KATP Subunits Research Articles

Effect of Type-2 Diabetes Mellitus on the Expression and Function of Smooth Muscle ATP-Sensitive Potassium Channels in Human Internal Mammary Artery Grafts.

Here we have shown for the first time altered expression of the vascular smooth muscle (VSM) KATP channel subunits in segments of the human internal mammary artery (HIMA) in patients with type-2 diabetes mellitus (T2DM). Functional properties of vascular KATP channels in the presence of T2DM, and the interaction between its subunits and endogenous ligands known to relax this vessel, were tested using the potassium (K) channels opener, pinacidil. HIMA is the most commonly used vascular graft in cardiac surgery. Previously it was shown that pinacidil relaxes HIMA segments through interaction with KATP (SUR2B/Kir6.1) vascular channels, but it is unknown whether pinacidil sensitivity is changed in the presence of T2DM, considering diabetes-induced vascular complications commonly seen in patients undergoing coronary artery bypass graft surgery (CABG). KATP subunits were detected in HIMA segments using Western blot and immunohistochemistry analyses. An organ bath system was used to interrogate endothelium-independent vasorelaxation caused by pinacidil. In pharmacological experiments, pinacidil was able to relax HIMA from patients with T2DM, with sensitivity comparable to our previous results. All three KATP subunits (SUR2B, Kir6.1 and Kir6.2) were observed in HIMA from patients with and without T2DM. There were no differences in the expression of the SUR2B subunit. The expression of the Kir6.1 subunit was lower in HIMA from T2DM patients. In the same group, the expression of the Kir6.2 subunit was higher. Therefore, KATP channels might not be the only method of pinacidil-induced dilatation of T2DM HIMA. T2DM may decrease the level of Kir6.1, a dominant subunit in VSM of HIMA, altering the interaction between pinacidil and those channels.

MiRNA Expression Profiles in Isolated Ventricular Cardiomyocytes: Insights into Doxorubicin-Induced Cardiotoxicity.

Doxorubicin (DOX), widely used as a chemotherapeutic agent for various cancers, is limited in its clinical utility by its cardiotoxic effects. Despite its widespread use, the precise mechanisms underlying DOX-induced cardiotoxicity at the cellular and molecular levels remain unclear, hindering the development of preventive and early detection strategies. To characterize the cytotoxic effects of DOX on isolated ventricular cardiomyocytes, focusing on the expression of specific microRNAs (miRNAs) and their molecular targets associated with endogenous cardioprotective mechanisms such as the ATP-sensitive potassium channel (KATP), Sirtuin 1 (SIRT1), FOXO1, and GSK3β. We isolated Guinea pig ventricular cardiomyocytes by retrograde perfusion and enzymatic dissociation. We assessed cell morphology, Reactive Oxygen Species (ROS) levels, intracellular calcium, and mitochondrial membrane potential using light microscopy and specific probes. We determined the miRNA expression profile using small RNAseq and validated it using stem-loop qRT-PCR. We quantified mRNA levels of some predicted and validated molecular targets using qRT-PCR and analyzed protein expression using Western blot. Exposure to 10 µM DOX resulted in cardiomyocyte shortening, increased ROS and intracellular calcium levels, mitochondrial membrane potential depolarization, and changes in specific miRNA expression. Additionally, we observed the differential expression of KATP subunits (ABCC9, KCNJ8, and KCNJ11), FOXO1, SIRT1, and GSK3β molecules associated with endogenous cardioprotective mechanisms. Supported by miRNA gene regulatory networks and functional enrichment analysis, these findings suggest that DOX-induced cardiotoxicity disrupts biological processes associated with cardioprotective mechanisms. Further research must clarify their specific molecular changes in DOX-induced cardiac dysfunction and investigate their diagnostic biomarkers and therapeutic potential.

Hypoxia Promotes Atrial Tachyarrhythmias via Opening of ATP-Sensitive Potassium Channels.

