Function Of ATP-sensitive Potassium Channels Research Articles

Loss-of-Function ABCC8 Mutations in Pulmonary Arterial Hypertension.

In pulmonary arterial hypertension (PAH), pathological changes in pulmonary arterioles progressively raise pulmonary artery pressure and increase pulmonary vascular resistance, leading to right heart failure and high mortality rates. Recently, the first potassium channelopathy in PAH, because of mutations in KCNK3, was identified as a genetic cause and pharmacological target. Exome sequencing was performed to identify novel genes in a cohort of 99 pediatric and 134 adult-onset group I PAH patients. Novel rare variants in the gene identified were independently identified in a cohort of 680 adult-onset patients. Variants were expressed in COS cells and function assessed by patch-clamp and rubidium flux analysis. We identified a de novo novel heterozygous predicted deleterious missense variant c.G2873A (p.R958H) in ABCC8 in a child with idiopathic PAH. We then evaluated all individuals in the original and a second cohort for rare or novel variants in ABCC8 and identified 11 additional heterozygous predicted damaging ABCC8 variants. ABCC8 encodes SUR1 (sulfonylurea receptor 1)-a regulatory subunit of the ATP-sensitive potassium channel. We observed loss of ATP-sensitive potassium channel function for all ABCC8 variants evaluated and pharmacological rescue of all channel currents in vitro by the SUR1 activator, diazoxide. Novel and rare missense variants in ABCC8 are associated with PAH. Identified ABCC8 mutations decreased ATP-sensitive potassium channel function, which was pharmacologically recovered.

Modulation of Excitability of Stellate Neurons in the Ventral Cochlear Nucleus of Mice by ATP-Sensitive Potassium Channels.

Major voltage-activated ionic channels of stellate cells in the ventral part of cochlear nucleus (CN) were largely characterized previously. However, it is not known if these cells are equipped with other ion channels apart from the voltage-sensitive ones. In the current study, it was aimed to study subunit composition and function of ATP-sensitive potassium channels (KATP) in stellate cells of the ventral cochlear nucleus. Subunits of KATP channels, Kir6.1, Kir6.2, SUR1, and SUR2, were expressed at the mRNA level and at the protein level in the mouse VCN tissue. The specific and clearly visible bands for all subunits but that for Kir6.1 were seen in Western blot. Using immunohistochemical staining technique, stellate cells were strongly labeled with SUR1 and Kir6.2 antibodies and moderately labeled with SUR2 antibody, whereas the labeling signals for Kir6.1 were too weak. In patch clamp recordings, KATP agonists including cromakalim (50µM), diazoxide (0.2mM), 3-Amino-1,2,4-triazole (ATZ) (1mM), 2,2-Dithiobis (5-nitro pyridine) (DTNP) (330µM), 6-Chloro-3-isopropylamino- 4H-thieno[3,2-e]-1,2,4-thiadiazine 1,1-dioxide (NNC 55-0118) (1µM), 6-chloro-3-(methylcyclopropyl)amino-4H-thieno[3,2-e]-1,2,4-thiadiazine 1,1-dioxide (NN414) (1µM), and H2O2 (0.88mM) induced marked responses in stellate cells, characterized by membrane hyperpolarization which were blocked by KATP antagonists. Blockers of KATP channels, glibenclamide (0.2mM), tolbutamide (0.1mM) as well as 5-hydroxydecanoic acid (1mM), and catalase (500IU/ml) caused depolarization of stellate cells, increasing spontaneous action potential firing. In conclusion, KATP channels seemed to be composed dominantly of Kir 6.2 subunit and SUR1 and SUR2 and activation or inhibition of KATP channels regulates firing properties of stellate cells by means of influencing resting membrane potential and input resistance.

Cardiac Ischemia/Reperfusion Injury: The Beneficial Effects of Exercise.

