Novel loss-of-function variants expand ABCC9-related intellectual disability and myopathy syndrome.

Loss-of-function mutation of ABCC9, the gene encoding the SUR2 subunit of ATP sensitive-potassium (KATP) channels, was recently associated with autosomal recessive ABCC9-related intellectual disability and myopathy syndrome (AIMS). Here we identify nine additional subjects, from seven unrelated families, harbouring different homozygous loss-of-function variants in ABCC9 and presenting with a conserved range of clinical features. All variants are predicted to result in severe truncations or in-frame deletions within SUR2, leading to the generation of non-functional SUR2-dependent KATP channels. Affected individuals show psychomotor delay and intellectual disability of variable severity, microcephaly, corpus callosum and white matter abnormalities, seizures, spasticity, short stature, muscle fatigability and weakness. Heterozygous parents do not show any conserved clinical pathology but report multiple incidences of intra-uterine fetal death, which were also observed in an eighth family included in this study. In vivo studies of abcc9 loss-of-function in zebrafish revealed an exacerbated motor response to pentylenetetrazole, a pro-convulsive drug, consistent with impaired neurodevelopment associated with an increased seizure susceptibility. Our findings define an ABCC9 loss-of-function-related phenotype, expanding the genotypic and phenotypic spectrum of AIMS and reveal novel human pathologies arising from KATP channel dysfunction.

Investigation of the role of KATP channels in the cytotoxic effect of cypermethrin on rat-derived aortic smooth muscle cells.

We investigate role of ATP sensitive potassium (KATP) channel in cytotoxic effect of cypermethrin on rat aortic smooth muscle cells. Cytotoxicity analysis was performed at 0, 0.1, 0.5, 10, 50, and 100 µM concentrations of cypermethrin and the cell index (CI) was calculated. KATP currents were recorded using patch clamp technique for 50 and 100 µM concentrations and channel conductivity was determined by obtaining current-voltage characteristics. No cytotoxic effect was observed in the first 72 hours. At the 96th hour, only at 100 µM concentration, the CI value decreased significantly compared to control group and at 120 and 144th hours, it was observed that the CI value decreased significantly at all concentrations. Currents and conductivities were significantly decreased at 50 and 100 µM concentrations. Results gave clues that cypermethrin causes a cytotoxic effect on vascular smooth muscles and that KATP channels may have a role in the emergence of this effect.

Molecular Dynamics Simulation of Kir6.2 Variants Reveals Potential Association with Diabetes Mellitus.

Diabetes mellitus (DM) represents a problem for the healthcare system worldwide. DM has very serious complications such as blindness, kidney failure, and cardiovascular disease. In addition to the very bad socioeconomic impacts, it influences patients and their families and communities. The global costs of DM and its complications are huge and expected to rise by the year 2030. DM is caused by genetic and environmental risk factors. Genetic testing will aid in early diagnosis and identification of susceptible individuals or populations using ATP-sensitive potassium (KATP) channels present in different tissues such as the pancreas, myocardium, myocytes, and nervous tissues. The channels respond to different concentrations of blood sugar, stimulation by hormones, or ischemic conditions. In pancreatic cells, they regulate the secretion of insulin and glucagon. Mutations in the KCNJ11 gene that encodes the Kir6.2 protein (a major constituent of KATP channels) were reported to be associated with Type 2 DM, neonatal diabetes mellitus (NDM), and maturity-onset diabetes of the young (MODY). Kir6.2 harbors binding sites for ATP and phosphatidylinositol 4,5-diphosphate (PIP2). The ATP inhibits the KATP channel, while the (PIP2) activates it. A Kir6.2 mutation at tyrosine330 (Y330) was demonstrated to reduce ATP inhibition and predisposes to NDM. In this study, we examined the effect of mutations on the Kir6.2 structure using bioinformatics tools and molecular dynamic simulations (SIFT, PolyPhen, SNAP2, PANTHER, PhD&SNP, SNP&Go, I-Mutant, MuPro, MutPred, ConSurf, HOPE, and GROMACS). Our results indicated that M199R, R201H, R206H, and Y330H mutations influence Kir6.2 structure and function and therefore may cause DM. We conclude that MD simulations are useful techniques to predict the effects of mutations on protein structure. In addition, the M199R, R201H, R206H, and Y330H variant in the Kir6.2 protein may be associated with DM. These results require further verification in protein-protein interactions, Kir6.2 function, and case-control studies.