Hypoxia-ischemia predisposes to atrial arrhythmia. Atrial ATP-sensitive potassium channel (KATP) modulation during hypoxia has not been explored. We investigated the effects of hypoxia on atrial electrophysiology in mice with global deletion of KATP pore-forming subunits. Whole heart KATP RNA expression was probed. Whole-cell KATP current and action potentials were recorded in isolated wild-type (WT), Kir6.1 global knockout (6.1-gKO), and Kir6.2 global knockout murine atrial myocytes. Langendorff-perfused hearts were assessed for atrial effective refractory period (ERP), conduction velocity, wavefront path length (WFPL), and arrhymogenicity under normoxia/hypoxia using a microelectrode array and programmed electrical stimulation. Heart histology was assessed. Expression patterns were essentially identical for all KATP subunit RNA across human heart, whereas in mouse, Kir6.1 and SUR2 (sulphonylurea receptor) were higher in ventricle than atrium, and Kir6.2 and SUR1 were higher in atrium. Compared with WT, Kir6.2 global knockout atrial myocytes had reduced tolbutamide-sensitive current and action potentials were more depolarized with slower upstroke and reduced peak amplitude. Action potential duration was prolonged in 6.1-gKO atrial myocytes, absent of changes in other ion channel gene expression or atrial myocyte hypertrophy. In Langendorff-perfused hearts, baseline atrial ERP was prolonged and conduction velocity reduced in both KATP knockout mice compared with WT, without histological fibrosis. Compared with baseline, hypoxia led to conduction velocity slowing, stable ERP, and WFPL shortening in WT and 6.1-gKO hearts, whereas WFPL was stable in Kir6.2 global knockout hearts due to ERP prolongation with conduction velocity slowing. Tolbutamide reversed hypoxia-induced WFPL shortening in WT and 6.1-gKO hearts through ERP prolongation. Atrial tachyarrhythmias inducible with programmed electrical stimulation during hypoxia in WT and 6.1-gKO mice correlated with WFPL shortening. Spontaneous arrhythmia was not seen. KATP block/absence leads to cellular and tissue level atrial electrophysiological modification. Kir6.2 global knockout prevents hypoxia-induced atrial WFPL shortening and atrial arrhythmogenicity to programmed electrical stimulation. This mechanism could be explored translationally to treat ischemically driven atrial arrhythmia.

Use of nicorandil is associated with increased risk of incident atrial fibrillation.

Background: Nicorandil will activate ATP-sensitive potassium channel (KATP). However, activation of potassium channels plays an important role in the mechanism of atrial fibrillation (AF) or atrial flutter (AFL). Whether use of nicorandil might contribute to initiation and/or perpetuation of AF/AFL remained unknown. We determined the relationship between use of nicorandil and risk of atrial fibrillation and determined its molecular mechanism.Methods: We performed a nested case-control study using a cohort from the National Health Insurance Research Database (NHIRD) of Taiwan. The association between nicorandil use and risk of atrial fibrillation/flutter was estimated by logistic regression model. We also performed molecular, cellular and animal studies to explain the association.Results: A total of 715 individuals who experienced AF/atrial flutter were matched to 72,215 controls. New use of nicorandil was found to be associated with increased risk for AF/AFL (odds ratio [OR], 2.34; 95% CI 1.07–5.13) compared to nitrate use. We found the expression of KATP subunits Kir6.2 and SUR2A in human and rat left atrial tissues. Furthermore, nicorandil directly shortened action potential duration (APD) in rat left atrium and shortened the QT interval of cultured human induced pluripotent stem cell (iPSC) derived cardiomyocytes (iPSC-CMs).Conclusions: Use of nicorandil was found to be associated with increased risk of AF/AFL. We also showed the expression of KATP subunits in human atria, and a possible mechanism that use of nicorandil increases the risk of AF through activation of KATP and shortening of atrial APD.