Cardiac ischemia reperfusion injury (IRI) occurs when the myocardium is revascularized after an episode of limited or absent blood supply. Many changes, including free radical production, calcium overload, protease activation, altered membrane lipids and leukocyte activation, contribute to IRI-induced myocardium damage. Aerobic exercise is the only countermeasure against IRI that can be sustained on a regular basis in clinical practice. Interestingly, both short-term (3-5days) and long-term (several weeks) exercise increase myocardial tolerance, reduce infarct size area and arrhythmias induced by IRI. Exercise protects the heart against IRI in a biphasic manner. The early phase of cardioprotection occurs between 30 min and 3h following an acute exercise bout, whilst the late phase is achieved within 24h after the exercise bout and persists for several days. As for the exercise intensity, although controversial data exists, it is feasible that the amount of cardioprotection is proportional to exercise intensity and only achieved above a critical threshold. It is known that aerobic exercise produces a cardioprotective phenotype, however the mechanisms responsible for this phenomenon remain unclear. Apparently, aerobic exercise-induced preconditioning is dependent on several factors that work together to protect the heart. Altered nitric oxide (NO) signaling, increased levels of heat shock proteins (HSPs), enhanced function of ATP-sensitive potassium channels, increased activation of opioids system, and enhanced antioxidant capacity may contribute to exercise-induced cardioprotection. Much has been discovered from animal models involving exercise-induced cardioprotection against cardiac IRI, however translating these findings to clinical practice still represents the major challenge in this field.

Disruption of ATP-sensitive potassium channel function in skeletal muscles promotes production and secretion of musclin

Sarcolemmal ATP-sensitive potassium (KATP) channels control skeletal muscle energy use through their ability to adjust membrane excitability and related cell functions in accordance with cellular metabolic status. Mice with disrupted skeletal muscle KATP channels exhibit reduced adipocyte size and increased fatty acid release into the circulation. As yet, the molecular mechanisms underlying this link between skeletal muscle KATP channel function and adipose mobilization have not been established. Here, we demonstrate that skeletal muscle-specific disruption of KATP channel function in transgenic (TG) mice promotes production and secretion of musclin. Musclin is a myokine with high homology to atrial natriuretic peptide (ANP) that enhances ANP signaling by competing for elimination. Augmented musclin production in TG mice is driven by a molecular cascade resulting in enhanced acetylation and nuclear exclusion of the transcription factor forkhead box O1 (FOXO1) – an inhibitor of transcription of the musclin encoding gene. Musclin production/secretion in TG is paired with increased mobilization of fatty acids and a clear trend toward increased circulating ANP, an activator of lipolysis. These data establish KATP channel-dependent musclin production as a potential mechanistic link coupling “local” skeletal muscle energy consumption with mobilization of bodily resources from fat. Understanding such mechanisms is an important step toward designing interventions to manage metabolic disorders including those related to excess body fat and associated co-morbidities.

Up-regulated ATP-sensitive potassium channels play a role in increased inflammation and plaque vulnerability in macrophages

ObjectiveIon channels expressed in monocytes/macrophages have been tightly attached to atherosclerosis by coupling cellular function with electrical activity. However, the function of ATP-sensitive potassium channels (KATP) in atherosclerosis has not been investigated directly. This study was performed to explore its role in atherosclerosis. Methods and resultsApoE−/− mice with collar placement and Ad5-CMV.p53 or lac Z gene transfer with or without intragastric administration glibenclamide were applied to establish the progressive atherosclerosis at different time points and detect the function of KATP channel in atherosclerosis. The expression and distribution of KATP subunits in plaques were examined and a correlation between KATP subunits expressed in macrophages, mainly Kir6.2 and SUR2A, and the vulnerability index of plaques was observed. In vitro, glibenclamide and pinacidil were used to detect the function and mechanism of KATP channels in RAW264.7 cells stimulated by LPS. And the data showed that glibenclamide could ameliorate the progress of atherosclerosis and reduce the production of inflammatory cytokines as well as the phosphorylation of p65 and ERK1/2, while inhibitors of p65 leaded to robust expression of KATP subunits in macrophages. ConclusionsWe concluded that KATP channels in monocytes/macrophages were up-regulated and correlated with increased inflammation in vulnerable plaques, while glibenclamide could rescue the progression. KATP channels may stimulate inflammatory reaction by MAPKs/NF-κB pathways in macrophages.