The cardioprotective role of sirtuins is mediated in part by regulating KATP channel surface expression.

Sirtuins are NAD+-dependent deacetylases with beneficial roles in conditions relevant to human health, including metabolic disease, type II diabetes, obesity, cancer, aging, neurodegenerative diseases, and cardiac ischemia. Since ATP-sensitive K+ (KATP) channels have cardioprotective roles, we investigated whether they are regulated by sirtuins. Nicotinamide mononucleotide (NMN) was used to increase cytosolic NAD+ levels and to activate sirtuins in cell lines, isolated rat and mouse cardiomyocytes or insulin-secreting INS-1 cells. KATP channels were studied with patch clamping, biochemistry techniques, and antibody uptake experiments. NMN led to an increase in intracellular NAD+ levels and an increase in the KATP channel current, without significant changes in the unitary current amplitude or open probability. An increased surface expression was confirmed using surface biotinylation approaches. The rate of KATP channel internalization was diminished by NMN, which may be a partial explanation for the increased surface expression. We show that NMN acts via sirtuins since the increased KATP channel surface expression was prevented by blockers of SIRT1 and SIRT2 (Ex527 and AGK2) and mimicked by SIRT1 activation (SRT1720). The pathophysiological relevance of this finding was studied using a cardioprotection assay with isolated ventricular myocytes, in which NMN protected against simulated ischemia or hypoxia in a KATP channel-dependent manner. Overall, our data draw a link between intracellular NAD+, sirtuin activation, KATP channel surface expression, and cardiac protection against ischemic damage.

KATP channel activity and slow oscillations in pancreatic beta cells are regulated by mitochondrial ATP production.

Pancreatic beta cells secrete insulin in response to plasma glucose. The ATP-sensitive potassium channel (KATP ) links glucose metabolism to islet electrical activity in these cells by responding to increased cytosolic [ATP]/[ADP]. It was recently proposed that pyruvate kinase (PK) in close proximity to beta cell KATP locally produces the ATP that inhibits KATP activity. This proposal was largely based on the observation that applying phosphoenolpyruvate (PEP) and ADP to the cytoplasmic side of excised inside-out patches inhibited KATP . To test the relative contributions of local vs. mitochondrial ATP production, we recorded KATP activity using mouse beta cells and INS-1 832/13 cells. In contrast to prior reports, we could not replicate inhibition of KATP activity by PEP + ADP. However, when the pH of the PEP solutions was not corrected for the addition of PEP, strong channel inhibition was observed as a result of the well-known action of protons to inhibit KATP . In cell-attached recordings, perifusing either a PK activator or an inhibitor had little or no effect on KATP channel closure by glucose, further suggesting that PK is not an important regulator of KATP . In contrast, addition of mitochondrial inhibitors robustly increased KATP activity. Finally, by measuring the [ATP]/[ADP] responses to imposed calcium oscillations in mouse beta cells, we found that oxidative phosphorylation could raise [ATP]/[ADP] even when ADP was at its nadir during the burst silent phase, in agreement with our mathematical model. These results indicate that ATP produced by mitochondrial oxidative phosphorylation is the primary controller of KATP in pancreatic beta cells. KEY POINTS: Phosphoenolpyruvate (PEP) plus adenosine diphosphate does not inhibit KATP activity in excised patches. PEP solutions only inhibit KATP activity if the pH is unbalanced. Modulating pyruvate kinase has minimal effects on KATP activity. Mitochondrial inhibition, in contrast, robustly potentiates KATP activity in cell-attached patches. Although the ADP level falls during the silent phase of calcium oscillations, mitochondria can still produce enough ATP via oxidative phosphorylation to close KATP . Mitochondrial oxidative phosphorylation is therefore the main source of the ATP that inhibits the KATP activity of pancreatic beta cells.

Rapamycin treatment unmasks a sex-specific pattern of scar expansion of the infarcted rat heart: The relationship between mTOR and KATP channel.