Functional Regulation of KATP Channels and Mutant Insight Into Clinical Therapeutic Strategies in Cardiovascular Diseases

ATP-sensitive potassium channels (KATP channels) play pivotal roles in excitable cells and link cellular metabolism with membrane excitability. The action potential converts electricity into dynamics by ion channel-mediated ion exchange to generate systole, involved in every heartbeat. Activation of the KATP channel repolarizes the membrane potential and decreases early afterdepolarization (EAD)-mediated arrhythmias. KATP channels in cardiomyocytes have less function under physiological conditions but they open during severe and prolonged anoxia due to a reduced ATP/ADP ratio, lessening cellular excitability and thus preventing action potential generation and cell contraction. Small active molecules activate and enhance the opening of the KATP channel, which induces the repolarization of the membrane and decreases the occurrence of malignant arrhythmia. Accumulated evidence indicates that mutation of KATP channels deteriorates the regulatory roles in mutation-related diseases. However, patients with mutations in KATP channels still have no efficient treatment. Hence, in this study, we describe the role of KATP channels and subunits in angiocardiopathy, summarize the mutations of the KATP channels and the functional regulation of small active molecules in KATP channels, elucidate the potential mechanisms of mutant KATP channels and provide insight into clinical therapeutic strategies.

Kir6.2-deficient mice develop somatosensory dysfunction and axonal loss in the peripheral nerves

SummaryGlucose-responsive ATP-sensitive potassium channels (KATP) are expressed in a variety of tissues including nervous systems. The depolarization of the membrane potential induced by glucose may lead to hyperexcitability of neurons and induce excitotoxicity. However, the roles of KATP in the peripheral nervous system (PNS) are poorly understood. Here, we determine the roles of KATP in the PNS using KATP-deficient (Kir6.2-deficient) mice. We demonstrate that neurite outgrowth of dorsal root ganglion (DRG) neurons was reduced by channel closers sulfonylureas. However, a channel opener diazoxide elongated the neurite. KATP subunits were expressed in mouse DRG, and expression of certain subunits including Kir6.2 was increased in diabetic mice. In Kir6.2-deficient mice, the current perception threshold, thermal perception threshold, and sensory nerve conduction velocity were impaired. Electron microscopy revealed a reduction of unmyelinated and small myelinated fibers in the sural nerves. In conclusion, KATP may contribute to the development of peripheral neuropathy.

The effect of signal pathways on the neural KATP subunits SUR1/Kir6.2 expression induced by Aβ1-42

The effect of signal pathways on the neural KATP subunits SUR1/Kir6.2 expression induced by Aβ1-42

Clinical and Genetic Characteristics of ABCC8 Nonneonatal Diabetes Mellitus: A Systematic Review.

Objectives Diabetes mellitus (DM) is a major chronic metabolic disease in the world, and the prevalence has been increasing rapidly in recent years. The channel of KATP plays an important role in the regulation of insulin secretion. The variants in ABCC8 gene encoding the SUR1 subunit of KATP could cause a variety of phenotypes, including neonatal diabetes mellitus (ABCC8-NDM) and ABCC8-induced nonneonatal diabetes mellitus (ABCC8-NNDM). Since the features of ABCC8-NNDM have not been elucidated, this study is aimed at concluding the genetic features and clinical characteristics. Methods We comprehensively reviewed the literature associated with ABCC8-NNDM in the following databases: MEDLINE, PubMed, and Web of Science to investigate the features of ABCC8-NNDM. Results Based on a comprehensive literature search, we found that 87 probands with ABCC8-NNDM carried 71 ABCC8 genetic variant alleles, 24% of whom carried inactivating variants, 24% carried activating variants, and the remaining 52% carried activating or inactivating variants. Nine of these variants were confirmed to be activating or inactivating through functional studies, while four variants (p.R370S, p.E1506K, p.R1418H, and p.R1420H) were confirmed to be inactivating. The phenotypes of ABCC8-NNDM were variable and could also present with early hyperinsulinemia followed by reduced insulin secretion, progressing to diabetes later. They had a relatively high risk of microvascular complications and low prevalence of nervous disease, which is different from ABCC8-NDM. Conclusions Genetic testing is essential for proper diagnosis and appropriate treatment for patients with ABCC8-NNDM. And further studies are required to determine the complex mechanism of the variants of ABCC8-NNDM.

Decreased KATP Channel Activity Contributes to the Low Glucose Threshold for Insulin Secretion of Rat Neonatal Islets.

