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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.

Accumulating evidence suggests that ATP‐sensitive potassium (KATP) channels play an important role in the selective degeneration of dopaminergic neurons in the substantia nigra (SN). Furthermore, the expression of the KATP channel subunit sulfonylurea receptor 1 (SUR1) is upregulated in the remaining nigral dopaminergic neurons in Parkinson's disease (PD). However, the mechanism underlying this selective upregulation of the SUR1 subunit and its subsequent roles in PD progression are largely unknown. In 3‐, 6‐, and 9‐month‐old A53T α‐synuclein transgenic (α‐SynA53T+/+) mice, only the SUR1 subunit and not SUR2B or Kir6.2 was upregulated, accompanied by neuronal damage. Moreover, the occurrence of burst firing in dopaminergic neurons was increased with the upregulation of the SUR1 subunit, whereas no changes in the firing rate were observed except in 9‐month‐old α‐SynA53T+/+ mice. After interference with SUR1 expression by injection of lentivirus into the SN, the progression of dopaminergic neuron degeneration was delayed. Further studies showed that elevated expression of the transcription factors FOXA1 and FOXA2 could cause the upregulation of the SUR1 subunit in α‐SynA53T+/+ mice. Our findings revealed the regulatory mechanism of the SUR1 subunit and the role of KATP channels in the progression of dopaminergic neuron degeneration, providing a new target for PD drug therapy. The elevated expression of SUR1 subunit of KATP channels in nigral dopaminergic neurons was regulated by the transcription factors FOXA1 and FOXA2 at the early stage of PD.The upregulated SUR1 promotes its transmembrane transport and then causes the membrane hyperpolarization, which may promote the occurrence of cluster discharges mediated by NMDA receptors and affect the activities of dopaminergic neurons.

The Insulin Secretory Granule Is the Major Site of K ATP Channels of the Endocrine Pancreas Xuehui Geng 1 , Lehong Li 1 , Simon Watkins 1 , Paul D. Robbins 2 and Peter Drain 1 1 Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 2 Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania Abstract With ATP sites on K ir 6.2 that inhibit activity and ADP sites on SUR1 that antagonize the inhibition, ATP-sensitive potassium channels (K ATP channels) are designed as exquisite sensors of adenine nucleotide levels that signal changes in glucose metabolism. If pancreatic K ATP channels localize to the insulin secretory granule, they would be well positioned to transduce changes in glucose metabolism into changes in granule transport and exocytosis. Tests for pancreatic K ATP channels localized to insulin secretory granules led to the following observations: fluorescent sulfonylureas that bind the pancreatic K ATP channel specifically label intracellular punctate structures in cells of the endocrine pancreas. The fluorescent glibenclamides colocalize with Ins-C-GFP, a live-cell fluorescent reporter of insulin granules. Expression of either SUR1-GFP or K ir 6.2-GFP fusion proteins, but not expression of GFP alone, directs GFP fluorescence to insulin secretory granules. An SUR1 antibody specifically labels insulin granules identified by anti-insulin. Two different K ir 6.2 antibodies specifically label insulin secretory granules identified by anti-insulin. Immunoelectron microscopy showed K ir 6.2 antibodies specifically label perimeter membrane regions of the secretory granule. Relatively little or no labeling of other structures, including the plasma membrane, was found. Our results demonstrate that the insulin secretory granule is the major site of K ATP channels of the endocrine pancreas. Footnotes Address correspondence and reprint requests to Peter Drain, Biomedical Science Tower South, Room 323, 3500 Terrace St., Pittsburgh, PA 15261. E-mail: drain{at}pitt.edu . Received for publication 30 August 2002 and accepted in revised form 10 December 2002. gK ATP , granule ATP-sensitive potassium channel; K ATP channel, ATP-sensitive potassium channel; K D , apparent dissociation constant. DIABETES

