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Background and purpose Sulfonylureas (SUs) have been suggested to have an insulin-independent blood glucose-decreasing activity due to an extrapancreatic effect. However, a lack of adequate in vivo evidence makes this statement controversial. Here, we aimed to evaluate whether glimepiride has extrapancreatic blood glucose-lowering activity in vivo. Experimental approach Sulfonylurea receptor 1 deficient (SUR1-/- ) rats were created by means of transcription activator-like effector nucleases (TALEN)-mediated gene targeting technology. Type 2 diabetic models were established by feeding a high-fat diet and administering a low-dose of streptozotocin. These rats were then randomly divided into four groups: glimepiride, gliclazide, metformin and saline. All rats were treated for 2 weeks. Key results Glimepiride decreased blood glucose levels and increased insulin sensitivity without elevating insulin levels. Gliclazide showed similar effects as glimepiride. Both agents were weaker than metformin. Further mechanistic investigations revealed that glimepiride increased hepatic glycogen synthesis and decreased gluconeogenesis, which were accompanied by the activation of Akt in the liver. Moreover, glimepiride increased both total and membrane glucose transporter 4 (GLUT4) levels in muscle and fat, which might be attributed to insulin receptor-independent IRS1/Akt activation. Conclusion and implications Glimepiride possesses an extrapancreatic blood glucose-lowering effect in vivo, which might be attributed to its direct effect on insulin-sensitive tissues. Therefore, the combination of glimepiride with multiple insulin injections should not be excluded per se.

ATP sensitive potassium channels (KATP ) are so named because they open as cellular ATP levels fall. This leads to membrane hyperpolarization and thus links cellular metabolism to membrane excitability. They also respond to MgADP and are regulated by a number of cell signaling pathways. They have a rich and diverse pharmacology with a number of agents acting as specific inhibitors and activators. KATP channels are formed of pore-forming subunits, Kir6.1 and Kir6.2, and a large auxiliary subunit, the sulfonylurea receptor (SUR1, SUR2A, and SUR2B). The Kir6.0 subunits are a member of the inwardly rectifying family of potassium channels and the sulfonylurea receptor is part of the ATP-binding cassette family of proteins. Four SURs and four Kir6.x form an octameric channel complex and the association of a particular SUR with a specific Kir6.x subunit constitutes the KATP current in a particular tissue. A combination of mutagenesis work combined with structural studies has identified how these channels work as molecular machines. They have a variety of physiological roles including controlling the release of insulin from pancreatic β cells and regulating blood vessel tone and blood pressure. Furthermore, mutations in the genes underlie human diseases such as congenital diabetes and hyperinsulinism. Additionally, opening of these channels is protective in a number of pathological conditions such as myocardial ischemia and stroke. © 2018 American Physiological Society. Compr Physiol 8:1463-1511, 2018.

ABCC8 encodes a subunit of the β-cell potassium channel (KATP ) whose loss of function is responsible for congenital hyperinsulinism (CHI). Patients with two recessive mutations of ABCC8 typically have severe diffuse forms of CHI unresponsive to diazoxide. Some dominant ABCC8 mutations are responsible for a subset of diffuse diazoxide-unresponsive forms of CHI. We report the analysis of 21 different ABCC8 mutations identified in 25 probands with diazoxide-unresponsive diffuse CHI and carrying a single mutation in ABCC8. Nine missense ABCC8 mutations were subjected to in vitro expression studies testing traffic efficiency and responses of mutant channels to activation by MgADP and diazoxide. Eight of the 9 missense mutations exhibited normal trafficking. Seven of the 8 mutants reaching the plasma membrane had dramatically reduced response to MgADP or to diazoxide (

The scarcity of human cadaveric pancreata limits large-scale application of islet transplantation for patients with diabetes. Islets isolated from pathogen-free pigs provide an economical and abundant alternative source assuming immunologic barriers are appropriate. Membrane receptors involved in insulin secretion that also have potential as imaging targets were investigated in isolated porcine islets. Quantitative (q)PCR revealed that porcine islets express mRNA transcripts for sulfonylurea receptor 1 (Sur1), inward rectifying potassium channel (Kir6.2, associated with Sur1), glucagon-like peptide 1 receptor (GLP1R), and adrenergic receptor alpha 2A (ADRα2A). Receptor function was assessed in static incubations with stimulatory glucose concentrations, and in the presence of receptor agonists. Glibenclamide, an anti-diabetic sulfonylurea, and exendin-4, a GLP-1 mimetic, potentiated glucose-stimulated insulin secretion >2-fold. Conversely, epinephrine maximally reduced insulin secretion 72 ± 9% (P < 0.05) and had a half maximal inhibitory concentration of 60 nm in porcine islets (95% confidence interval of 45-830 nm). The epinephrine action was inhibited by the ADRα2A antagonist yohimbine. Our findings demonstrate that porcine islets express and are responsive to both stimulatory and inhibitory membrane localized receptors, which can be used as imaging targets after transplantation or to modify insulin secretion.

Existing concepts and models for glucose‐stimulated insulin secretion (GSIS) are overviewed and a newer perspective has been formulated toward the physiological understanding of GSIS. A conventional model has been created on the basis of in vitro data on application of a square wave high glucose in the absence of any other stimulatory inputs. Glucose elicits rapid insulin release through an adenosine triphosphate‐sensitive K+ channel (KATP channel)‐dependent mechanism, which is gradually augmented in a KATP channel‐independent manner. Biphasic GSIS thus occurs. In the body, the β‐cells are constantly exposed to stimulatory signals, such as glucagon‐like peptide 1 (GLP‐1), parasympathetic inputs, free fatty acid (FFA), amino acids and slightly suprathreshold levels of glucose, even at fasting. GLP‐1 increases cellular cyclic adenosine monophosphate, parasympathetic stimulation activates protein kinase C, and FFA, amino acids and glucose generate metabolic amplification factors. Plasma glucose concentration gradually rises postprandially under such tonic stimulation. We hypothesize that these stimulatory inputs together make the β‐cells responsive to glucose independently from its action on KATP channels. Robust GSIS in patients with a loss of function mutation of the sulfonylurea receptor, a subunit of KATP channels, is compatible with this hypothesis. Furthermore, pre‐exposure of the islets to an activator of protein kinase A and/or C makes β‐cells responsive to glucose in a KATP channel‐ and Ca2+‐independent manner. We hypothesize that GSIS occurs in islet β‐cells without glucose regulation of KATP channels in vivo, for which priming with cyclic adenosine monophosphate, protein kinase C and non‐glucose nutrients are required. To understand the physiology of GSIS, comprehensive integration of accumulated knowledge is required.

Aims/Introduction The adenosine triphosphate (ATP)-sensitive potassium (KATP) channel is a key component of insulin secretion in pancreatic β-cells. Activating mutations in ABCC8 encoding for the sulfonylurea receptor subunit of the KATP channel have been associated with the development of neonatal diabetes mellitus (NDM). The aim was to investigate clinical and functional characterization of the Pro1198Leu ABCC8 gene mutation associated with permanent NDM (PNDM). Materials and Methods The coding regions and conserved splice sites of KCNJ11, ABCC8 and INS were screened for mutations in a 12-year-old girl diagnosed with PNDM. The functional property of the mutant channel identified was examined with patch-clamp experiments in COS-1 cells. We also investigated the difference of effectiveness between two groups of oral sulfonylureas in vitro and in the patient. Results We identified a heterozygous missense mutation (c.3593 C>T, Pro1198Leu) in ABCC8. The mutated residue (P1198) is located within a putative binding site of sulfonylureas, such as tolbutamide or gliclazide. In patch-clamp experiments, the mutant channel was less ATP sensitive than the wild type. Furthermore, the sensitivity to tolbutamide was also reduced in the mutant channel. In addition to the tolbutamide/gliclazide binding site, glibenclamide is thought to also bind to another site. Glibenclamide was more effective than other sulfonylureas in vitro and in the patient. The treatment of the patient was finally able to be switched from insulin injection to oral glibenclamide. Conclusions We identified the Pro1198Leu ABCC8 mutation in a PNDM patient, and clarified the functional and clinical characterization. The present findings provide new information for understanding PNDM.