Heterogeneity and Function of ATP-Sensitive Potassium Channels in Canine Hearts

<h3>Background</h3> In contrast to the consensus notion that cardiac ATP-sensitive potassium (K_ATP) channels are composed of Kir6.2 as the pore-forming subunit and SUR2A as the regulatory subunit, recent studies have revealed that rodent atrial K_ATP channels are primarily regulated by SUR1 subunit. In addition, a gain-of-function mutation of Kir6.1 subunit has recently been linked to a form of Brugada syndrome. Heterogeneity of K_ATP channels within and between heart chambers may differentially predispose certain myocytes to action potential (AP) shortening. <h3>Methods</h3> To assess the heterogeneity of cardiac K_ATP channels in large animals, whole-cell and excised patch-clamp recordings were performed on myocytes isolated from dog atrium, ventricular epicardium (Epi), mid myocardium (Mid), and endocardium (Endo). To measure the consequences of K_ATP channel opening on dog myocytes, dynamic current clamp was applied via a custom analog circuit to simulate a progressively increasing linear K⁺ conductance. <h3>Results</h3> Mean K_ATP conductance (activated by metabolic inhibitors) did not differ significantly between chambers (49 ± 9 pA/pF in atrium vs 43 ± 5 pA/pF in ventricle); however, within the ventricle the current was smallest in Endo (29 ± 5 pA/pF) vs Epi (53 ± 11 pA/pF, <i>P</i> <.05) and Mid (46 ± 10 pA/pF, <i>P</i> <.05). Pinacidil-activated (SUR2A-specific) current was large in both atrium (40 ± 4 pA/pF) and ventricle (47 ± 7 pA/pF). Diazoxide-activated (SUR1-specific) current in atrium (13 ± 6 pA/pF) was significantly larger than in ventricle (3 ± 2 pA/pF, <i>P</i> <.05). In current clamp, myocytes with longer action potential duration (APD) (Mid) were more susceptible to injected "K_ATP" current, a result that is reproduced <i>in silico</i> using a second-generation canine ventricular AP model to simulate Epi, Mid, and Endo (HRd2010). The results indicate that increased shortening is caused by replenishment of repolarizing current that is otherwise lacking in cells with long APDs. <h3>Conclusions</h3> If similarly activated, the heterogeneous makeup of K_ATP channels between and within heart chambers is likely to cause proarrhythmic dispersion of repolarization; however, the enhanced shortening of long APDs by K_ATP current would tend to reduce dispersion and may consequently be protective.

Optical Mapping of Cardiac ATP Sensitive Potassium Channel Function under Metabolic Inhibition

ATP-sensitive potassium channel (KATP) activation can drastically shorten action potential duration (APD) in metabolically compromised myocytes. We showed previously that SUR1 with Kir6.2 forms the functional channel in mouse atria while Kir6.2 and SUR2A predominate in ventricles. SUR1 is more sensitive to metabolic stress than SUR2A, raising the possibility that KATP in atria and ventricles may respond differently to metabolic stress. We performed optical mapping with voltage-sensitive dyes on Langendorff-perfused hearts from C57BL wild-type (WT), Kir6.2 deficient (Kir6.2-/-), and SUR1 deficient (SUR1-/-) mice to examine APD during metabolic inhibition (MI, 0mM glucose+2mM sodium cyanide). In WT hearts, significant shortening of atrial APD after variable delay occurred before ventricular APD shortening after a variable delay. Atrial APD shortened by 60.5±2.7% at 5.5±1.3 min (n=6, p<0.01) earlier than comparable ventricular APD shortening (56.4±10.0% shortening 20.33 min after onset of MI). Interestingly, prolongation (48.6±5.2%, p<0.01) preceded ventricular APD shortening. In SUR1-/- hearts (n=6), atrial APD shortening was abolished but ventricular shortening (65.0±15.4%, p<0.01) was maintained. Atrioventricular block occurred earlier in WT (3.85±1.21 minutes before 50% shortening of ventricular APD) than in SUR1-/- (1.08±3.98 minutes before 50% shortening of ventricular APD, p=0.15, NS). In Kir6.2-/- hearts, two disparate responses to MI were observed; 3 of 5 hearts displayed slight shortening in the ventricles (24±3%, p<0.05) and atria (39.0±1.9%, p<0.05) but this shortening occurred later and to much less extent than in WT (p<0.05). Prolongation of ventricular APD was observed in the remaining hearts (327% and 489% prolongation). The results confirm that Kir6.2 contributes to APD shortening in both atria and ventricle during metabolic stress, and SUR1 is required for atrial APD shortening. Moreover, the results show the atrial action potential is more susceptible to MI with SUR1 underlying atrial KATP.