Inhibition of the mammalian target of rapamycin (mTOR) with the macrolide rapamycin or pharmacological suppression of KATP channel opening translated to scar expansion of the myocardial infarcted (MI) adult female rodent heart. The present study tested the hypotheses that rapamycin-mediated scar expansion was sex-specific and that mTOR signaling directly influenced KATP channel subunit expression/activity. Scar size was significantly larger in post-MI male rats as compared to the previous data reported in post-MI female rats. The reported scar expansion of rapamycin-treated post-MI female rats was not observed following the administration of the macrolide to post-MI male rats. Protein levels of the KATP channel subunits Kir6.2 and SUR2A and phosphorylation of the serine2448 residue of mTOR were similar in the normal heart of adult male and female rats. By contrast, greater tuberin inactivation characterized by the increased phosphorylation of the threonine1462 residue and reduced raptor protein levels were identified in the normal heart of adult female rats. Rapamycin pretreatment of phorbol 12,13-dibutyrate (PDBu)-treated neonatal rat ventricular cardiomyocytes (NNVMs) suppressed hypertrophy, inhibited p70S6K phosphorylation, and attenuated SUR2A protein upregulation. In the presence of low ATP levels, KATP channel activity detected in untreated NNVMs was significantly attenuated in PDBu-induced hypertrophied NNVMs via a rapamycin-independent pathway. Thus, rapamycin administration to post-MI rats unmasked a sex-specific pattern of scar expansion and mTOR signaling in PDBu-induced hypertrophied NNVMs significantly increased SUR2A protein levels. However, the biological advantage associated with SUR2A protein upregulation was partially offset by an mTOR-independent pathway that attenuated KATP channel activity in PDBu-induced hypertrophied NNVMs.

Fibroblast growth factor 10 ameliorates renal ischaemia-reperfusion injury by attenuating mitochondrial damage.

Ischaemia-reperfusion (I/R) injury is one of the leading causes of acute kidney injury (AKI). Its pathologic mechanism is quite complex, involving oxidative stress, inflammatory response, autophagy, and apoptosis. Fibroblast growth factor 10 (FGF10) and 5-hydroxydecanoate (5-HD) play essential roles in kidney injury. Rats were divided into four groups: (i) sham group, sham-operated animals with an unconstructed renal artery; (ii) I/R group, kidneys were subjected to 50 min of ischaemia followed by reperfusion for 2 days; (iii) I/R + FGF10 group, animals treated with 0.5 mg/kg FGF10 (i.p.) 1 h before ischaemia; and (iv) 5-HD group, animals treated with 5 mg/kg 5-HD (i.m.) 30 min before FGF10 treatment. Renal injury, apoptosis damage, mitochondrial oxidative damage, mitochondrial membrane potential (MMP), and expression of the ATP-sensitive K+ (KATP) channel subunit Kir6.2 were evaluated. FGF10 treatment significantly alleviated I/R-induced elevation in the serum creatinine level and the number of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling-positive tubular cells in the kidney. In addition, FGF10 dramatically ameliorated renal mitochondrial-related damage, including reducing mitochondrial-dependent apoptosis, alleviating oxidative stress, maintaining the mitochondrial membrane potential, and opening the mitochondrial KATP channels. The protective effect of FGF10 was significantly compromised by the ATP-dependent potassium channel blocker 5-HD. Our data suggest that FGF10 offers effective protection against I/R and improves animal survival by attenuating mitochondrial damage.

Palmitoylation of the KATP channel Kir6.2 subunit promotes channel opening by regulating PIP2 sensitivity.