BACKGROUND Chitin produced by membrane-inserted chitin synthases is an important constituent of the arthropod cuticle and midgut peritrophic matrix. Chitin synthesis inhibitors are common insecticides in pest control. As the target of sulfonylurea-derived insecticides such as diflubenzuron, the ABC transporter sulfonylurea receptor (Sur) has been postulated to be an essential cofactor of chitin synthesis. However, direct evidence for this assumption is missing. RESULTS Here, a study has been made of the phenotype of Drosophila melanogaster larvae suffering completely eliminated Sur function. Taken together, it is found that cuticle architecture is normal and chitin amounts are not diminished in the cuticle of these animals, indicating that Sur is dispensable for chitin synthesis. CONCLUSION The data obtained suggest that there must exist another sulfonylurea-sensitive ABC transporter that either instead of Sur is the true sulfonylurea-sensitive transporter involved in chitin synthesis or is able to substitute Sur function during cuticle formation. Identification and characterisation of this factor is pivotal for understanding the mode of action of sulfonylurea as insecticide. © 2013 Society of Chemical Industry

Objective: Disability or death occurs more frequently in patients with hemorrhagic transformation (HT) after ischemic stroke. In rat models of stroke, sulfonylurea (SU) drugs such as glibenclamide (adopted US name, glyburide) confer protection against swelling and HT through actions on the novel SUR1-regulated NCCa-ATP channel. Here, we sought to determine whether the use of SU drugs in patients with diabetes mellitus (DM) presenting with acute ischemic stroke might influence the incidence of HT. Methods: We retrospectively analyzed data on 220 patients with DM who presented with acute ischemic stroke, 43 of whom were managed with and continued to receive SU drugs, and 177 of whom were managed without (controls). Results: During a median length of stay in hospital of 11 days, 20 control patients (11%) experienced symptomatic HT (sHT), whereas no patient in the SU group experienced sHT (p = 0.016). No patient in the SU group died, compared to 18 (10%) in the control group (p = 0.027). Similarly favorable outcomes were observed after matching for baseline imbalances and excluding outliers. In support of the proposed mechanism, we present a case of sHT in which an analysis of brain tissues obtained intraoperatively showed prominent upregulation of SUR1, the target of SU drugs, in microvessels and neurons. Interpretation: We conclude that, in diabetic patients with acute ischemic stroke, prior and continued use of SU drugs is associated with reduced sHT compared to those whose treatment regimen does not include SU drugs. ANN NEUROL 2012;72:799–806

Hemorrhagic transformation (HT) associated with recombinant tissue plasminogen activator (rt-PA) complicates and limits its use in stroke. Here, we provide a focused review on the involvement of matrix metalloproteinase 9 (MMP-9) in rt-PA-associated HT in cerebral ischemia, and we review emerging evidence that the selective inhibitor of the sulfonylurea receptor 1 (Sur1), glibenclamide (U.S. adopted name, glyburide), may provide protection against rt-PA-associated HT in cerebral ischemia. Glyburide inhibits activation of MMP-9, ameliorates edema formation, swelling, and symptomatic hemorrhagic transformation, and improves preclinical outcomes in several clinically relevant models of stroke, both without and with rt-PA treatment. A retrospective clinical study comparing outcomes in diabetic patients with stroke treated with rt-PA showed that those who were previously on and were maintained on a sulfonylurea fared significantly better than those whose diabetes was managed without sulfonylureas. Inhibition of Sur1 with injectable glyburide holds promise for ameliorating rt-PA-associated HT in stroke.

Mutations in the pancreatic ATP sensitive K(+) channel proteins [sulfonyluea receptor 1 (SUR1) and inward rectifier K(+) channel Kir6.2 (Kir6.2), encoded by ATP-binding cassette transporter subfamily C member 8 (ABCC8) and potassium channel J11 (KCNJ11), respectively], are the most common cause of neonatal diabetes. We describe the clinical presentation and molecular characterization of Asian Indian children with neonatal diabetes mellitus and monogenic syndromes of diabetes. We sequenced KCNJ11, ABCC8 and insulin (INS) genes in 33 unrelated Indian probands with onset of diabetes below one year of age. A total of 12 mutations were identified which included ABCC8 mutations in seven, KCNJ11 mutations in three and INS mutations in two children. The Asp212Tyr mutation in ABCC8 was novel. We also detected two novel mutations (Val67Met and Leu19Arg) in children with syndromic forms of diabetes like Berardinelli Seip syndrome [1-acyl-sn-glycerol-3-phosphate acyltransferase beta (AGPAT2)] and Fanconi Bickel syndrome [solute carrier family 2A2 (SLC2A2)]. Children carrying the KCNJ11 (Cys42Arg, Arg201Cys) and ABCC8 (Val86Ala, Asp212Tyr) mutations have been successfully switched over from insulin therapy to oral sulfonylurea. Our study is the first large genetic screening study of neonatal diabetes in India.

It is well known that sulphonylureas (SUs), commonly used in the treatment of type 2 diabetes mellitus, stimulate insulin secretion by closing ATP-sensitive K(+) (K(ATP) ) channels in pancreatic β-cells by binding to the SU receptor SUR1. SUs are now known also to activate cAMP sensor Epac2 (cAMP-GEFII) to Rap1 signalling, which promotes insulin granule exocytosis. For SUs to exert their full effects in insulin secretion, they are required to activate Epac2 as well as to inhibit the β-cell K(ATP) channels. As Epac2 is also necessary for potentiation of glucose-induced insulin secretion by cAMP-increasing agents, such as incretin, Epac2 is a target of both cAMP and SUs. The distinct effects of various SUs appear to be because of their different actions on Epac2/Rap1 signalling as well as K(ATP) channels. Differently from other SUs, gliclazide is unique in that it is specific for β-cell K(ATP) channel and does not activate Epac2.

Activating mutations in different domains of the ABCC8 gene-coded sulfonylurea receptor 1 (SUR1) cause neonatal diabetes. Here we show that a diabetogenic mutation in an unexplored helix preceding the ABC core of SUR1 dramatically increases open probability of (SUR1/Kir6.2)4 channel (KATP) by reciprocally changing rates of its transitions to and from the long-lived, inhibitory ligand-stabilized closed state. This kinetic mechanism attenuates ATP and sulfonylurea inhibition, but not Mg-nucleotide stimulation, of SUR1/Kir6.2. The results suggest a key role for L0 helix in KATP gating and together with previous findings from mutant KATP clarify why many patients with neonatal diabetes require high doses of sulfonylureas.

ATP-sensitive K(+) (K(ATP)) channels are composed of pore-forming subunits, typically Kir6.2 in neurons, and regulatory sulfonylurea receptor subunits. In dorsal striatum, activity-dependent H(2)O(2) produced from glutamate receptor activation inhibits dopamine release via K(ATP) channels. Sources of modulatory H(2)O(2) include striatal medium spiny neurons, but not dopaminergic axons. Using fast-scan cyclic voltammetry in guinea-pig striatal slices and immunohistochemistry, we determined the time window for H(2)O(2)/K(ATP)-channel-mediated inhibition and assessed whether modulatory K(ATP) channels are on dopaminergic axons. Comparison of paired-pulse suppression of dopamine release in the absence and presence of glibenclamide, a K(ATP)-channel blocker, or mercaptosuccinate, a glutathione peroxidase inhibitor that enhances endogenous H(2)O(2) levels, revealed a time window for inhibition of 500-1000 ms after stimulation. Immunohistochemistry demonstrated localization of Kir6.2 K(ATP)-channel subunits on dopaminergic axons. Consistent with the presence of functional K(ATP) channels on dopaminergic axons, K(ATP)-channel openers, diazoxide and cromakalim, suppressed single-pulse evoked dopamine release. Although cholinergic interneurons that tonically regulate dopamine release also express K(ATP) channels, diazoxide did not induce the enhanced frequency responsiveness of dopamine release seen with nicotinic-receptor blockade. Together, these studies reveal subsecond regulation of striatal dopamine release by endogenous H(2)O(2) acting at K(ATP) channels on dopaminergic axons, including a role in paired-pulse suppression.

SUR2A is an ATP-binding protein that serves as a regulatory subunit of cardioprotective ATP-sensitive K(+) (K(ATP) ) channels. Based on signalling pathway regulating SUR2A expression and SUR2A role in regulating numbers of fully assembled K(ATP) channels, we have suggested that nicotinamide-rich diet could improve physical endurance by stimulating SUR2A expression. We have found that mice on nicotinamide-rich diet significantly improved physical endurance, which was associated with significant increase in expression of SUR2A. Transgenic mice with solely overexpressed SUR2A on control diet had increased physical endurance in a similar manner as the wild-type mice on nicotinamide-rich diet. The experiments focused on action membrane potential and intracellular Ca(2+) concentration have demonstrated that increased SUR2A expression was associated with the activation of sarcolemmal K(ATP) channels and steady Ca(2+) levels in cardiomyocytes in response to β-adrenergic stimulation. In contrast, the same challenge in the wild-type was characterized by a lack of the channel activation and rise in intracellular Ca(2+) . Nicotinamide-rich diet was ineffective to increase physical endurance in mice lacking K(ATP) channels. This study has shown that nicotinamide-rich diet improves physical endurance by increasing expression of SUR2A and that this is a sole mechanism of the nicotinamide-rich diet effect. The obtained results suggest that oral nicotinamide is a regulator of SUR2A expression and has a potential as a drug that can improve physical endurance in conditions where this effect would be desirable.