Background Parkinson's disease (PD) is a common neurodegenerative disorder. Microglia‐mediated neuroinflammation has emerged as an involving mechanism at the initiation and development of PD. Activation of adenosine triphosphate (ATP)‐sensitive potassium (KATP) channels can protect dopaminergic neurons from damage. Sodium butyrate (NaB) shows anti‐inflammatory and neuroprotective effects in some animal models of brain injury and regulates the KATP channels in islet β cells. In this study, we aimed to verify the anti‐inflammatory effect of NaB on PD and further explored potential molecular mechanisms. Methods We established an in vitro PD model in BV2 cells using 1‐methyl‐4‐phenylpyridinium (MPP+). The effects of MPP+ and NaB on BV2 cell viability were detected by cell counting kit‐8 assays. The morphology of BV2 cells with or without MPP+ treatment was imaged via an optical microscope. The expression of Iba‐1 was examined by the immunofluorescence staining. The intracellular ATP content was estimated through the colorimetric method, and Griess assay was conducted to measure the nitric oxide production. The expression levels of pro‐inflammatory cytokines and KATP channel subunits were evaluated by reverse transcription–quantitative polymerase chain reaction and western blot analysis. Results NaB (5 mM) activated the KATP channels through elevating Kir6.1 and Kir6.1 expression in MPP+‐challenged BV2 cells. Both NaB and pinacidil (a KATP opener) suppressed the MPP+‐induced activation of BV2 cells and reduced the production of nitrite and pro‐inflammatory cytokines in MPP+‐challenged BV2 cells. Conclusion NaB treatment alleviates the MPP+‐induced inflammatory responses in microglia via activation of KATP channels. Sodium butyrate (NaB) retrains the activation of the proinflammatory phenotype of microglia by promoting KATP channel opening through enhancing the expression of Kir6.1 and Kir6.2.

We have demonstrated that ATP‐sensitive potassium (KATP) channel agonists attenuated fibrosis; however, the mechanism remained unclear. Since RhoA has been identified as a mediator of cardiac fibrosis, we sought to determine whether the anti‐fibrotic effects of KATP channel agonists were mediated via regulating macrophage phenotype and fibroblast differentiation by a RhoA/RhoA‐kinase‐dependent pathway. Wistar male rats after induction of myocardial infarction were randomized to either vehicle, nicorandil, an antagonist of KATP channel glibenclamide, an antagonist of ROCK fasudil, or a combination of nicorandil and glibenclamide or fasudil and glibenclamide starting 24 hrs after infarction. There were similar infarct sizes among the infarcted groups. At day 3 after infarction, post‐infarction was associated with increased RhoA/ROCK activation, which can be inhibited by administering nicorandil. Nicorandil significantly increased myocardial IL‐10 levels and the percentage of regulatory M2 macrophages assessed by immunohistochemical staining, Western blot, and RT‐PCR compared with vehicle. An IL‐10 receptor antibody increased myofibroblast infiltration compared with nicorandil alone. At day 28 after infarction, nicorandil was associated with attenuated cardiac fibrosis. These effects of nicorandil were functionally translated in improved echocardiographically derived cardiac performance. Fasudil showed similarly increased expression of M2 macrophages as nicorandil. The beneficial effects of nicorandil on fibroblast differentiation were blocked by adding glibenclamide. However, glibenclamide cannot abolish the attenuated fibrosis of fasudil, implying that RhoA/RhoA‐kinase is a downstream effector of KATP channel activation. Nicorandil polarized macrophages into M2 phenotype by inhibiting RhoA/RhoA‐kinase pathway, which leads to attenuated myofibroblast‐induced cardiac fibrosis after myocardial infarction.

As activated microglia (MG) is an early sign that often precedes and triggers neuronal death, inhibition of microglial activation and reduction of subsequent neurotoxicity may offer therapeutic benefit. The present study demonstrates that rat primary cultured MG expressed Kir6.1 and SUR2 subunits of KATP channel, which was identical to that expressed in BV‐2 microglial cell line. The classic KATP channel opener pinacidil and selective mitochondrial KATP (mito‐KATP) channel opener diazoxide prevented rotenone‐induc microglial activation and production of pro‐inflammatory factors (tumour necrosis factor[TNF]‐α and prostaglandin E2[PGE2]). And the effects of pinacidil and diazoxide were reversed by mito‐KATP blocker 5‐hydroxydecanoate (5‐HD), indicating that mito‐KATP channels participate in the regulation of microglial activation. Moreover, the underlying mechanisms involved the stabilization of mitocho drial membrane potential and inhibition of p38/c‐Jun‐N‐terminal kinase (JNK) activation in microglia. Furthermore, the in vivo study confirmed that diazoxide exhibited neuroprotective effects against rotenone along with the inhibition of microglial activation and neuroinflammation. Thus, microglial mito‐KATP channel might be a novel prospective target for the treatment of neuroinflammation‐related degenerative disorders such as Parkinson's disease.