A physiological role for long-chain acyl-CoA esters to activate ATP-sensitive K+ (KATP) channels is well established. Circulating palmitate is transported into cells and converted to palmitoyl-CoA, which is a substrate for palmitoylation. We found that palmitoyl-CoA, but not palmitic acid, activated the channel when applied acutely. We have altered the palmitoylation state by preincubating cells with micromolar concentrations of palmitic acid or by inhibiting protein thioesterases. With acyl-biotin exchange assays we found that Kir6.2, but not sulfonylurea receptor (SUR)1 or SUR2, was palmitoylated. These interventions increased the KATP channel mean patch current, increased the open time, and decreased the apparent sensitivity to ATP without affecting surface expression. Similar data were obtained in transfected cells, rat insulin-secreting INS-1 cells, and isolated cardiac myocytes. Kir6.2ΔC36, expressed without SUR, was also positively regulated by palmitoylation. Mutagenesis of Kir6.2 Cys166 prevented these effects. Clinical variants in KCNJ11 that affect Cys166 had a similar gain-of-function phenotype, but was more pronounced. Molecular modeling studies suggested that palmitoyl-C166 and selected large hydrophobic mutations make direct hydrophobic contact with Kir6.2-bound PIP2 Patch-clamp studies confirmed that palmitoylation of Kir6.2 at Cys166 enhanced the PIP2 sensitivity of the channel. Physiological relevance is suggested since palmitoylation blunted the regulation of KATP channels by α1-adrenoreceptor stimulation. The Cys166 residue is conserved in some other Kir family members (Kir6.1 and Kir3, but not Kir2), which are also subject to regulated palmitoylation, suggesting a general mechanism to control the open state of certain Kir channels.

Pyridoxal-5-phosphate induced cardioprotection in aging associated with up-expression of cystathionine-γ-lyase, 3-mercaptopyruvate sulfurtransferase, and ATP-sensitive potassium channels.

BACKGROUND: In the present work, we investigated the cardioprotective potential of pyridoxal-5-phosphate (PLP) in old rats as a cofactor of enzymes that synthesize hydrogen sulphide (H2 S). MATERIALS AND METHODS: PLP was administered per os in a dose of 0.7 mg per kg daily for 2 weeks. Rats were divided into three groups (adult, old and old +PLP) of 20 animals. The cardiac mRNA levels of genes encoding H2 S-synthesizing enzymes cystathionine-γ-lyase (CSE) and 3-mercaptopyruvate sulfurtransferase (3-MST), uncoupling proteins (UCP3), subunits of ATP-sensitive potassium (KATP ) channels were determined using real-time polymerase chain reaction analysis. We also studied the effect of PLP-administration on the content of H2 S, oxidative stress, the activities of inducible and constitutive NO-synthase (iNOS, cNOS), arginase and nitrate reductase in the heart homogenates as well as cardiac resistance to ischemia-reperfusion in Langendorff-isolated heart model. RESULTS: It was shown that PLP restored mRNA levels of CSE, 3-MST and UCP3 genes, and H2 S content and also significantly increased the expression of SUR2 and Kir6.1 (2.2 and 3.3 times, respectively) in the heart of old rats. PLP significantly reduced the formation of superoxide, malondialdehyde, diene conjugates as well as the activity of iNOS and arginase. PLP significantly increased constitutive synthesis of NO and prevented reperfusion disturbances of the heart function after ischemia. CONCLUSIONS: Thus, PLP-administration in old rats was associated with up-expression of CSE, 3-MST, UCP3 and SUR2 and Kir6.1 subunits of KATP channels, and also increased cNOS activity and reduced oxidative stress and prevented reperfusion dysfunction of the heart in ischemia-reperfusion.

Iptakalim alleviates synaptic damages via targeting mitochondrial ATP-sensitive potassium channel in depression.

Synaptic plasticity damages play a crucial role in the onset and development of depression, especially in the hippocampus, which is more susceptible to stress and the most frequently studied brain region in depression. And, mitochondria have a major function in executing the complex processes of neurotransmission and plasticity. We have previously demonstrated that Iptakalim (Ipt), a new ATP-sensitive potassium (K-ATP) channel opener, could improve the depressive-like behavior in mice. But the underlying mechanisms are not well understood. The present study demonstrated that Ipt reversed depressive-like phenotype in vivo (chronic mild stress-induced mice model of depression) and in vitro (corticosterone-induced cellular model). Further study showed that Ipt could upregulate the synaptic-related proteins postsynaptic density 95 (PSD 95) and synaptophysin (SYN), and alleviated the synaptic structure damage. Moreover, Ipt could reverse the abnormal mitochondrial fission and fusion, as well as the reduced mitochondrial ATP production and collapse of mitochondrial membrane potential in depressive models. Knocking down the mitochondrial ATP-sensitive potassium (Mito-KATP) channel subunit MitoK partly blocked the above effects of Ipt. Therefore, our results reveal that Ipt can alleviate the abnormal mitochondrial dynamics and function depending on MitoK, contributing to improve synaptic plasticity and exert antidepressive effects. These findings provide a candidate compound and a novel target for antidepressive therapy.