Non-technical summary Neonatal diabetes is a rare genetic form of diabetes that develops within the first 6 months of life. It is often caused by genetic defects (mutations) in a specialised membrane protein known as the KATP channel. This protein acts as a tiny pore in the membrane of the insulin-secreting cells in the pancreas and its opening and closing is regulated by blood glucose levels. Low blood glucose holds the pore open and prevents insulin secretion whereas high blood sugar leads to channel closure and insulin secretion. We identified a novel mutation (Kir6.2-W68R) that prevents channel closure and insulin secretion, and so results in neonatal diabetes. Detailed molecular studies showed that channel opening and closing is disrupted, suggesting Kir6.2-W68 may act as a channel ‘gatekeeper’. Our results also indicated that the mutant channel could be shut by sulphonylurea drugs, which enabled the patient to transfer from insulin injections to tablet therapy. Abstract We identified a novel heterozygous mutation, W68R, in the Kir6.2 subunit of the ATP-sensitive potassium (KATP) channel, in a patient with transient neonatal diabetes. This tryptophan is absolutely conserved in mammalian Kir channels. The functional effects of mutations at residue 68 of Kir6.2 were studied by heterologous expression in Xenopus oocytes, and by homology modelling. We found the Kir6.2-W68R mutation causes a small reduction in ATP inhibition in the heterozygous state and an increase in the whole-cell KATP current. This can explain the clinical phenotype of the patient. The effect of the mutation was not charge or size dependent, the order of potency for ATP inhibition being W

Specialized hypothalamic neurons responding to rising extracellular glucose via increases or decreases in their electrical activity [glucose-excited (GE) and glucose-inhibited (GI) cells, respectively] have been reported in the hypothalamic arcuate, ventromedial and lateral nuclei. The hypothalamic paraventricular nucleus (PVN) is an important neurosecretory and preautonomic output nucleus. We tested whether parvocellular PVN neurons also possess glucosensing properties, using patch-clamp recording and immunocytochemistry. Putative neurosecretory (p-NS) and preautonomic (p-PA) cells were identified electrophysiologically. Although parvocellular neurons were insensitive to transitions from 10 to 2.5 mm glucose, approximately 68% of p-PA cells responded directly to glucopenia (mimicked by a step to 0.2 mm glucose) with an increased membrane conductance. Of these, approximately 24% hyperpolarized (accompanied by an outward current) and thus were GE, approximately 26% depolarized (with an inward current, thus GI) and approximately 18% did not change membrane potential. The concentration dependence of the glucose response was similar for both GE and GI cells (EC(50) of 0.67-0.7 mm), but was steep, with Hill slopes of 3-4. The K(ATP) channel blockers glibenclamide and tolbutamide did not prevent, while the K(ATP) channel opener diazoxide did not mimic, the effects of low glucose on GE neurons. Moreover, the K(ATP) sulfonylurea receptor SUR1 was not detected in glucosensitive neurons. We conclude that the PVN contains previously unknown GE and GI cells that could participate in regulation of autonomic functions. GE neurons in the PVN sense ambient glucose via a unique mechanism, probably independent of K(ATP) channels, in contrast to neurons in other hypothalamic nuclei.

Unusually among ATP-binding cassette proteins, the sulfonylurea receptor (SUR) acts as a channel regulator. ATP-sensitive potassium channels are octameric complexes composed of four pore-forming Kir6.2 subunits and four regulatory SUR subunits. Two different genes encode SUR1 (ABCC8) and SUR2 (ABCC9), with the latter being differentially spliced to give SUR2A and SUR2B, which differ only in their C-terminal 42 amino acids. ATP-sensitive potassium channels containing these different SUR2 isoforms are differentially modulated by MgATP, with Kir6.2/SUR2B being activated more than Kir6.2/SUR2A. We show here that purified SUR2B has a lower ATPase activity and a 10-fold lower K(m) for MgATP than SUR2A. Similarly, the isolated nucleotide-binding domain (NBD) 2 of SUR2B was less active than that of SUR2A. We further found that the NBDs of SUR2B interact, and that the activity of full-length SUR cannot be predicted from that of either the isolated NBDs or NBD mixtures. Notably, deletion of the last 42 amino acids from NBD2 of SUR2 resulted in ATPase activity resembling that of NBD2 of SUR2A rather than that of NBD2 of SUR2B: this might indicate that these amino acids are responsible for the lower ATPase activity of SUR2B and the isolated NBD2 of SUR2B. We suggest that the lower ATPase activity of SUR2B may result in enhanced duration of the MgADP-bound state, leading to channel activation.

Background and purpose: ATP-sensitive potassium channels (K(ATP) channels) in beta cells are a major target for insulinotropic drugs. Here, we studied the effects of selected stimulatory and inhibitory pharmacological agents in islets lacking K(ATP) channels. Experimental approach: We compared insulin secretion (IS) and cytosolic calcium ([Ca(2+)](c)) changes in islets isolated from control mice and mice lacking sulphonylurea receptor1 (SUR1), and thus K(ATP) channels in their beta cells (Sur1KO). Key results: While similarly increasing [Ca(2+)](c) and IS in controls, agents binding to site A (tolbutamide) or site B (meglitinide) of SUR1 were ineffective in Sur1KO islets. Of two non-selective blockers of potassium channels, quinine was inactive, whereas tetraethylammonium was more active in Sur1KO compared with control islets. Phentolamine, efaroxan and alinidine, three imidazolines binding to K(IR)6.2 (pore of K(ATP) channels), stimulated control islets, but only phentolamine retained weaker stimulatory effects on [Ca(2+)](c) and IS in Sur1KO islets. Neither K(ATP) channel opener (diazoxide, pinacidil) inhibited Sur1KO islets. Calcium channel blockers (nimodipine, verapamil) or diphenylhydantoin decreased [Ca(2+)](c) and IS in both types of islets, verapamil and diphenylhydantoin being more efficient in Sur1KO islets. Activation of alpha(2)-adrenoceptors or dopamine receptors strongly inhibited IS while partially (clonidine > dopamine) lowering [Ca(2+)](c) (control > Sur1KO islets). Conclusions and implications: Those drugs retaining effects on IS in islets lacking K(ATP) channels, also affected [Ca(2+)](c), indicating actions on other ionic channels. The greater effects of some inhibitors in Sur1KO than in control islets might be relevant to medical treatment of congenital hyperinsulinism caused by inactivating mutations of K(ATP) channels.

The female myocardium, relative to that of the male, exhibits sustained resistance to ischaemic tissue injury, a phenomenon termed sex-specific cardioprotection (SSC). SSC is dependent upon the sarcolemmal KATP channel (sarcKATP), and protein kinase C (PKC). Here we investigate whether PKC-mediated regulation of sarcKATP concentration can explain this endogenous form of protection. Hearts from male (M) and female (F) rats were Langendorff-perfused for 30 min prior to either regional ischaemia–reperfusion (I/R), or global ischaemia (GISC). For both protocols, pre-ischaemic blockade of PKC was achieved by chelerythrine (Chel) in male (M + C) and female (F + C) hearts. Additional female hearts underwent sarcKATP antagonism during I/R by HMR-1098 (HMR), either alone or in combination with Chel (HMR + Chel). GISC hearts were fractionated to assess cellular distribution of PKCɛ and sarcKATP. Sex-specific infarct resistance was apparent under control I/R (F, 23 ± 3%vs. M, 36 ± 4%, P < 0.05) and abolished by Chel (F + C, 36 ± 3%). Female infarct resistance was susceptible to sarcKATP blockade (Control, 16 ± 2%vs. HMR, 27 ± 3%), and PKC blockade had no additional effect (HMR + Chel, 26 ± 2%). The prevalence of Kir6.2 and SUR2 was higher in the sarcolemmal fractions of females (Kir6.2: F, 1.24 ± 0.07 vs. M, 1.02 ± 0.06; SUR2: F, 3.16 ± 0.22 vs. M, 2.45 ± 0.09; ratio units), but normalized by Chel (Kir6.2: F, 1.06 ± 0.07 vs. M, 0.99 ± 0.06; SUR2: F, 2.99 ± 0.09 vs. M, 2.82 ± 0.22, M; ratio units). Phosphorylation of sarcolemmal PKCɛ was reduced by Chel (p-PKCɛ/PKCɛ: control, 0.43 ± 0.02; Chel, 0.29 ± 0.01; P < 0.01). We conclude that PKC-mediated regulation of sarcKATP may account for the physiologically sustainable dependence of SSC upon both PKC and sarcKATP, and that this regulation involves PKC-permitted enrichment of the female sarcolemma with sarcKATP. As such, the PKC-sarcKATP axis may represent a target for sustainable prophylactic induction of cardioprotection.

The ATP-sensitive potassium (K(ATP)) channel is expressed in most excitable tissues and plays a critical role in numerous physiological processes by coupling intracellular energetics to electrical activity. The channel is comprised of four Kir6.x subunits associated with four regulatory sulfonylurea receptors (SUR). Intracellular ATP acts on Kir6.x to inhibit channel activity, while MgADP stimulates channel activity through SUR. Changes in the cytosolic [ATP] to [ADP] ratio thus determine channel activity. Multiple mutations in Kir6.x and SUR genes have implicated K(ATP) channels in various diseases ranging from diabetes and hyperinsulinism to cardiac arrhythmias and cardiovascular disease. Continuing studies of channel physiology and pathology will bring new insights to the molecular basis of K(ATP) channel function, leading to a better understanding of the role that K(ATP) channels play in both health and disease.