Transitional hypoglycemia in normal newborns occurs in the first 3 days of life and has clinical features consistent with hyperinsulinism. We found a lower threshold for glucose-stimulated insulin secretion from freshly isolated embryonic day (E) 22 rat islets, which persisted into the first postnatal days. The threshold reached the adult level by postnatal day (P) 14. Culturing P14 islets also decreased the glucose threshold. Freshly isolated P1 rat islets had a lower threshold for insulin secretion in response to 2-aminobicyclo-(2, 2, 1)-heptane-2-carboxylic acid, a nonmetabolizable leucine analog, and diminished insulin release in response to tolbutamide, an inhibitor of β-cell KATP channels. These findings suggested that decreased KATP channel function could be responsible for the lower glucose threshold for insulin secretion. Single-cell transcriptomic analysis did not reveal a lower expression of KATP subunit genes in E22 compared with P14 β cells. The investigation of electrophysiological characteristics of dispersed β cells showed that early neonatal and cultured cells had fewer functional KATP channels per unit membrane area. Our findings suggest that decreased surface density of KATP channels may contribute to the observed differences in glucose threshold for insulin release.

Novel cholesterol-dependent regulation of cardiac KATP subunit expression revealed using histone deacetylase inhibitors.

We recently discovered that the histone deacetylase inhibitor, trichostatin A (TSA), increases expression of the sulfonylurea receptor 2 (SUR2; Abcc9) subunit of the ATP‐sensitive K+ (KATP) channel in HL‐1 cardiomyocytes. Interestingly, the increase in SUR2 was abolished with exogenous cholesterol, suggesting that cholesterol may regulate channel expression. In the present study, we tested the hypothesis that TSA increases SUR2 by depleting cholesterol and activating the sterol response element binding protein (SREBP) family of transcription factors. Treatment of HL‐1 cardiomyocytes with TSA (30 ng/ml) caused a time‐dependent increase in SUR2 mRNA expression that correlates with the time course of cholesterol depletion assessed by filipin staining. Consistent with the cholesterol‐dependent regulation of SREBP increasing SUR2 mRNA expression, we observe a significant increase in SREBP cleavage and translocation to the nucleus following TSA treatment that is inhibited by exogenous cholesterol. Further supporting the role of SREBP in mediating the effect of TSA on KATP subunit expression, SREBP1 significantly increased luciferase reporter gene expression driven by the upstream SUR2 promoter. Lastly, HL‐1 cardiomyocytes treated with the SREBP inhibitor PF429242 significantly suppresses the effect of TSA on SUR2 gene expression. These results demonstrate that SREBP is an important regulator of KATP channel expression and suggest a novel method by which hypercholesterolemia may exert negative effects on the cardiovascular system, namely, by suppressing expression of the KATP channel.

Linking lymphatic function to disease.

