasmic reticulum (SR) contribute to initiation, termination and modulation of phase-2 EADs during pacing in relation to bifurcation phenomena in non-paced model cells. Parameter-dependent dynamical behaviors of the non-paced model cell were determined by calculating stabilities of equilibrium points (EPs) and limit cycles, and bifurcation points to construct bifurcation diagrams. Action potentials (APs) and EADs during pacing were reproduced by numerical simulations for constructing phase diagrams of the paced model cell dynamics. Results are summarized as follows: (1) A modified version of the ten Tusscher-Panfilov model with accelerated ICaL inactivation could reproduce bradycardia-related EADs in LQTS type 2 and β-adrenergic stimulation-induced EADs in LQTS type 1. (2) Two types of EADs with different initiation mechanisms, ICaL reactivation–dependent and spontaneous SR Ca2+ release–mediated EADs, were detected. (3) Termination of EADs (AP repolarization) during pacing depended on the slow activation of IKs. (4) Spontaneous SR Ca2+ releases occurred at higher Ca2+ uptake rates, attributable to the instability of steady-state intracellular Ca2+ concentrations. Dynamical mechanisms of EAD formation and termination in the paced model cell are closely related to stability changes (bifurcations) in dynamical behaviors of the non-paced model cell, but they are model-dependent. Nevertheless, the modified ten Tusscher-Panfilov model would be useful for systematically investigating possible dynamical mechanisms of EAD-related arrhythmias in LQTS.

Augmentation of the Delayed Rectifier Potassium Current by ETA Endothelin Receptor in Guinea Pig Atrial Myocytes

The role of ETA endothelin receptor (ETAR) in the regulation of the delayed rectifier potassium current (IK) was examined in guinea pig atrial myocytes. Application of ET-1 (10 nM) together with an ETB-receptor-selective antagonist, BQ-788 (300 nM), significantly increased the voltage-dependent activation of IK without affecting its half-activation voltage or the slope factor, while it suppressed the calcium current (ICaL) and displaced the time-independent background current to the outward direction. The data suggests that the augmentation of IK contributes to the ETA-receptor-mediated shortening of action potential duration, and hence to the negative inotropic response, in atria.

Concerted suppression of Ih and activation of IK(M) by ivabradine, an HCN-channel inhibitor, in pituitary cells and hippocampal neurons

Ivabradine (IVA), a heart-rate reducing agent, is recognized as an inhibitor of hyperpolarization-activated cation current (Ih) and also reported to ameliorate inflammatory or neuropathic pain. However, to what extent this agent can perturb another types of membrane ion currents in neurons or endocrine cells remains to be largely unknown. Therefore, the Ih or other types of ionic currents in pituitary tumor (GH3) cells and in hippocampal mHippoE-14 neurons was studied with or without the presence of IVA or other related compounds. The IVA addition caused a time- and concentration-dependent reduction in the amplitude of Ih with an IC50 value of 0.64 μM and a KD value of 0.68 μM. IVA (0.3 μM) shifted the Ih activation curve to a more negative potential by approximately 8 mV, despite no concomitant change in the gating charge. Additionally, IVA was found to increase M-type K+ current (IK(M)) together with a rightward shift in the activation curve. In cell-attached current recordings, IVA (3 μM) applied to the bath increased the open probability of M-type K+ channels; however, it did not modify single-channel conductance of the channel. In current-clamp voltage recordings, IVA suppressed the firing of spontaneous action potentials in GH3 cells; and, further addition of linopirdine attenuated its suppression of firing. In hippocampal mHippoE-14 neurons, IVA also effectively increased IK(M) amplitude. In summary, both inhibition of Ih and activation of IK(M) caused by IVA can synergistically combine to influence electrical behaviors in different types of electrically excitable cells occurring in vivo.

Effects of β-subunit on gating of a potassium ion channel: Molecular simulations of cardiac IKs activation

Dynamic conformational changes of ion channel proteins during activation gating determine their function as carriers of current. The relationship between these molecular movements and channel function over the physiological timescale of the action potential (AP) has not been fully established due to limitations of existing techniques. We constructed a library of possible cardiac IKs protein conformations and applied a combination of protein segmentation and energy linearization to study this relationship computationally. Simulations reproduced the effects of the beta-subunit (KCNE1) on the alpha-subunit (KCNQ1) dynamics and function, observed in experiments. Mechanistically, KCNE1 increased the probability of “visiting” conducting pore conformations on activation trajectories, thereby increasing IKs current. KCNE1 slowed IKs activation by impeding the voltage sensor (VS) movement and reducing its coupling to pore opening. Conformational changes along activation trajectories determined that the S4-S5 linker (S4S5L) plays an important role in these modulatory effects by KCNE1. Integration of these molecular structure-based IKs dynamics into a model of human cardiac ventricular myocyte, revealed that KCNQ1-KCNE1 interaction is essential for normal AP repolarization.

VEGF-A inhibits agonist-mediated Ca2+ responses and activation of IKCa channels in mouse resistance artery endothelial cells.

Prolonged exposure to vascular endothelial growth factor A (VEGF-A) inhibits agonist-mediated endothelial cell Ca2+ release and subsequent activation of intermediate conductance Ca2+ -activated K+ (IKCa ) channels, which underpins vasodilatation as a result of endothelium-dependent hyperpolarization (EDH) in mouse resistance arteries. Signalling via mitogen-activated protein/extracellular signal-regulated kinase kinase (MEK) downstream of VEGF-A was required to attenuate endothelial cell Ca2+ responses and the EDH-vasodilatation mediated by IKCa activation. VEGF-A exposure did not modify vasodilatation as a result of the direct activation of IKCa channels, nor the pattern of expression of inositol 1,4,5-trisphosphate receptor 1 within endothelial cells of resistance arteries. These results indicate a novel role for VEGF-A in resistance arteries and suggest a new avenue for investigation into the role of VEGF-A in cardiovascular diseases. Vascular endothelial growth factor A (VEGF-A) is a potent permeability and angiogenic factor that is also associated with the remodelling of the microvasculature. Elevated VEGF-A levels are linked to a significant increase in the risk of cardiovascular dysfunction, although it is unclear how VEGF-A has a detrimental, disease-related effect. Small resistance arteries are central determinants of peripheral resistance and endothelium-dependent hyperpolarization (EDH) is the predominant mechanism by which these arteries vasodilate. Using isolated, pressurized resistance arteries, we demonstrate that VEGF-A acts via VEGF receptor-2 (R2) to inhibit both endothelial cell (EC) Ca2+ release and the associated EDH vasodilatation mediated by intermediate conductance Ca2+ -activated K+ (IKCa ) channels. Importantly, VEGF-A had no direct effect against IKCa channels. Instead, the inhibition was crucially reliant on the downstream activation of the mitogen-activated protein/extracellular signal-regulated kinase kinase 1/2 (MEK1/2). The distribution of EC inositol 1,4,5-trisphosphate (IP3 ) receptor-1 (R1) was not affected by exposure to VEGF-A and we propose an inhibition of IP3 R1 through the MEK pathway, probably via ERK1/2. Inhibition of EC Ca2+ via VEGFR2 has profound implications for EDH-mediated dilatation of resistance arteries and could provide a mechanism by which elevated VEGF-A contributes towards cardiovascular dysfunction.