Potassium channels in the plasma membrane of the pancreatic beta cells are critical in maintaining glucose homeostasis by responding to ATP and coupling metabolic changes to insulin secretion. These channels consist of subunits denoted the sulfonylurea receptor SUR1 and the inwardly rectifying ion channel KIR6.2, which are encoded by the genes ABCC8 and KCNJ11, respectively. Activating mutations in the subunit genes can result in monogenic diabetes, whereas inactivating mutations are the most common cause of congenital hyperinsulinism of infancy (CHI). Twenty-six Norwegian probands with CHI were analyzed for alterations in ABCC8 and KCNJ11. Fifteen probands (58%) had mutations in the ABCC8 gene. Nine patients were homozygous or compound heterozygous for the mutations, indicating diffuse pancreatic disease. In five patients, heterozygous and paternally inherited mutations were found, suggesting focal disease. One patient had a de novo mutation likely to cause a milder, dominant form of CHI. Altogether, 16 different ABCC8 mutations (including the novel alterations W231R, C267X, IVS6-3C>G, I462V, Q917X and T1531A) were identified. The mutations IVS10+1G>T, R1493W and V21D occurred in five, three and two families, respectively. KCNJ11 mutations were not found in any patients. Based on our mutation screening, we estimate the minimum birth prevalence of ABCC8-CHI in Norway to 1:70,000 during the past decade. Our results considerably extend the knowledge of the molecular genetics behind CHI in Scandinavia.

ATP-sensitive K(+) (K(ATP)) channels couple cell metabolism to cell electrical activity. Wild-type (Kir6.2/SUR1) K(ATP) channels heterologously expressed in Xenopus oocytes give rise to very small inward currents in cell-attached patches. A large increase in the current is observed on patch excision into zero ATP solution. This is presumably due to loss of intracellular ATP leading to unblock of K(ATP) channels. In contrast, channels containing Kir6.2 mutations associated with reduced ATP-sensitivity display non-zero cell-attached currents. Unexpectedly, these cell-attached currents are significantly smaller (by approximately 40%) than those observed when excised patches are exposed to physiological ATP concentrations (1-10 mm). Cramming the patch back into the oocyte cytoplasm restores mutant K(ATP) current amplitude to that measured in the cell-attached mode. This implies that the magnitude of the cell-attached current is regulated not only by intracellular ATP but also by another cytoplasmic factor/s. This factor seems to require the nucleotide-binding domains of SUR1 to be effective. Thus a mutant Kir6.2 (Kir6.2DeltaC-I296L) expressed in the absence of SUR1 exhibited currents of similar magnitude in cell-attached patches as in inside-out patches exposed to 10 mm MgATP. Similar results were found when Kir6.2-I296L was coexpressed with an SUR1 mutant that is insensitive to MgADP or MgATP activation. This suggests the oocyte contains a cytoplasmic factor that reduces nucleotide binding/hydrolysis at the NBDs of SUR1. In conclusion, our results reveal a novel regulatory mechanism for the K(ATP) channel. This was not evident for wild-type channels because of their high sensitivity to block by ATP.

Background and purpose: Dilatation of cerebral and dural arteries causes a throbbing, migraine-like pain, indicating that these structures are involved in migraine. Clinical trials suggest that adenosine 5 0 -triphosphate-sensitive K þ (KATP) channel opening may cause migraine by dilatating intracranial arteries, including the middle meningeal artery (MMA). We studied the KATP channel expression profile in rat MMA and examined the potential inhibitory effects of the KATP channel blocker PNU-37883A on KATP channel opener-induced relaxation of the rat MMA, using the three KATP channel openers levcromakalim, pinacidil and P-1075. Experimental approach: mRNA and protein expression of KATP channel subunits in the rat MMA were studied by quantitative real-time PCR and western blotting, respectively. The in vivo and in vitro effects of the KATP channel drugs on rat MMA were studied in the genuine closed cranial window model and in myograph baths, respectively. Key results: Expression studies indicate that inwardly rectifying K þ (Kir)6.1/sulphonylurea receptor (SUR)2B is the major KATP channel complex in rat MMA. PNU-37883A (0.5 mg kg � 1 ) significantly inhibited the in vivo dilatory effect of levcromakalim (0.025 mg kg � 1 ), pinacidil (0.38 mg kg � 1 ) and P-1075 (0.016 mg kg � 1 ) in rat MMA. In vitro PNU-37883A significantly inhibited the dilatory responses of the three KATP channel openers in rat MMA at 10 � 7 and 3 � 10 � 7 M. Conclusions and implications: We suggest that Kir6.1/SUR2B is the major functional KATP channel complex in the rat MMA. Furthermore, we demonstrate the potent in vivo and in vitro blocking potentials of PNU-37883A on KATP channel openerinduced relaxation of the rat MMA. British Journal of Pharmacology (2008) 154, 72–81; doi:10.1038/bjp.2008.86; published online 10 March 2008

The monocarboxylate, lactate, is produced by astrocytic glycolysis and is trafficked to neurons as a substrate fuel for aerobic respiration. This molecule is a critical monitored metabolic variable in hindbrain detection of cellular energy imbalance, because diminished uptake and/or oxidative catabolism of lactate in this part of the brain activates neural mechanisms that increase systemic glucose availability. Lactate-sensitive chemosensory neurons occur in the hindbrain dorsal vagal complex (DVC). Estradiol (E) enhances expression of the neuronal monocarboxylate transporter MCT2 in the DVC during insulin-induced hypoglycemia (IIH), evidence that this hormone may promote local lactate utilization during systemic glucose shortages. We investigated the hypothesis that E regulates basal and IIH-associated patterns of DVC MCT2 and neuronal glucose transporter gene expression and that caudal fourth ventricular (CV4) lactate infusion exerts divergent effects on blood glucose levels and DVC energy transducer gene profiles in hypoglycemic E- vs. oil (O)-implanted ovariectomized (OVX) rats. Insulin-induced decrements in circulating glucose were significantly augmented by lactate, albeit to a greater extent in the presence of E. DVC MCT2, GLUT3, GLUT4, glucokinase (GCK), and sulfonylurea receptor-1 (SUR1) mRNA levels did not differ between saline-injected OVX + E and OVX + O rats. IIH elevated MCT2 and GLUT3 gene profiles in both E- and O-implanted groups, but up-regulation of MCT2 transcripts was reversed by CV4 lactate infusion during hypoglycemia in E- but not O-implanted animals. DVC GLUT4 and GK mRNA were decreased by insulin alone in OVX + O but not OVX + E, but were suppressed by lactate plus insulin treatment in the latter group. Expression of the SUR1 subunit of the energy-dependent potassium channel K(ATP) was significantly decreased by IIH in both E- and O-treated rats and further suppressed in response to lactate delivery during hypoglycemia in OVX + E. These data reveal that E does not control baseline DVC substrate fuel transporter or energy transducer gene profiles or local MCT2, GLUT3, or SUR1 transcriptional responses to IIH but prevents IIH-associated decreases in GLUT4 and GCK mRNA in this brain site. The results also show that, in the presence of E, intensifying effects of CV4 lactate infusion on hypoglycemia are correlated with reversal of IIH enhancement of DVC MCT2 gene expression, augmented IIH inhibition of SUR1 transcripts, and reductions in GLUT4 and GCK mRNA levels relative to baseline. This work implies that IIH may enhance specific neuronal lactate and glucose transport mechanisms in the female rat DVC and that, in the presence of E, caudal hindbrain lactate repletion may normalize neuronal lactate but not glucose internalization by local neurons. The results also suggest that putative IIH-associated reductions in K(ATP)-mediated regulation of membrane voltage in this brain site may be causally related to diminished glucose availability.

The ATP-sensitive potassium (K(ATP)) channel couples glucose metabolism to insulin secretion in pancreatic beta-cells. It comprises regulatory sulfonylurea receptor 1 and pore-forming Kir6.2 subunits. Binding and/or hydrolysis of Mg-nucleotides at the nucleotide-binding domains of sulfonylurea receptor 1 stimulates channel opening and leads to membrane hyperpolarization and inhibition of insulin secretion. We report here the first purification and functional characterization of sulfonylurea receptor 1. We also compared the ATPase activity of sulfonylurea receptor 1 with that of the isolated nucleotide-binding domains (fused to maltose-binding protein to improve solubility). Electron microscopy showed that nucleotide-binding domains purified as ring-like complexes corresponding to approximately 8 momomers. The ATPase activities expressed as maximal turnover rate [in nmol P(i).s(-1).(nmol protein)(-1)] were 0.03, 0.03, 0.13 and 0.08 for sulfonylurea receptor 1, nucleotide-binding domain 1, nucleotide-binding domain 2 and a mixture of nucleotide-binding domain 1 and nucleotide-binding domain 2, respectively. Corresponding K(m) values (in mm) were 0.1, 0.6, 0.65 and 0.56, respectively. Thus sulfonylurea receptor 1 has a lower K(m) than either of the isolated nucleotide-binding domains, and a lower maximal turnover rate than nucleotide-binding domain 2. Similar results were found with GTP, but the K(m) values were lower. Mutation of the Walker A lysine in nucleotide-binding domain 1 (K719A) or nucleotide-binding domain 2 (K1385M) inhibited the ATPase activity of sulfonylurea receptor 1 by 60% and 80%, respectively. Beryllium fluoride (K(i) 16 microm), but not MgADP, inhibited the ATPase activity of sulfonylurea receptor 1. In contrast, both MgADP and beryllium fluoride inhibited the ATPase activity of the nucleotide-binding domains. These data demonstrate that the ATPase activity of sulfonylurea receptor 1 differs from that of the isolated nucleotide-binding domains, suggesting that the transmembrane domains may influence the activity of the protein.