The Role of Mitochondrial KATP Channels in the Infarct-Reducing Effect of Normobaric Hypoxia.

We studied the role of KATP channels in the infarct-limiting effect of short-term normobaric hypoxia. Male Wistar rats were subjected to a 45-min coronary artery occlusion followed by a 120-min reperfusion. Normobaric hypoxia was simulated 30 min before coronary artery occlusion: 6 sessions of hypoxia (8% O2, 10 min) and reoxygenation (21% O2, 10 min). The following drugs were administered to rats: glibenclamide, 5-hydroxydecanoate, and HMR1098. It was found that normobaric hypoxia contributes to a decrease in myocardial infarct size by 36%. Preliminary administration of glibenclamide or 5-hydroxydecanoate eliminated the infarct-reducing effect of normobaric hypoxia. Activator of mitochondrial KATP channel diazoxide limited the infarct size. These findings suggest that mitochondrial KATP channels are involved into the cardioprotective effect of normobaric hypoxia.

δ2-Opioid Receptors as a Target in Designing New Cardioprotective Drugs: the Role of Protein Kinase C, AMPK, and Sarcolemmal KATP Channels.

In rats anesthetized with α-chloralose, coronary artery occlusion (45 min) and reperfusion (120 min) were modeled. The selective δ2-opioid receptor agonist deltorphin II was administered 5 min before reperfusion. Protein kinase C inhibitor chelerythrine, AMP-activated protein kinase inhibitor compound C, and ATP-sensitive K+ channel blockers glibenclamide, 5-hydroxydecanoate, and HMR 1098 were administered 10 min before reperfusion. It was found that the infarct-limiting effect of deltorphin II is associated with activation of protein kinase C and opening of sarcolemmal ATP-sensitive K+ channel.

ATP-sensitive K+ channels control the spontaneous firing of a glycinergic interneuron in the auditory brainstem.

KEY POINTS: Cartwheel neurons provide potent inhibition to fusiform neurons in the dorsal cochlear nucleus (DCN). Most cartwheel neurons fire action potentials spontaneously, but the ion channels responsible for this intrinsic activity are unknown. We investigated the ion channels responsible for the intrinsic firing of cartwheel neurons and the stable resting membrane potential found in a fraction of these neurons (quiet neurons). Among the ion channels controlling membrane potential of cartwheel neurons, the presence of open ATP-sensitive potassium channels (KATP ) is responsible for the existence of quiet neurons. Our results pinpoint KATP channel modulation as a critical factor controlling the firing of cartwheel neurons. Hence, it is a crucial channel influencing the balance of excitation and inhibition in the DCN. ABSTRACT: Cartwheel neurons from the dorsal cochlear nucleus (DCN) are glycinergic interneurons and the primary source of inhibition on the fusiform neurons, the DCN's principal excitatory neuron. Most cartwheel neurons present spontaneous firing (active neurons), producing a steady inhibitory tone on fusiform neurons. In contrast, a small fraction of these neurons do not fire spontaneously (quiet neurons). Hyperactivity of fusiform neurons is seen in animals with behavioural evidence of tinnitus. Because of its relevance in controlling the excitability of fusiform neurons, we investigated the ion channels responsible for the spontaneous firing of cartwheel neurons in DCN slices from rats. We found that quiet neurons presented an outward conductance not seen in active neurons, which generates a stable resting potential. This current was sensitive to tolbutamide, an ATP-sensitive potassium channel (KATP ) antagonist. After inhibition with tolbutamide, quiet neurons start to fire spontaneously, while the active neurons were not affected. On the other hand, in active neurons, KATP agonist diazoxide activated a conductance similar to quiet neurons' KATP conductance and stopped spontaneous firing. According to the effect of KATP channels on cartwheel neuron firing, glycinergic neurotransmission in DCN was increased by tolbutamide and decreased by diazoxide. Our results reveal a role of KATP channels in controlling the spontaneous firing of neurons not involved in fuel homeostasis.