Lymphatic function is critical for maintaining interstitial fluid balance and is linked to multiple pathological conditions. Over the past 15 years, lymphatic research has re-emerged as an area of emphasis for vascular biologists wanting to understand the causes of oedema and the poor fluid drainage that are common denominators for various diseases (Breslin et al. 2018). Research has focused on two big questions: (1) How does fluid enter the lymphatic system? (2) How does fluid move through an initial lymphatic network? Interstitial fluid is thought to enter the lymphatic system through specialized intercellular junctions between endothelial cells of the initial lymphatic vessels. The fluid then passes into upstream collecting lymphatic vessels, also termed contractile vessels. These vessel segments, referred to as lympangions, are separated by intraluminal valves that prevent backflow of lymph. While current research has expanded to include coordination of lymphangion development, the active mechanism responsible for fluid flow from initial lymphatics to the collecting vessels and the involvement of immune cells, the mechanisms that control the frequency and force of contractions by lymphatic smooth muscle cells remain critical foci of investigation. Spontaneous smooth muscle cell contraction involves cell-surface receptors, ion fluxes and actin-myosin dynamics. As these mechanisms are essential for driving collecting vessel constriction and lymph flow, the discovery of new information has led to advancements in our understanding of how the lymphatic system works (Fig. 1). Basic science discoveries now motivate the challenge of linking new information to diseases. Perhaps, the more important question for lymphatic researchers and clinicians should be related to how our advanced understanding can impact treatment. In the current issue of The Journal of Physiology, work by Davis et al. (2020) provides an example of meeting this challenge by linking ATP sensitive smooth muscle specific potassium channel (KATP) dysfunction to Cantú syndrome, a rare disease associated with genetic mutation that alters potassium channel function and leads to lymphoedema. In the context of lymphatic function, the genetic mutation is considered to cause the potassium channels to be preferentially in the open state (Davis et al. 2020). The results by Davis et al. suggest that gain-of-function KATP subunit mutant mice also exhibit lymphatic contractile dysfunction, and that the inhibition of over-activity represents a therapeutic approach for reversing the effects. While a few studies have implicated the presence and a role for KATP channels in lymphatic vessels, the work by Davis et al. contributes cell-specific information regarding their role and a link to a disease state. The incorporation of studies with mutant mice, isolated vessels from humans, in vitro experiments, and multiple methodological approaches serve to provide novel mechanistic understanding of how a disease scenario can influence lymphatic fluid flow and in turn highlights why clinicians should be mindful about new basic research findings. The authors do not have any conflicts of interests on the submission. All authors have read and approved the final version of this manuscript and agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All persons designated as authors qualify for authorship, and all those who qualify for authorship are listed. Funding was provided by NIH 1R01AG049821 awarded to W.L.M. and NIH 1R01GM120774 awarded to J.W.B.

Propofol inhibits stromatoxin-1-sensitive voltage-dependent K+ channels in pancreatic β-cells and enhances insulin secretion.

BackgroundProper glycemic control is an important goal of critical care medicine, including perioperative patient care that can influence patients’ prognosis. Insulin secretion from pancreatic β-cells is generally assumed to play a critical role in glycemic control in response to an elevated blood glucose concentration. Many animal and human studies have demonstrated that perioperative drugs, including volatile anesthetics, have an impact on glucose-stimulated insulin secretion (GSIS). However, the effects of the intravenous anesthetic propofol on glucose metabolism and insulin sensitivity are largely unknown at present.MethodsThe effect of propofol on insulin secretion under low glucose or high glucose was examined in mouse MIN6 cells, rat INS-1 cells, and mouse pancreatic β-cells/islets. Cellular oxygen or energy metabolism was measured by Extracellular Flux Analyzer. Expression of glucose transporter 2 (GLUT2), potassium channels, and insulin mRNA was assessed by qRT-PCR. Protein expression of voltage-dependent potassium channels (Kv2) was also assessed by immunoblot. Propofol’s effects on potassium channels including stromatoxin-1-sensitive Kv channels and cellular oxygen and energy metabolisms were also examined.ResultsWe showed that propofol, at clinically relevant doses, facilitates insulin secretion under low glucose conditions and GSIS in MIN6, INS-1 cells, and pancreatic β-cells/islets. Propofol did not affect intracellular ATP or ADP concentrations and cellular oxygen or energy metabolism. The mRNA expression of GLUT2 and channels including the voltage-dependent calcium channels Cav1.2, Kir6.2, and SUR1 subunit of KATP, and Kv2 were not affected by glucose or propofol. Finally, we demonstrated that propofol specifically blocks Kv currents in β-cells, resulting in insulin secretion in the presence of glucose.ConclusionsOur data support the hypothesis that glucose induces membrane depolarization at the distal site, leading to KATP channel closure, and that the closure of Kv channels by propofol depolarization in β-cells enhances Ca2+ entry, leading to insulin secretion. Because its activity is dependent on GSIS, propofol and its derivatives are potential compounds that enhance and initiate β-cell electrical activity.

Aβ1-42 increases the expression of neural KATP subunits Kir6.2/SUR1 via the NF-κB, p38 MAPK and PKC signal pathways in rat primary cholinergic neurons.