Sulfonylurea receptors SUR1 and SUR2 are the regulatory subunits of K(ATP) channels. Their differential affinity for hypoglycemic sulfonylureas provides a basis for the selectivity of these compounds for different K(ATP) channel isoforms. Sulfonylureas have a 100- to 1000-fold greater affinity for SUR1 vs. SUR2. Structure-activity studies suggested a bipartite binding pocket. Chimeric SUR1 approximately SUR2 receptors have shown TMD2, the third bundle of transmembrane helices, to be part of an "A" site that confers SUR1 selectivity for sulfonylureas. The purpose of this study is to determine the position of the "B" site. Previous photoaffinity labeling studies have placed the B site on the amino-terminal third of SUR and colabeled the associated K(IR). In our study, deletion of TMD0, the first bundle of transmembrane helices, did not compromise labeling. Further deletions into the cytoplasmic linker, L0, eliminated binding and labeling. Alanine substitutions in L0 identified a limited number of conserved residues, Y230 and W232, important for affinity labeling. A fragment of K(IR)6.2, missing M2 and the entire carboxyl terminal, assembles with SUR1 and is affinity labeled, while deletion of 10 or more amino-terminal residues compromises labeling. These studies indicate that the B site involves L0 and the K(IR) amino terminus, elements that are critical for control of channel gating.

Background and purpose: It has been postulated that isoflurane, a volatile anaesthetic, produces vasodilatation through activation of ATP-sensitive K+ (KATP) channels. However, there is no direct evidence for the activation of vascular KATP channels by isoflurane. This study was conducted to examine the effect of isoflurane on vascular KATP channels and compare it with that on cardiac KATP channels. Experimental approach: Effects of isoflurane on KATP channels were examined in aortic smooth muscle cells and cardiomyocytes of the mouse using patch clamp techniques. Effects of the anaesthetic on the KATP channels with different combinations of the inward rectifier pore subunits (Kir6.1 and Kir6.2) and sulphonylurea receptor subunits (SUR2A and SUR2B) reconstituted in a heterologous expression system were also examined. Key results: Isoflurane increased the coronary flow in Langendorff-perfused mouse hearts in a concentration-dependent manner, which was abolished by 10 μM glibenclamide. In enzymically-dissociated aortic smooth muscle cells, isoflurane evoked a glibenclamide-sensitive current (i.e. KATP current). In isolated mouse ventricular cells, however, isoflurane failed to evoke the KATP current unless the KATP current was preactivated by the K+ channel opener pinacidil. Although isoflurane readily activated the Kir6.1/SUR2B channels (vascular type), the volatile anesthetic could not activate the Kir6.2/SUR2A channels (cardiac type) expressed in HEK293 cells. Isoflurane activated a glibenclamide-sensitive current in HEK293 cells expressing Kir6.2/SUR2B channels. Conclusion and implications: Isoflurane activates KATP channels in vascular smooth muscle cells and produces coronary vasodilation in mouse hearts. SUR2B may be important for the activation of vascular-type KATP channels by isoflurane. British Journal of Pharmacology (2006) 149, 573–580. doi:10.1038/sj.bjp.0706891

The Epac family of cAMP-regulated guanine nucleotide exchange factors (cAMPGEFs, also known as Epac1 and Epac2) mediate stimulatory actions of the second messenger cAMP on insulin secretion from pancreatic beta cells. Because Epac2 is reported to interact in vitro with the isolated nucleotide-binding fold-1 (NBF-1) of the beta-cell sulphonylurea receptor-1 (SUR1), we hypothesized that cAMP might act via Epac1 and/or Epac2 to inhibit beta-cell ATP-sensitive K+ channels (K(ATP) channels; a hetero-octomer of SUR1 and Kir6.2). If so, Epac-mediated inhibition of K(ATP) channels might explain prior reports that cAMP-elevating agents promote beta-cell depolarization, Ca2+ influx and insulin secretion. Here we report that Epac-selective cAMP analogues (2'-O-Me-cAMP; 8-pCPT-2'-O-Me-cAMP; 8-pMeOPT-2'-O-Me-cAMP), but not a cGMP analogue (2'-O-Me-cGMP), inhibit the function of K(ATP) channels in human beta cells and rat INS-1 insulin-secreting cells. Inhibition of K(ATP) channels is also observed when cAMP, itself, is administered intracellularly, whereas no such effect is observed upon administration N6-Bnz-cAMP, a cAMP analogue that activates protein kinase A (PKA) but not Epac. The inhibitory actions of Epac-selective cAMP analogues at K(ATP) channels are mimicked by a cAMP agonist (8-Bromoadenosine-3', 5'-cyclic monophosphorothioate, Sp-isomer, Sp-8-Br-cAMPS), but not a cAMP antagonist (8-Bromoadenosine-3', 5'-cyclic monophosphorothioate, Rp-isomer, Rp-8-Br-cAMPS), and are abrogated following transfection of INS-1 cells with a dominant-negative Epac1 that fails to bind cAMP. Because both Epac1 and Epac2 coimmunoprecipitate with full-length SUR1 in HEK cell lysates, such findings delineate a novel mechanism of second messenger signal transduction in which cAMP acts via Epac to modulate ion channel function, an effect measurable as the inhibition of K(ATP) channel activity in pancreatic beta cells.

Potassium channels that are inhibited by intracellular ATP (ATP(i)) were first identified in ventricular myocytes, and are referred to as ATP-sensitive K+ channels (i.e. K(ATP) channels). Subsequently, K+ channels with similar characteristics have been demonstrated in many other tissues (pancreatic beta-cells, skeletal muscle, central neurones, smooth muscle). Approximately one decade ago, K(ATP) channels were cloned and were found to be composed of at least two subunits: an inwardly rectifying K+ channel six family (Kir6.x) that forms the ion conducting pore and a modulatory sulphonylurea receptor (SUR) that accounts for several pharmacological properties. Various types of native K(ATP) channels have been identified in a number of visceral and vascular smooth muscles in single-channel recordings. However, little attention has been paid to the molecular properties of the subunits in K(ATP) channels and it is important to determine the relative expression of K(ATP) channel components which give rise to native K(ATP) channels in smooth muscle. The aim of this review is to briefly discuss the current knowledge available for K(ATP) channels with the main interest in the molecular basis of native K(ATP) channels, and to discuss their possible linkage with physiological functions in smooth muscle.

ATP-sensitive K+ channels composed of the pore-forming protein Kir6.2 and the sulphonylurea receptor SUR1 are inhibited by ATP and activated by Phosphatidylinositol Bisphosphate (PIP2). Residues involved in binding of these ligands to the Kir6.2 cytoplasmic domain have been identified, and it has been hypothesized that gating mechanisms involve conformational changes in the regions of the bundle crossing and/or the selectivity filter of Kir6.2. Regulation of Kir6.2 by SUR1, however, is not well-understood, even though this process is ATP and PIP2 dependent. In this study, we investigated the relationship between channel regulation by SUR1 and PIP2 by comparing a number of single and double mutants known to affect open probability (Po), PIP2 affinity, and sulphonylurea and MgADP sensitivity. When coexpressed with SUR1, the Kir6.2 mutant C166A, which is characterized by a Po value close to 0.8, exhibits no sulphonylurea or MgADP sensitivity. However, when Po was reduced by combining mutations at the PIP2-sensitive residues R176 and R177 with C166A, sulphonylurea and MgADP sensitivities were restored. These effects correlated with a dramatic decrease in PIP2 affinity, as assessed by PIP2-induced channel reactivation and inhibition by neomycin, an antagonist of PIP2 binding. Based on macroscopic and single-channel data, we propose a model in which entry into the high-Po bursting state by the C166A mutation or by SUR1 depends on the interaction of PIP2 with R176 and R177 and, to a lesser extent, R54. In conjunction with this PIP2-dependent process, SUR1 also regulates channel activity via a PIP2-independent, but MgADP-dependent process.

ATP-sensitive K+ (KATP) channels are hetero-octamers of inwardly rectifying K+ channel (Kir6.2) and sulphonylurea receptor subunits (SUR1 in pancreatic β-cells, SUR2A in heart). Heterozygous gain-of-function mutations in Kir6.2 cause neonatal diabetes, which may be accompanied by epilepsy and developmental delay. However, despite the importance of KATP channels in the heart, patients have no obvious cardiac problems. We examined the effects of adenine nucleotides on KATP channels containing wild-type or mutant (Q52R, R201H) Kir6.2 plus either SUR1 or SUR2A. In the absence of Mg2+, both mutations reduced ATP inhibition of SUR1- and SUR2A-containing channels to similar extents, but when Mg2+ was present ATP blocked mutant channels containing SUR1 much less than SUR2A channels. Mg-nucleotide activation of SUR1, but not SUR2A, channels was markedly increased by the R201H mutation. Both mutations also increased resting whole-cell KATP currents through heterozygous SUR1-containing channels to a greater extent than for heterozygous SUR2A-containing channels. The greater ATP inhibition of mutant Kir6.2/SUR2A than of Kir6.2/SUR1 can explain why gain-of-function Kir6.2 mutations manifest effects in brain and β-cells but not in the heart.