KATP channels and NO dilate redundantly intramuscular arterioles during electrical stimulation of the skeletal muscle in mice.

Functional hyperemia is fundamental to provide enhanced oxygen delivery during exercise in skeletal muscle. Different mechanisms are suggested to contribute, mediators from skeletal muscle, transmitter spillover from the neuromuscular synapse as well as endothelium-related dilators. We hypothesized that redundant mechanisms that invoke adenosine, endothelial autacoids, and KATP channels mediate the dilation of intramuscular arterioles in mice. Arterioles (maximal diameter: 20-42 µm, n = 65) were studied in the cremaster by intravital microscopy during electrical stimulation of the motor nerve to induce twitch or tetanic skeletal muscle contractions (10 or 100 Hz). Stimulation for 1-60 s dilated arterioles rapidly up to 65% of dilator capacity. Blockade of nicotinergic receptors blocked muscle contraction and arteriolar dilation. Exclusive blockade of adenosine receptors (1,3-dipropyl-8-(p-sulfophenyl)xanthine) or of NO and prostaglandins (nitro-L-arginine and indomethacin, LN + Indo) exerted only a minor attenuation. Combination of these blockers, however, reduced the dilation by roughly one-third during longer stimulation periods (> 1 s at 100 Hz). Blockade of KATP channels (glibenclamide) which strongly reduced adenosine-induced dilation reduced responses upon electrical stimulation only moderately. The attenuation was strongly enhanced if glibenclamide was combined with LN + Indo and even observed during brief stimulation. LN was more efficient than indomethacin to abrogate dilations if combined with glibenclamide. Arteriolar dilations induced by electrical stimulation of motor nerves require muscular contractions and are not elicited by acetylcholine spillover from neuromuscular synapses. The dilations are mediated by redundant mechanisms, mainly activation of KATP channels and release of NO. The contribution of K+ channels and hyperpolarization sets the stage for ascending dilations that are crucial for a coordinated response in the network.

Cardiac dysfunction in spontaneously hypertensive old rats is associated with a significant decrease of SUR2 expression.

ATP-sensitive potassium (KATP) channels are participants of mechanisms of pathological myocardial remodeling containment. The aim of our work was to find the association of changes in the expression of Kir6.1, Kir6.2, SUR1, and SUR2 subunits of KATP channels with changes in heart function and structure during aging under conditions of the constant increase of vascular pressure. The experiments were carried out on young and old spontaneously hypertensive rats (SHR) and Wistar rats. The expression levels of KATP channels subunits were determined using reverse transcription and quantitative PCR. It is shown that the mRNA expression level of Kir6.1 in young SHR rats is significantly lower (6.3-fold, p = 0.035) than that of young Wistar rats that may be one of the causes of arterial hypertension in SHR. At the same time, mRNA expression of both Kir6.1 and Kir6.2 in old SHR rats was significantly higher (6.8-fold, p = 0.003, and 5.9-fold, p = 0.006, respectively) than in young hypertensive animals. In both groups of old animals, SUR2 expression was significantly reduced compared to young animals, in Wistar rats at 3.87-fold (p = 0.028) and in SHR rats at 48.2-fold (p = 0.033). Changes in SUR1 expression were not significant. Thus, significant changes in the cardiovascular system, including impaired function and structure of the heart in old SHR rats, were associated with a significant decrease in SUR2 expression that may be one of the mechanisms of heart failure decompensation. Therefore, it can be assumed that increased expression of SUR2 may be one of the protective mechanisms against pathological myocardial remodeling.

Thromboxane-prostanoid receptor activation blocks ATP-sensitive potassium channels in rat aortas.