The sulphonylurea receptor (SUR) subunits of K ATP channels are the targets for several classes of therapeutic drugs. Sulphonylureas close K ATP channels in pancreatic β-cells and are used to stimulate insulin release in type 2 diabetes, whereas the K ATP channel opener nicorandil acts as an antianginal agent by opening K ATP channels in cardiac and vascular smooth muscle. The predominant type of SUR varies between tissues: SUR1 in β-cells, SUR2A in cardiac muscle, and SUR2B in smooth muscle. Sulphonylureas and related drugs exhibit differences in tissue specificity, as the drugs interact to varying degrees with different types of SUR. Gliclazide and tolbutamide are β-cell selective and reversible. Glimepiride, glibenclamide, and repaglinide, however, inhibit cardiac and smooth muscle K ATP channels in addition to those in β-cells and are only slowly reversible. Similar properties have been observed by recording K ATP channel activity in intact cells and in Xenopus oocytes expressing cloned K ATP channel subunits. While K ATP channels in cardiac and smooth muscle are largely closed under physiological conditions (but open during ischaemia), they are activated by antianginal agents such as nicorandil. Under these conditions, they may be inhibited by sulphonylureas that block SUR2-type K ATP channels (e.g., glibenclamide). Care should, therefore, be taken when choosing a sulphonylurea if potential interactions with cardiac and smooth muscle K ATP channels are to be avoided.

ATP‐sensitive potassium (K ATP ) channels are required for maintenance of homeostasis during the metabolically demanding adaptive response to stress. However, in disease, the effect of cellular remodeling on K ATP channel behavior and associated tolerance to metabolic insult is unknown. Here, transgenic expression of tumor necrosis factor α induced heart failure with typical cardiac structural and energetic alterations. In this paradigm of disease remodeling, K ATP channels responded aberrantly to metabolic signals despite intact intrinsic channel properties, implicating defects proximal to the channel. Indeed, cardiomyocytes from failing hearts exhibited mitochondrial and creatine kinase deficits, and thus a reduced potential for metabolic signal generation and transmission. Consequently, K ATP channels failed to properly translate cellular distress under metabolic challenge into a protective membrane response. Failing hearts were excessively vulnerable to metabolic insult, demonstrating cardiomyocyte calcium loading and myofibrillar contraction banding, with tolerance improved by K ATP channel openers. Thus, disease‐induced K ATP channel metabolic dysregulation is a contributor to the pathobiology of heart failure, illustrating a mechanism for acquired channelopathy.

Local control of blood flow in the heart is important yet poorly understood. Here we show that ATP-sensitive K⁺ channels (KATP), hugely abundant in cardiac ventricular myocytes, sense the local myocyte metabolic state and communicate a negative feedback signal-correction upstream electrically. This electro-metabolic voltage signal is transmitted instantaneously to cellular elements in the neighboring microvascular network through gap junctions, where it regulates contractile pericytes and smooth muscle cells and thus blood flow. As myocyte ATP is consumed in excess of production, [ATP]i decreases to increase the openings of KATP channels, which biases the electrically active myocytes in the hyperpolarization (negative) direction. This change leads to relative hyperpolarization of the electrically connected cells that include capillary endothelial cells, pericytes, and vascular smooth muscle cells. Such hyperpolarization decreases pericyte and vascular smooth muscle [Ca2+]i levels, thereby relaxing the contractile cells to increase local blood flow and delivery of nutrients to the local cardiac myocytes and to augment ATP production by their mitochondria. Our findings demonstrate the pivotal roles of local cardiac myocyte metabolism and KATP channels and the minor role of inward rectifier K⁺ (Kir2.1) channels in regulating blood flow in the heart. These findings establish a conceptually new framework for understanding the hugely reliable and incredibly robust local electro-metabolic microvascular regulation of blood flow in heart.

KATP channels are metabolic sensors that translate intracellular ATP/ADP balance into membrane excitability. The molecular composition of KATP includes an inward-rectifier potassium channel (Kir) and an ABC transporter–like sulfonylurea receptor (SUR). Although structures of KATP have been determined in many conformations, in all cases, the pore in Kir is closed. Here, we describe human pancreatic KATP (hKATP) structures with an open pore at 3.1- to 4.0-Å resolution using single-particle cryo-electron microscopy (cryo-EM). Pore opening is associated with coordinated structural changes within the ATP-binding site and the channel gate in Kir. Conformational changes in SUR are also observed, resulting in an area reduction of contact surfaces between SUR and Kir. We also observe that pancreatic hKATP exhibits the unique (among inward-rectifier channels) property of PIP₂-independent opening, which appears to be correlated with a docked cytoplasmic domain in the absence of PIP₂