This study was conducted to examine the role of myocardial ATP-sensitive potassium (KATP) channels in exercise-induced protection from ischaemia–reperfusion (I–R) injury. Female rats were either sedentary (Sed) or exercised for 12 weeks (Tr). Hearts were excised and underwent a 1–2 h regional I–R protocol. Prior to ischaemia, hearts were subjected to pharmacological blockade of the sarcolemmal KATP channel with HMR 1098 (SedHMR and TrHMR), mitochondrial blockade with 5-hydroxydecanoic acid (5HD; Sed5HD and Tr5HD), or perfused with buffer containing no drug (Sed and Tr). Infarct size was significantly smaller in hearts from Tr animals (35.4 ± 2.3 versus 44.7 ± 3.0% of the zone at risk for Tr and Sed, respectively). Mitochondrial KATP blockade did not abolish the training-induced infarct size reduction (30.0 ± 3.4 versus 38.0 ± 2.6 in Tr5HD and Sed5HD, respectively); however, sarcolemmal KATP blockade completely eradicated the training-induced cardioprotection. Infarct size was 71.2 ± 3.3 and 64.0 ± 2.4% of the zone at risk for TrHMR and Sed HMR. The role of sarcolemmal KATP channels in Tr-induced protection was also supported by significant increases in both subunits of the sarcolemmal KATP channel following training. LV developed pressure was better preserved in hearts from Tr animals, and was not influenced by addition of HMR 1098. 5HD decreased pressure development regardless of training status, from 15 min of ischaemia through the duration of the protocol. This mechanical dysfunction was likely to be due to a 5HD-induced increase in myocardial Ca2+ content following I–R. The major findings of the present study are: (1) unlike all other known forms of delayed cardioprotection, infarct sparing following chronic exercise was not abolished by 5HD; (2) pharmacological blockade of the sarcolemmal KATP channel nullified the cardioprotective benefits of exercise training; and (3) increased expression of sarcolemmal KATP channels was observed following chronic training.

ATP-sensitive potassium (K(ATP)) channels conduct potassium ions across cell membranes and thereby couple cellular energy metabolism to membrane electrical activity. Here, we report the heterologous expression and purification of a functionally active K(ATP) channel complex composed of pore-forming Kir6.2 and regulatory SUR1 subunits, and determination of its structure at 18 A resolution by single-particle electron microscopy. The purified channel shows ATP-ase activity similar to that of ATP-binding cassette proteins related to SUR1, and supports Rb(+) fluxes when reconstituted into liposomes. It has a compact structure, with four SUR1 subunits embracing a central Kir6.2 tetramer in both transmembrane and cytosolic domains. A cleft between adjacent SUR1s provides a route by which ATP may access its binding site on Kir6.2. The nucleotide-binding domains of adjacent SUR1 appear to interact, and form a large docking platform for cytosolic proteins. The structure, in combination with molecular modelling, suggests how SUR1 interacts with Kir6.2.

Pharmacological modulation of the mitochondrial ATP-sensitive K+ channel (mitoKATP) sensitive to diazoxide and 5-hydroxydecanoate (5-HD) represents an attractive strategy to protect cells against ischaemia/reperfusion- and stroke-related injury. To re-evaluate a functional role for the mitoKATP in brain, we used Percoll-gradient-purified brain nonsynaptosomal mitochondria in a light absorbance assay, in radioisotope measurements of matrix volume, and in measurements of respiration, membrane potential (ΔΨ) and depolarization-induced K+ efflux. The changes in mitochondrial morphology were evaluated by transmission electron microscopy (TEM). Polyclonal antibodies raised against certain fragments of known sulphonylurea receptor subunits, SUR1 and SUR2, and against different epitopes of K+ inward rectifier subunits Kir 6.1 and Kir 6.2 of the ATP-sensitive K+ channel of the plasma membrane (cellKATP), were employed to detect similar subunits in brain mitochondria. A variety of plausible blockers (ATP, 5-hydroxydecanoate, glibenclamide, tetraphenylphosphonium cation) and openers (diazoxide, pinacidil, chromakalim, minoxidil, testosterone) of the putative mitoKATP were applied to show the role of the channel in regulating matrix volume, respiration, and ΔΨ and K+ fluxes across the inner mitochondrial membrane. None of the pharmacological agents applied to brain mitochondria in the various assays pinpointed processes that could be unequivocally associated with mitoKATP activity. In addition, immunoblotting analysis did not provide explicit evidence for the presence of the mitoKATP, similar to the cellKATP, in brain mitochondria. On the other hand, the depolarization-evoked release of K+ suppressed by ATP could be re-activated by carboxyatractyloside, an inhibitor of the adenine nucleotide translocase (ANT). Moreover, bongkrekic acid, another inhibitor of the ANT, inhibited K+ efflux similarly to ATP. These observations implicate the ANT in ATP-sensitive K+ transport in brain mitochondria.

Mounting evidence reveals that ATP-sensitive potassium (K(ATP)) channel openers (KCOs) exert significant neuroprotection in vivo and in vitro in several models of Parkinson's disease (PD). However, the mechanisms are not well understood. In this study, we demonstrated that SH-SY5Y cells expressed mRNA and proteins for Kir6.1, Kir6.2, SUR1 and SUR2 subunits of K(ATP) channels. Moreover, our results showed that 1-methyl-4-phenyl-pyridinium ion (MPP+) induced up-regulation of mRNA for the Kir6.2 subunit and down-regulation of SUR1. It was further found that pretreatment with iptakalim, a novel K(ATP) channel opener, could attenuate increased extracellular glutamate level and decreased cell survival in SH-SY5Y cell culture after exposure to MPP+. Trans-pyrrolidine-2, 4-dicarboxylic acid (t-PDC), a glutamate transporter inhibitor, partially blocked the effect of iptakalim decreasing extracellular glutamate level. Additionally, iptakalim prevented MPP+-induced inhibition of glutamate uptake in primary cultured astrocytes. The beneficial effects of iptakalim on glutamate uptake of astrocytes were abolished by selective mitochondrial K(ATP) (mitoK(ATP)) channel blocker 5-HD. These results suggest (i) K(ATP) channel dysfunction may be involved in the mechanisms of MPP+-induced cytotoxicity and (ii) iptakalim may modulate glutamate transporters and subsequently alleviate the increase of extracellular glutamate levels induced by MPP+ through opening mitoK(ATP) channels, thereby protecting SH-SY5Y cells against MPP+-induced cytotoxicity.

Summary Objective Persistent hyperinsulinaemic hypoglycaemia of infancy (PHHI) is a disorder of glucose metabolism that is characterized by dysregulated secretion of insulin from pancreatic β-cells. This disease has been reported to be associated with mutations of the sulfonylurea receptor SUR1 (ABCC8) or the inward-rectifying potassium channel Kir6.2 (KCNJ11), which are two subunits of the pancreatic β-cell ATP-sensitive potassium channel. Patients and Methods In 14 Japanese PHHI patients, all exons of SUR1 and Kir6.2 genes were analysed by polymerase chain reaction (PCR) and direct sequencing. Four patients responded to diazoxide, and nine patients underwent a subtotal pancreatectomy. Histologically, seven patients were diagnosed to have a focal form and two a diffuse form of the disease. Results We found nine novel mutations in the SUR1 gene and two in the Kir6.2 gene. In the SUR1 gene mutations, three were nonsense mutations (Y512X, Y1354X and G1469X), one was a one-base deletion in exon 7, and two were missense mutations in the nucleotide-binding domain 2 (K1385Q, R1487K). The other three mutations occurred in introns 14, 29 and 36, which might cause aberrant splicing of RNA. Two siblings in one family were heterozygotes for a missense mutation, K1385Q, which was maternally inherited. In Kir6.2 gene screening, one patient was found to be a compound heterozygote of a missense mutation (R34H) and a one-base deletion (C344fs/ter). Conclusion The novel mutations reported here could be pathological candidates for PHHI in Japan. They also reveal that SUR1 and Kir6.2 mutations in the Japanese population exhibit heterogeneity and that they occurred at a frequency similar to other genetic populations.

K-ATP channels consist of two structurally different subunits: a pore-forming subunit of the Kir6.0-family (Kir6.1 or Kir6.2) and a sulfonylurea receptor (SUR1, SUR2, SUR2A, SUR2B) with regulatory activity. The functional diversity of K-ATP channels in brain is broad and of fundamental importance for neuronal activity. Here, using immunocytochemistry with monospecific antibodies against the Kir6.1 and Kir6.2 subunits, we analyze the regional and cellular distribution of both proteins in the adult rat brain. We find Kir6.2 to be widely expressed in all brain regions, suggesting that the Kir6.2 subunit forms the pore of the K-ATP channels in most neurons, presumably protecting the cells during cellular stress conditions such as hypoglycemia or ischemia. Especially in hypothalamic nuclei, in particular the ventromedial and arcuate nucleus, neurons display Kir6.2 immunoreactivity only, suggesting that Kir6.2 is the pore-forming subunit of the K-ATP channels in the glucose-responsive neurons of the hypothalamus. In contrast, Kir6.1-like immunolabeling is restricted to astrocytes (Thomzig et al. [2001] Mol Cell Neurosci 18:671-690) in most areas of the rat brain and very weak or absent in neurons. Only in distinct nuclei or neuronal subpopulations is a moderate or even strong Kir6.1 staining detected. The biological functions of these K-ATP channels still need to be elucidated.