K+ channel activation is one of the major mechanisms involved in vasodilation. Vasoconstrictor agonists such as angiotensin II promote ATP-dependent potassium channels (KATP ) dysfunction. This study evaluates whether thromboxane-prostanoid (TP receptor) activation by the agonist U46619 increases reactive oxygen species (ROS) production in rat aortas, which could contribute to KATP channel dysfunction and impaired NO-dependent vasodilation. TP receptor activation with the selective agonist U46619 increased ROS in endothelial cells (ECs) and vascular smooth muscle cells (VSMCs), but the TP receptor antagonist SQ29548 abolished this effect. ECs and VSMCs incubation with ROS scavengers like Tiron or PEG-Catalase impaired U46619-induced ROS production. U46619 at the concentrations of 0.1 and 1 µmol/L induced contractions with similar amplitude. KATP channel activation with pinacidil-induced relaxation was lower for the contractions induced with 0.1 or 1 µmol/L U46619 than with 10 nmol/L U46619. Acetylcholine-induced relaxation provided similar results. In aortas pre-contracted with 10 nmol/L U46619, neither Tiron (100 µmol/L) nor catalase (300 U/mL) affected pinacidil-induced relaxation. However, in aortas pre-contracted with 0.1 µmol/L U46619, catalase potentiated pinacidil-induced relaxation. Pinacidil potentiated acetylcholine-induced relaxation in aortas pre-contracted with 0.1 and 1 µmol/L U46619. Incubation with 10 nmol/L U46619 increased NO concentration in ECs. Taken together, these results show that high concentrations of the TP receptor agonist U46619 impair KATP channels, which is probably due to ROS production. It is likely that hydrogen peroxide is the ROS.

Diazoxide Needs Mitochondrial Connexin43 to Exert Its Cytoprotective Effect in a Cellular Model of CoCl2-Induced Hypoxia.

Hypoxia is the leading cause of death in cardiomyocytes. Cells respond to oxygen deprivation by activating cytoprotective programs, such as mitochondrial connexin43 (mCx43) overexpression and the opening of mitochondrial KATP channels, aimed to reduce mitochondrial dysfunction. In this study we used an in vitro model of CoCl2-induced hypoxia to demonstrate that mCx43 and KATP channels cooperate to induce cytoprotection. CoCl2 administration induces apoptosis in H9c2 cells by increasing mitochondrial ROS production, intracellular and mitochondrial calcium overload and by inducing mitochondrial membrane depolarization. Diazoxide, an opener of KATP channels, reduces all these deleterious effects of CoCl2 only in the presence of mCx43. In fact, our results demonstrate that in the presence of radicicol, an inhibitor of Cx43 translocation to mitochondria, the cytoprotective effects of diazoxide disappear. In conclusion, these data confirm that there exists a close functional link between mCx43 and KATP channels.

Analysis of KATP Channels Opening Probability of Hippocampus Cells Treated with Kainic Acid.

BACKGROUND: Kainic acid (KA)-induced seizures may be a valuable tool in the assessment of anti-epileptic drug efficacy in complex partial seizures. This study investigated the effects of KA on ATP-sensitive K+ (KATP) channels opening probability (NPo), which plays a crucial role in neuronal activities. METHODS: For the optimisation and validation protocol, ß-cells were plated onto 35 mm plastic petri dishes and maintained in RPMI-1640 media supplemented with 10 mM glucose, 10% FCS and 25 mM of N-2-hydroxyethylpiperazine-N-ethanesulfonic acid (HEPES). The treatment effects of 10 mM glucose and 30 µM fluoxetine on KATP channels NPo of ß-cells were assessed via cell-attached patch-clamp recordings. For hippocampus cell experiments, hippocampi were harvested from day 17 of maternal Lister-hooded rat foetus, and then transferred to a Ca2+ and Mg2+-free HEPES-buffered Hank's salt solution (HHSS). The dissociated cells were cultured and plated onto a 25 mm round cover glasses coated with poly-d-lysine (0.1 mg/mL) in a petri dish. The KATP channels NPo of hippocampus cells when perfused with 1 mM and 10 mM of KA were determined. RESULTS: NPo of ß-cells showed significant decreasing patterns (P < 0.001) when treated with 10 mM glucose 0.048 (0.027) as well as 30 µM fluoxetine 0.190 (0.141) as compared to basal counterpart. In hippocampus cell experiment, a significant increase (P < 0.001) in mean NPo 2.148 (0.175) of neurons when applied with 1 mM of KA as compared to basal was observed. CONCLUSION: The two concentrations of KA used in the study exerted contrasting effects toward the mean of NPo. It is hypothesised that KA at lower concentration (1 mM) opens more KATP channels, leading to hyperpolarisation of the neurons, which may prevent neuronal hyper excitability. No effect was shown in 10 mM KA treatment, suggesting that only lower than 10 mM KA produced significant changes in KATP channels. This implies further validation of KA concentration to be used in the future.