Chronic sustained and intermittent hypoxia reduce function of ATP-sensitive potassium channels in nucleus of the solitary tract

Activation of neuronal ATP-sensitive potassium (K(ATP)) channels is an important mechanism that protects neurons and conserves neural function during hypoxia. We investigated hypoxia (bath gassed with 95% N(2)-5% CO(2) vs. 95% O(2)-5% CO(2) in control)-induced changes in K(ATP) current in second-order neurons of peripheral chemoreceptors in the nucleus of the solitary tract (NTS). Hypoxia-induced K(ATP) currents were compared between normoxic (Norm) rats and rats exposed to 1 wk of either chronic sustained hypoxia (CSH) or chronic intermittent hypoxia (CIH). Whole cell recordings of NTS second-order neurons identified after 4-(4-(dihexadecylamino)styryl)-N-methylpyridinium iodide (DiA) labeling of the carotid bodies were obtained in a brain stem slice. In Norm cells (n = 9), hypoxia (3 min) induced an outward current of 12.7 +/- 1.1 pA with a reversal potential of -73 +/- 2 mV. This current was completely blocked by the K(ATP) channel blocker tolbutamide (100 muM). Bath application of the K(ATP) channel opener diazoxide (200 muM, 3 min) evoked an outward current of 21.8 +/- 5.8 pA (n = 6). Hypoxia elicited a significantly smaller outward current in both CSH (5.9 +/- 1.4 pA, n = 11; P < 0.01) and CIH (6.8 +/- 1.7 pA, n = 6; P < 0.05) neurons. Diazoxide elicited a significantly smaller outward current in CSH (3.9 +/- 1.0 pA, n = 5; P < 0.05) and CIH (2.9 +/- 0.9 pA, n = 3; P < 0.05) neurons. Western blot analysis showed reduced levels of K(ATP) potassium channel subunits Kir6.1 and Kir6.2 in the NTS from CSH and CIH rats. These results suggest that hypoxia activates K(ATP) channels in NTS neurons receiving monosynaptic chemoreceptor afferent inputs. Chronic exposure to either sustained or intermittent hypoxia reduces K(ATP) channel function in NTS neurons. This may represent a neuronal adaptation that preserves neuronal excitability in crucial relay neurons in peripheral chemoreflex pathways.

Hypoxia-induced vascular smooth muscle relaxation: increased ATP-sensitive K+ efflux or decreased voltage-sensitive Ca2+ influx?

controversy continues regarding the cellular mechanisms of hypoxic vasodilation. The vascular endothelium may contribute to hypoxic dilations by releasing relaxing factors that act on the smooth muscle ([4][1], [6][2]–[8][3]). In contrast, numerous studies have suggested a role of the vascular

Regulation of ATP-sensitive potassium channel function by protein kinase A-mediated phosphorylation in transfected HEK293 cells

ATP-sensitive potassium (K(ATP)) channels regulate insulin secretion, vascular tone, heart rate and neuronal excitability by responding to transmitters as well as the internal metabolic state. K(ATP) channels are composed of four pore-forming alpha-subunits (Kir6.2) and four regulatory beta-subunits, the sulfonylurea receptor (SUR1, SUR2A or SUR2B). Whereas protein kinase A (PKA) phosphorylation of serine 372 of Kir6.2 has been shown biochemically by others, we found that the phosphorylation of T224 rather than S372 of Kir6.2 underlies the catalytic subunits of PKA (c-PKA)- and the D1 dopamine receptor-mediated stimulation of K(ATP) channels expressed in HEK293 cells. Specific changes in the kinetic properties of channels treated with c-PKA, as revealed by single-channel analysis, were mimicked by aspartate substitution of T224. The T224D mutation also reduced the sensitivity to ATP inhibition. Alteration of channel gating and a decrease in the apparent affinity for ATP inhibition thus underlie the positive regulation of K(ATP) channels by PKA phosphorylation of T224 in Kir6.2, which may represent a general mechanism for K(ATP) channel regulation in different tissues.