To explore how the sulfonylurea receptor (SUR1) is involved in docking and fusion of insulin granules, dynamic motion of single insulin secretory granules near the plasma membrane was examined in SUR1 knock-out (Sur1KO) beta-cells by total internal reflection fluorescence microscopy. Sur1KO beta-cells exhibited a marked reduction in the number of fusion events from previously docked granules. However, the number of docked granules declined during stimulation as a consequence of the release of docked granules into the cytoplasm vs. fusion with the plasma membrane. Thus, the impaired docking and fusion results in decreased insulin exocytosis from Sur1KO beta-cells.

ATP-sensitive K(+) (K(ATP)) channel activation is implicated in the vascular hyporeactivity occurring in septic shock. However, channel inhibition with the sulphonylurea receptor (SUR) antagonist, glibenclamide (Glib) fails to reverse lipopolysaccharide (LPS)-induced vascular hyporeactivity in vitro. We investigated whether inhibitors that act by binding to the K(ATP) channel pore could be effective. Ring segments of endothelium-intact rat mesenteric artery were incubated with LPS in culture media for either 6 or 20 h before contractile responses to phenylephrine were assessed in the absence or presence of K(ATP) channel inhibitors. The pore-forming subunit inhibitors barium chloride (BaCl(2); 300 microM) and PNU-37883A (1 microM) significantly reversed hyporeactivity at both time points, although less so at 20 h. In contrast, the SUR inhibitors, Glib (10 microM), tolbutamide (Tolb) (1 mM) and PNU-99963 (1 microM) were ineffective. In LPS-incubated tissues, Glib and Tolb antagonised contractions to the thromboxane A2 mimetic, U46619 (9,11-dideoxy-9alpha, 11alpha-methanoepoxy prostaglandin F(2alpha)) (10(-7) M), whereas the pinacidil-derived inhibitor, PNU-99963, did not. Contractions to 60 mM KCl were unaffected by LPS at 6 h, but were significantly depressed by LPS at 20 h, suggesting that K(+)-channel-independent pathways contribute to hyporeactivity at the later time point. The inducible nitric oxide synthase (iNOS) inhibitor, 1400 W (10 microM) and Tolb inhibited the production of nitrite induced by LPS, whereas BaCl(2) and PNU-37883A had no effect. In conclusion, K(ATP) channels contribute to LPS-induced vascular hyporeactivity via the iNOS pathway in rat mesenteric artery. The effectiveness of pore inhibitors over SUR inhibitors of the K(ATP) channel suggests altered SUR function following LPS administration, which cannot be explained by thromboxane receptor inhibition.

In this study we have investigated the voltage dependence of ATP-dependent K+ current (I(K(ATP))) in atrial and ventricular myocytes from hearts of adult rats and in CHO cells expressing Kir6.2 and SUR2A. The current-voltage relation of 2,4-dinitrophenole (DNP) -induced I(K(ATP)) in atrial myocytes and expressed current in CHO cells was linear in a voltage range between 0 and -100 mV. In ventricular myocytes, the background current-voltage relation of which is dominated by a large constitutive inward rectifier (I(K1)), the slope conductance of I(K(ATP)) was reduced at membrane potentials negative to E(K) (around -50 mV), resulting in an outwardly rectifying I-V relation. Overexpression of Kir2.1 by adenoviral gene transfer, a subunit contributing to I(K1) channels, in atrial myocytes resulted in a large I(K1)-like background current. The I-V relation of I(K(ATP)) in these cells showed a reduced slope conductance negative to E(K) similar to ventricular myocytes. In atrial myocytes with an increased background inward-rectifier current through Kir3.1/Kir3.4 channels (I(K(ACh))), irreversibly activated by intracellular loading with GTP-gamma-S, the I-V relation of I(K(ATP)) showed a reduced slope negative to E(K), as in ventricular myocytes and atrial myocytes overexpressing Kir2.1. It is concluded that the voltage dependencies of membrane currents are not only dependent on the molecular composition of the charge-carrying channel complexes but can be affected by the activity of other ion channel species. We suggest that the interference between inward I(K(ATP)) and other inward rectifier currents in cardiac myocytes reflects steady-state changes in K+ driving force due to inward K+ current.

ATP-binding cassette (ABC) transporters are membrane proteins responsible for cellular detoxification processes in plants and animals. Recent evidence shows that this class of transporters may also be involved in many other cellular processes. Because of their homology with human multidrug resistance-associated proteins (MRP), cystic fibrosis transmembrane conductance regulator (CFTR) and sulfonylurea receptor (SUR), some plant ABC transporters have been implicated in the regulation of ion channel activities. This paper describes an investigation of the AtMRP4 gene and its role in stomatal regulation. Reporter gene studies showed that AtMRP4 is highly expressed in stomata and that the protein is localized to the plasma membrane. Stomatal aperture in three independent atmrp4 mutant alleles was larger than in wild-type plants, both in the light and in the dark, resulting in increased water loss but no change in the photosynthetic rate. In baker's yeast, AtMRP4 shows ATP-dependent, vanadate-sensitive transport of methotrexate (MTX), an antifolate and a substrate of mammalian MRPs. Treatment with MTX reduced stomatal opening in wild-type plants, but had no effect in atmrp4 mutants. These results indicate the involvement of AtMRP4 in the complex regulation of stomatal aperture.

C-terminal fragments of the sulphonylurea receptor SUR2A can alter the functional expression of cloned ATP-sensitive K(+) channels (K(ATP)). To investigate the protective role of K(ATP) channels during metabolic stress we transfected SUR2A fragments into adult rat cardiac myocytes. A fragment comprising residues 1294-1358, the A-fragment, reduced sarcolemmal K(ATP) currents by over 85% after 2 days (pinacidil-activated current densities were: vector alone 7.04 +/- 1.22; and A-fragment 0.94 +/- 0.07 pA pF(-1), n= 6,6, P < 0.001). An inactive fragment (1358-1545, current density 6.30 +/- 0.85 pA pF(-1), n= 6) was used as a control. During metabolic inhibition (CN and iodoacetate) of isolated myocytes stimulated at 1 Hz, the A-fragment delayed action potential shortening and contractile failure, but accelerated rigor contraction and increased Ca(2+) loading. On reperfusion, A-fragment-transfected cells also showed increased intracellular Ca(2+) and the proportion of cells recovering contractile function was reduced from 40.0 to 9.5% (P < 0.01). The protective effect of pretreatment with 2,4-dinitrophenol, measured from increased functional recovery and reduced Ca(2+) loading, was abolished by the A-fragment. Our data are consistent with a role for K(ATP) channels in causing action potential failure and reduced Ca(2+) loading during metabolic stress, and with a major role in protection by preconditioning. The effects of the A-fragment may arise entirely from reduced expression of the sarcolemmal K(ATP) channel, but we also discuss the possibility of mitochondrial effects.

1. ATP-sensitive K(+) channels (K(ATP) channels) are composed of pore-forming subunits (Kir6.x) and of regulatory subunits, the sulphonylurea receptors (SURx). Synthetic openers of K(ATP) channels form a chemically heterogeneous class of compounds that are of interest in several therapeutic areas. We have investigated the interaction of a novel dihydropyridine opener, A-312110 ((9R)-9-(4-fluoro-3-iodophenyl)-2,3,5,9-tetrahydro-4H-pyrano[3,4-b]thieno [2,3-e]pyridin-8(7H)-one-1,1-dioxide), with SURs and Kir6/SUR channels in comparison to the cyanoguanidine opener P1075. 2. In the presence of 1 mM MgATP, A-312110 bound to SUR2A (the SUR in cardiac and skeletal muscle) and to SUR2B (smooth muscle) with K(i) values of 14 and 18 nM; the corresponding values for P1075 were 16 and 9 nM, respectively. Decreasing the MgATP concentration reduced the affinity of A312110 binding to SUR2A significantly more than that to SUR2B; for P1075, the converse was true. At SUR1 (pancreatic beta-cell), both openers showed little binding up to 100 microM. 3. In the presence of MgATP, both openers inhibited [(3)H]glibenclamide binding to the SUR2 subtypes in a biphasic manner. In the absence of MgATP, the high-affinity component of the inhibition curves was absent. 4. In inside-out patches, the two openers activated the Kir6.2/SUR2A and Kir6.2/SUR2B channels with similar potency (approximately 50 nm). Both were almost 2 x more efficacious in opening the Kir6.2/SUR2B than the Kir6.2/SUR2A channel. 5. The results show that the novel dihydropyridine A-312110 is a potent K(ATP) channel opener with binding and channel-opening properties similar to those of P1075.