ATP-sensitive potassium channels (KATP) may mediate a potential neuroprotective role in Alzheimer's disease (AD). Given that exposure to Aβ1-42 in cultured primary cholinergic neurons for 72 h significantly upregulates the expression of KATP subunits Kir6.2/SUR1, we aim to study the underlying signal transduction mechanisms that are involved in Aβ1-42-induced upregulation of KATP subunits Kir6.2/SUR1. In the present study, we first identified the primary cultured rat cortical and hippocampal neurons using immunocytochemistry. 0.5 μM NF-κB inhibitor SN-50, 2 μM p38MAPK inhibitor SB203580 or 2 μM PKC inhibitor Chelerythrine chloride (CTC) were then added in three separate groups, followed by 2 μM Aβ1-42 30 min later in all 3 groups. Western Blot was performed 72 h later to detect the expression of KATP subunits Kir6.2/SUR1. We found that Aβ1-42 significantly increased the level of KATP subunits Kir6.2/SUR1 expression at 72 h when compared with the control group ( p < 0.05). However, when compared with the Aβ1-42 group, the level of KATP subunits Kir6.2/SUR1 expression at 72 h significantly decreased in the SN50 + Aβ1-42 group, SB203580 + Aβ1-42 group, and the CTC + Aβ1-42 group ( p < 0.05). Our findings suggest that the NF-κB, p38 MAPK, and PKC signal pathways are partially involved in the upregulation of KATP subunits Kir6.2/SUR1 expression induced by Aβ1-42 cytotoxicity in neurons, which supports a potential theoretical basis of targeting these signal pathways in the treatment of AD.

2-Deoxy-D-Glucose Exhibits Anti-seizure Effects by Mediating the Netrin-G1-KATP Signaling Pathway in Epilepsy.

Epilepsy is a disorder of the brain characterized by an enduring predisposition to generate epileptic seizures. The glycolytic inhibitor 2-deoxy-D-glucose (2-DG) has been reported to exert antiepileptic effects by upregulating KATP subunits (kir6.1 and kir6.2). We evaluated whether 2-DG exhibits anti-seizure effect by mediating the netrin-G1-KATP signaling pathway in epilepsy. In a mouse epilepsy model induced by lithium chloride-pilocarpine, 2-DG intervention increased the mRNA and protein expression levels of kir6.1 and kir6.2, and these increases were significantly reversed after knocking down netrin-G1 expression. Similarly, in cultured neurons with a magnesium-free medium, we found that the frequency of spontaneous postsynaptic potentials (SP) was increased, and in the meanwhile, expression levels of kir6.1 and kir6.2 were increased after pretreatment with 2DG. These effects were remarkably reversed after knocking down netrin-G1. Thus, our findings show that 2DG exhibits anti-seizure effects through the netrin-G1-KATP signaling pathway.

Activation mechanism of ATP-sensitive K+ channels explored with real-time nucleotide binding.

The response of ATP-sensitive K+ channels (KATP) to cellular metabolism is coordinated by three classes of nucleotide binding site (NBS). We used a novel approach involving labeling of intact channels in a native, membrane environment with a non-canonical fluorescent amino acid and measurement (using FRET with fluorescent nucleotides) of steady-state and time-resolved nucleotide binding to dissect the role of NBS2 of the accessory SUR1 subunit of KATP in channel gating. Binding to NBS2 was Mg2+-independent, but Mg2+ was required to trigger a conformational change in SUR1. Mutation of a lysine (K1384A) in NBS2 that coordinates bound nucleotides increased the EC50 for trinitrophenyl-ADP binding to NBS2, but only in the presence of Mg2+, indicating that this mutation disrupts the ligand-induced conformational change. Comparison of nucleotide-binding with ionic currents suggests a model in which each nucleotide binding event to NBS2 of SUR1 is independent and promotes KATP activation by the same amount.