ATP-sensitive potassium channel blockade enhances spontaneous alternation performance in the rat: a potential mechanism for glucose-mediated memory enhancement

Peripheral and central injections of d-glucose enhance learning and memory in rats, and block memory impairments produced by morphine. The mechanism(s) for these effects is (are) as yet unknown. One mechanism by which glucose might act on memory and other brain functions is by regulating the ATP-sensitive potassium channel. This channel may couple glucose metabolism and neuronal excitability, with channel blockade increasing the likelihood of stimulus-evoked neurotransmitter release. The present experiments explored the effects of intra-septal injections of glucose and the ATP-sensitive potassium channel blocker glibenclamide on spontaneous alternation behavior in the rat. Intra-septal injections of glucose (20 nmol) or glibenclamide (10 nmol), 30 min prior to plus-maze spontaneous alternation performance, significantly enhanced alternation scores compared to rats receiving vehicle injections. Glibenclamide enhanced spontaneous alternation performance in an inverted-U dose–response manner. Individually sub-effective doses of glucose (5 nmol) and glibenclamide (5 nmol) significantly enhanced plus-maze alternation scores when co-injected into the septal area. Glibenclamide (10 nmol), when co-administered with morphine (4 nmol) 30 min prior to Y-maze spontaneous alternation performance, attenuated the performance-impairing effects of morphine alone. The present findings show that intra-septal injections of the direct ATP-sensitive potassium channel blocker glibenclamide, both alone and in conjunction with a sub-effective dose of glucose, enhance spontaneous alternation performance and attenuate the performance-impairing effects of morphine. The similarity of the results obtained with glibenclamide and glucose, together with their similar actions on ATP-sensitive potassium channel function, suggests that glucose may modulate memory-dependent behavior in the rat by regulating the ATP-sensitive potassium channel.

Differential Efficacy of Vasodilators in Hypercholesterolaemic Rabbits

The effects of hypercholesterolaemia on the endothelium-dependent and -independent vascular reactivity of the superior mesenteric artery has been examined in anaesthetized rabbits in-vivo. Rabbits were fed with either standard or cholesterol-enriched diet for 24 weeks. Plasma lipids and changes in the endothelin content of plasma and vascular tissue were measured in the superior mesenteric artery and in the thoracic aorta. The functional severity of atherosclerosis was determined by examining vascular responses in the isolated thoracic aorta. The blood flow in the superior mesenteric artery was measured by transit-time flowmetry and drugs were injected through an intra-abdominal aortic catheter. Acetylcholine (5, 10, 20 microg kg(-1)) elicited dose-dependent, mesenteric vasodilation in normocholesterolaemic rabbits. In hypercholesterolaemic animals the response to acetylcholine was completely abolished and even became a vasoconstriction. Endothelin levels in plasma and in the vascular tissue were significantly elevated in hypercholesterolaemic animals compared with controls. Cromakalim at a dose of 3 microg kg(-1), elicited similar mesenteric vasodilation in hypercholesterolaemic and normocholesterolaemic animals. These experiments show that the endothelium-dependent responses of the superior mesenteric artery to acetylcholine are functionally impaired by prolonged hypercholesterolaemia, that this altered vascular reactivity is associated with the elevation of endothelin levels in the circulation and in vascular tissues, and that in hypercholesterolaemia the mesenteric vasodilator effect of the K+-channel opener cromakalim is entirely preserved, suggesting that severe hypercholesterolaemia does not depress the function of ATP-sensitive potassium channels in mesenteric vascular smooth muscle.

Attenuation of vascular relaxation after development of tachyphylaxis to peroxynitrite in vivo.

Peroxynitrite, formed endogenously by the near diffusion-limited reaction of nitric oxide with superoxide anion, induces vascular relaxation. This effect is subject to rapid tachyphylaxis, suggesting that peroxynitrite may alter subsequent vasorelaxant responses. The present study examined the effects of peroxynitrite on mean arterial pressure and hindquarter, renal, and mesenteric vascular resistances in pentobarbital-anesthetized rats. Peroxynitrite induced dose-dependent decreases in mean arterial pressure and hindquarter and mesenteric vascular resistances. The repetitive administration of peroxynitrite resulted in the rapid development of tachyphylaxis, with subsequent doses producing progressively smaller effects. After the development of tachyphylaxis to peroxynitrite, the hemodynamic effects produced by the systemic administration of acetylcholine and prostacyclin were significantly attenuated, whereas the hemodynamic responses to bradykinin and the nitric oxide donor (Z)-1-¿N-methyl-N-[6(N-methylammoniohexyl)amino]¿diazen-1-++ +ium-1, 2-diolate (MAHMA NONOate) remained unchanged. These results demonstrate that 1) peroxynitrite is a potent vasorelaxant in vivo, 2) peroxynitrite-mediated vasodilatation is subject to the development of rapid tachyphylaxis, and 3) peroxynitrite alters the vascular smooth muscle response to prostacyclin, perhaps via inactivation of vascular smooth muscle ATP-sensitive potassium channel function.