'Resistance is futile?' - paradoxical inhibitory effects of KATP channel closure in glucagon-secreting α-cells.

By secreting insulin and glucagon, the ß- and α-cells of the pancreatic islets play a central role in the regulation of systemic metabolism. Both cells are equipped with ATP-regulated potassium (KATP ) channels that are regulated by the intracellular ATP/ADP ratio. In ß-cells, KATP channels are active at low (non-insulin-releasing) glucose concentrations. An increase in glucose leads to KATP channel closure, membrane depolarization and electrical activity that culminates in elevation of [Ca2+ ]i and initiation of exocytosis of the insulin-containing secretory granules. The α-cells are also equipped with KATP channels but they are under strong tonic inhibition at low glucose, explaining why α-cells are electrically active under hypoglycaemic conditions and generate large Na+ - and Ca2+ -dependent action potentials. Closure of residual KATP channel activity leads to membrane depolarization and an increase in action potential firing but this stimulation of electrical activity is associated with inhibition rather than acceleration of glucagon secretion. This paradox arises because membrane depolarization reduces the amplitude of the action potentials by voltage-dependent inactivation of the Na+ channels involved in action potential generation. Exocytosis in α-cells is tightly linked to the opening of voltage-gated P/Q-type Ca2+ channels, the activation of which is steeply voltage-dependent. Accordingly, the inhibitory effect of the reduced action potential amplitude exceeds the stimulatory effect resulting from the increased action potential frequency. These observations highlight a previously unrecognised role of the action potential amplitude as a key regulator of pancreatic islet hormone secretion.

Differential expression of genes participating in cardiomyocyte electrophysiological remodeling via membrane ionic mechanisms and Ca2+-handling in human heart failure.

Excitation-contraction coupling in normal cardiac function is performed with well balanced and coordinated functioning but with complex dynamic interactions between functionally connected membrane ionic currents. However, their genomic investigations provide essential information on the regulation of diseases by their transcripts. Therefore, we examined the gene expression levels of the most important voltage-gated ionic channels such as Na+-channels (SCN5A), Ca2+-channels (CACNA1C and CACNA1H), and K+-channels, including transient outward (KCND2, KCNA2, KCNA5, KCNA8), inward rectifier (KCNJ2, KCNJ12, KCNJ4), and delayed rectifier (KCNB1) in left ventricular tissues from either ischemic or dilated cardiomyopathy (ICM or DCM). We also examined the mRNA levels of ATP-dependent K+-channels (KCNJ11, ABCC9) and ERG-family channels (KCNH2). We further determined the mRNA levels of ryanodine receptors (RyR2; ARVC2), phospholamban (PLB or PLN), SR Ca2+-pump (SERCA2; ATP2A1), an accessory protein FKBP12 (PPIASE), protein kinase A (PPNAD4), and Ca2+/calmodulin-dependent protein kinase II (CAMK2G). The mRNA levels of SCN5A, CACNA1C, and CACNA1H in both groups decreased markedly in the heart samples with similar significance, while KvLQT1 genes were high with depressed Kv4.2. The KCNJ11 and KCNJ12 in both groups were depressed, while the KCNJ4 level was significantly high. More importantly, the KCNA5 gene was downregulated only in the ICM, while the KCNJ2 was upregulated only in the DCM. Besides, mRNA levels of ARVC2 and PLB were significantly high compared to the controls, whereas others (ATP2A1, PPIASE, PPNAD4, and CAMK2G) were decreased. Importantly, the increases of KCNB1 and KCNJ11 were more prominent in the ICM than DCM, while the decreases in ATP2A1 and FKBP1A were more prominent in DCM compared to ICM. Overall, this study was the first to demonstrate that the different levels of changes in gene profiles via different types of cardiomyopathy are prominent particularly in some K+-channels, which provide further information about our knowledge of how remodeling processes can be differentiated in HF originated from different pathological conditions.