Nucleotide Modulation of KATP Channels Disentangled with FRET

ATP-sensitive K+ channel (KATP) gating is modulated via adenine nucleotide (ATP or ADP) binding to three classes of nucleotide binding site (NBS). The first site is located on the pore-forming Kir subunit of KATP (Kir6.2 in pancreatic β-cells); binding to this NBS is inhibitory. Two stimulatory NBSs are located on the cytoplasmic nucleotide binding domains of the accessory sulfonylurea receptor subunit (SUR1 in β-cells), part of the ABC transporter family. Despite decades of investigation, the details of nucleotide modulation, the affinity and specificity for each NBS, the interactions between the various NBSs, and the mechanism whereby nucleotide binding events are transmitted to the channel pore are largely unknown. To fully understand the nucleotide effects on KATP, we have labelled each NBS (one at a time) with the fluorescent, non-canonical amino acid ANAP and measured nucleotide binding at each site using FRET between ANAP and trinitrophenyl (TNP) nucleotide derivatives in unroofed membrane fragments. This has allowed us to dissect nucleotide binding to each site, evaluate mechanistically mutations expected to affect nucleotide binding (e.g. SUR1-K1384A, Kir6.2-G334D) or channel gating (e.g. Kir6.2-C166S), and investigate the action of clinically important drugs that inhibit or potentiate KATP (sulfonylureas and K+ channel openers). We have also combined this method with patch-clamp electrophysiology (either in separate experiments or simultaneously with patch-clamp fluorometry) to determine the functional consequences of binding at each NBS. This method will not only yield insights into KATP channel activity, but is readily extended to other channels, ABC transporters, and virtually any protein with a suitable fluorescent ligand.

Kir6.2, the Pore-Forming Subunit of ATP-Sensitive K+ Channels, Is Overexpressed in Human Posttraumatic Brain Contusions.

Brain contusions (BCs) are one of the most frequent lesions in patients with moderate and severe traumatic brain injury (TBI). BCs increase their volume due to peri-lesional edema formation and/or hemorrhagic transformation. This may have deleterious consequences and its mechanisms are still poorly understood. We previously identified de novo upregulation sulfonylurea receptor (SUR) 1, the regulatory subunit of adenosine triphosphate (ATP)-sensitive potassium (KATP) channels and other channels, in human BCs. Our aim here was to study the expression of the pore-forming subunit of KATP, Kir6.2, in human BCs, and identify its localization in different cell types. Protein levels of Kir6.2 were detected by western blot (WB) from 33 contusion specimens obtained from 32 TBI patients aged 14-74 years. The evaluation of Kir6.2 expression in different cell types was performed by immunofluorescence in 29 contusion samples obtained from 28 patients with a median age of 42 years. Control samples were obtained from limited brain resections performed to access extra-axial skull base tumors or intraventricular lesions. Contusion specimens showed an increase of Kir6.2 expression in comparison with controls. Regarding cellular location of Kir6.2, there was no expression of this channel subunit in blood vessels, either in control samples or in contusions. The expression of Kir6.2 in neurons and microglia was also analyzed, but the observed differences were not statistically significant. However, a significant increase of Kir6.2 was found in glial fibrillary acidic protein (GFAP)-positive cells in contusion specimens. Our data suggest that further research on SUR1-regulated ionic channels may lead to a better understanding of key mechanisms involved in the pathogenesis of BCs, and may identify novel targeted therapeutic strategies.

MiR-20 regulates myocardiac ischemia by targeting KATP subunit Kir6.1

This study aimed to examine the functional role of microRNA-20 (miR-20) and its potential target, Kir6.1, in ischemic myocardiocytes. The expression of miR-20 was detected by real-time PCR. Myocardiocytes were stained with terminal deoxynucleotidyl transferase dUTP nick end labeling (TU-NEL) reagent for apoptosis evaluation. Western blotting was used to detect the Kir6.1 protein in ischemic myocardiocytes transfected with miR-20 mimics or inhibitors. Luciferase reporter gene assay was performed to confirm the targeting effect of miR-20 on KCNJ8. The results showed that miR-20 was remarkably down-regulated, while the KATP subunit Kir6.1 was significantly up-regulated, during myocardial ischemia. The miR-20 overexpression promoted the apoptosis of ischemic myocardiocytes, but showed no such effect on normal cells. Under ischemic condition, myocardiocytes transfected with miR-20 mimics expressed less Kir6.1. On the contrary, inhibiting miR-20 increased the expression of Kir6.1 in the cells. Co-transfection of miR-20 mimics with the KCNJ8 3'-UTR plasmid into HEK293 cells consistently produced less luciferase activity than transfection of the plasmid alone. It was concluded that miR-20 may regulate myocardiac ischemia by targeting KATP subunit Kir6.1 to accelerate the cell apoptosis. Therefore miR-20 may serve as a therapeutic target for myocardial ischemic disease.

High glucose stimulates cell proliferation and Collagen IV production in rat mesangial cells through inhibiting AMPK-KATP signaling.

The present study investigated the putative mechanisms underlying effects of KATP channel on high glucose (HG)-induced mesangial cell proliferation and tissue inhibitors of metalloproteinases (TIMP)-2 and Collagen IV production. Rat mesangial cells were subjected to whole cell patch clamp to record the KATP channel currents under high glucose (HG, 30mM) condition. Cell proliferation was measured using a CCK-8 assay. The production of TIMP-2 and Collagen IV and AMP-activated protein kinase (AMPK)-signaling pathway activity was assessed by ELISA and Western blotting, respectively. AMPK agonist (AICAR) was used to analyze the role of this kinase. The expression of KATP subunit (Kir6.1, Kir6.2, SUR1, SUR2A and SUR2B) was examined using quantitative real-time PCR (RT-PCR). We found that HG was significant decreases in the expression of Kir6.1, SUB2A and SUB2B, three subunits of KATP, TIMP-2 production, KATP channel activity and AMPK activity, while it promoted the cell proliferation and Collagen IV production in rat mesangial cells. Pretreatment with KATP selective opener (diazoxide, DZX) significantly inhibited HG-induced mesangial cell proliferation, Collagen IV production and decrease in KATP channel activity in rat mesangial cells, which were reversed by pretreatment of 5-hydroxydecanoate, a selective inhibitor of KATP. Moreover, AICAR pretreatment inhibited HG-induced decrease in KATP channel activity. Taken together, activating AMPK-KATP signaling may protect against HG-induced mesangial cell proliferation and Collagen IV production, and, thereby, provides new insights into the molecular mechanisms underlying early diabetic nephropathy (DN).

KATP channels in high glucose-induced rat mesangial cell proliferation and release of MMP-2 and fibronectin

ATP-sensitive potassium (KATP) channels are well characterized in cardiac, pancreatic and many other muscle cells. The purpose of this study was to determine if KATP channels play a role in diabetic nephropathy (DN). In the present study, functional expression of the KATP channel was examined in rat mesangial cells with or without high glucose (HG) stimulation. The mesangial cell proliferation and the release of matrix metalloproteinase (MMP)-2 and fibronectin in response to high glucose with a selective opener of KATP (diazoxide, DZX), or with a selective inhibitor of KATP (5-hydroxydecanoate, 5-HD) were also measured. The cell proliferation was observed using Cell Counting Kit-8 assay, and the mRNA expressions of KATP subunit, including Kir6.1, Kir6.2, sulfonylurea receptor 1 (SUR1), SUR2A and SUR2B, were assessed using quantitative real-time PCR. MMP-2 and fibronectin release was measured by ELISA. The present study clarified expression of SUR subunit of KATP in plasma. HG treatment could cause increased cell proliferation and release of MMP-2 and fibronectin in a dose-dependent manner. HG also significantly decreased the expression of Kir6.1, SUR2A and SUR2B. Pretreatment of DZX markedly decreased the expression of SUR1, SUR2A and SUR2B, but had no effect on Kir6.1 expression compared with HG alone, while these changes were inhibited by 5-HD pretreatment. Moreover, DZX also inhibited cell proliferation and release of MMP-2 and fibronectin in HG-induced rat mesangial cells, and that was corrected by 5-HD. These data suggest that HG stimulates mesangial cell proliferation and cellular matrix release via inhibiting KATP channel activity, leading us to propose that KATP channel dysfunction may be involved in the development of DN.