Endosulfine (EDSF) belongs to a highly conserved cAMP-regulated phosphoprotein (ARPP) family and was first isolated from ovine brain as a possible endogenous ligand for sulfonylurea receptors. To explore its involvement in brain functions, we investigated regional distribution of α-EDSF gene expression in the rat brain, and its regulation under physiological and pathological conditions. The majority of α-EDSF gene was expressed in the pyramidal neurons, which represent the principal excitatory neurons in various brain regions. Down-regulation of α-EDSF mRNA was detected in the rat hippocampus during long-term memory consolidation following a spatial learning experience, whereas swimming-related stress caused persistent up-regulation of α-EDSF gene expression in several brain regions. These changes, however, were absent from brains of diabetic rats that were subjected to the same behavioral treatments. Intracerebroventricular injection of streptozocin with a toxic dose induced severe learning deficits and brain structure alteration accompanied by a massive increase of α-EDSF mRNA in the somatosensory cortex. These results suggest that α-EDSF gene expression is differentially regulated by distinct brain processes involving excitatory neuronal activities.

The sulfonylurea receptor (SUR), an ATP-binding cassette (ABC) protein, assembles with a potassium channel subunit (Kir6) to form the ATP-sensitive potassium channel (K(ATP)) complex. Although SUR is an important regulator of Kir6, the specific SUR domain that associates with Kir6 is still unknown. All functional ABC proteins contain two transmembrane domains but some, including SUR and MRP1 (multidrug resistance protein 1), contain an extra N-terminal transmembrane domain called TMD0. The functions of any TMD0s are largely unclear. Using Xenopus oocytes to coexpress truncated SUR constructs with Kir6, we demonstrated by immunoprecipitation, single-oocyte chemiluminescence and electrophysiological measurements that the TMD0 of SUR1 strongly associated with Kir6.2 and modulated its trafficking and gating. Two TMD0 mutations, A116P and V187D, previously correlated with persistent hyperinsulinemic hypoglycemia of infancy, were found to disrupt the association between TMD0 and Kir6.2. These results underscore the importance of TMD0 in K(ATP) channel function, explaining how specific mutations within this domain result in disease, and suggest how an ABC protein has evolved to regulate a potassium channel.

1 Openers of ATP-sensitive K(+) channels (K(ATP) channels) are thought to act by enhancing the ATPase activity of sulphonylurea receptors (SURs), the regulatory channel subunits. At higher concentrations, some openers activate K(ATP) channels also in the absence of MgATP. Here, we describe binding and effect of structurally diverse openers in the absence of Mg(2+) and presence of EDTA. 2 Binding of openers to SUR2B was measured using a mutant with high affinity for [(3)H]glibenclamide ([(3)H]GBC). In the absence of Mg(2+), 'typical' openers (benzopyrans, cyanoguanidines and aprikalim) inhibited [(3)H]GBC binding with K(i) values approximately 200 x higher than in the presence of MgATP. Minoxidil sulphate and nicorandil were inactive, whereas binding of diazoxide was unaffected by MgATP. 3 In the absence/presence of MgATP, N-cyano-N'-(1,1-dimethylpropyl)-N"-3-pyridylguanidine (P1075) activated the Kir6.2/SUR2B channel in inside-out patches with EC(50)=2000/67nM and E(max)=32/134%. In the absence of Mg(2+), responses were variable with only a small part of the variability being explained by a decrease in channel responsiveness with time after patch excision and to differences in the ATP sensitivity between patches. 4 The rank order of efficacy of the openers was P1075>rilmakalim approximately nicorandil>diazoxide>minoxidil sulphate. 5 The data show that structurally diverse openers are able to bind to, and to activate the Kir6.2/SUR2B channel by a pathway independent of ATP hydrolysis. These effects are observed at concentrations used to define the biochemical mechanism of the openers in the presence of MgATP and allow the openers to be classified into 'typical' and 'atypical' KCOs with diazoxide standing apart.

1. We investigated the mechanism of action of two novel nonsulphonylurea ATP-sensitive potassium channel (K(ATP)) inhibitors, PNU-99963 and PNU-37883A, on four types of cloned K(ATP) channels. 2. Whole-cell currents were recorded in a symmetrical potassium (140 mM) gradient in HEK-293 cells stably expressing Kir6.2/SUR1, Kir6.2/SUR2A, Kir6.2/SUR2B or Kir6.1/SUR2B. 3. PNU-99963 potently inhibited the four K(ATP) channel clones. The concentration at which half-maximum current was inhibited (IC(50)) was 66, 41, 43 and 11 nM for Kir6.2/SUR1, Kir6.2/SUR2A, Kir6.2/SUR2B and Kir6.1/SUR2B, respectively. In contrast, PNU-99963 up to a concentration of 3 microM had no significant effect on current generated in HEK-293 cells by transiently expressing Kir6.2Delta26, a C-terminal truncated pore-forming subunit of Kir6.2. 4. PNU-37883A inhibited four types of K(ATP) channels, but to different extents. Inhibition of the putative smooth muscle K(ATP) channel types, Kir6.2/SUR2B (IC(50); 15 microM) and Kir6.1/SUR2B (IC(50); 6 microM), was significantly greater than inhibition of either the pancreatic beta cell or cardiac K(ATP) channel clones. Moreover, PNU-37883A significantly inhibited currents generated by expressing Kir6.2Delta26 alone, with an IC(50) of 5 microM, which was significantly increased to 38 microM when Kir6.2Delta26 was expressed with SUR2B. 5. In conclusion, two structurally different nonsulphonylurea compounds, PNU-99963 and PNU-37883A, inhibit K(ATP) channels via different mechanisms, namely through the sulphonylurea receptor (SUR) and the pore-forming subunits, respectively, although SUR2B reduced the inhibitory effect of PNU-37883A. While PNU-99963 potently inhibits all the four cloned K(ATP) channels, PNU-37883A has a degree of selectivity towards both smooth muscle K(ATP) channels, but could not discriminate between them.

We recently showed that activation of ATP-sensitive potassium (KATP) channels in PC12 cells induces protection against the neurotoxic effect of rotenone, a mitochondrial complex I inhibitor. In this study, we sought to determine the locus of the KATP channels that mediate this protection in PC12 cells. We found that pretreatment of PC12 cells with diazoxide, a mitochondrial KATP channel selective opener, dose-dependently increases cell viability against rotenone-induced cell death as indicated in trypan blue exclusion assays. The protective effect of this preconditioning is attenuated by 5-hydroxydecanoic acid (5-HD), a selective mitochondrial KATP channel antagonist but not in the presence of HMR-1098, a selective plasma membrane KATP potassium channel antagonist. In contrast, P-1075, a selective plasma membrane KATP channel opener, does not induce protection. Using specific antibodies against SUR1 and Kir6.1, we detected immunoreactive proteins of apparent molecular masses 155 and 50 kDa, corresponding to those previously reported for SUR1 and Kir6.1, respectively, in the mitochondria-enriched fraction of PC12 cells. In addition, whole cell patch-clamp studies revealed that inward currents in PC12 cells are insensitive to P-1075, HMR-1098, glibenclamide and diazoxide, indicating that functional plasma membrane KATP channels are negligible. Taken together, our results demonstrate for the first time that activation of mitochondrial KATP channels elicits protection against rotenone-induced cell death.

1. This study reports on the identification and characterization of a 1,4-dihydropyridine analogue, 9-(3,4-dichlorophenyl)-3,3,6,6-tetramethyl-3,4,6,7,9,10-hexahydro-1,8(2H,5H)-acridinedione (A-184209) as a novel inhibitor of ATP-sensitive K(+) channels. 2. A-184209 inhibited membrane potential changes evoked by the prototypical cyanoguanidine ATP-sensitive K(+) channel opener (KCO) P1075 in both vascular (A10) and urinary bladder smooth muscle cells with IC(50) values of 1.44 and 2.24 micro M respectively. 3. P1075-evoked relaxation of 25 mM K(+) stimulated aortic strips was inhibited by A-184209 in an apparently competitive fashion with a pA(2) value of 6.34. 4. The potencies of A-184209 to inhibit P1075-evoked decreases in membrane potential responses in cardiac myocytes (IC(50)=0.53 micro M) and to inhibit 2-deoxyglucose-evoked cation efflux pancreatic RINm5F cells (IC(50)=0.52 micro M) were comparable to the values for inhibition of smooth muscle K(ATP) channels. 5. On the other hand, a structural analogue of A-184209 that lacked the gem-dimethyl substituent, 9-(3,4-dichlorophenyl)-3,4,6,7,9,10-hexahydro-1,8(2H,5H)-acridinedione (A-184208), was found to be a K(ATP) channel opener, evoking membrane potential responses in A10 smooth muscle cells (EC(50)=385 nM) and relaxing aortic smooth muscle strips (IC(50)=101 nM) in a glyburide-sensitive manner. 6. Radioligand binding studies demonstrated that A-184209 displaced SUR1 binding defined by [(3)H]glyburide binding to RINm5F cell membranes with a K(i) value of 0.11 micro M whereas A-184208 was ineffective. On the other hand, both A-184209 (K(i)=1.34 micro M) and A-184208 (K(i)=1.14 micro M) displaced binding of the KCO radioligand, [(125)I]A-312110 in guinea-pig bladder membranes with similar affinities. 7. These studies demonstrate that A-184209 is a novel and structurally distinct compound that inhibits K(ATP) channels in smooth muscle with potencies comparable to glyburide. The structural overlap between DHP openers and blockers, together with their differential interaction with ligand binding sites, support the notion that both openers and blockers bind to similar or very closely coupled sites on the sulfonylurea receptor and that subtle changes in the pharmacophore itself could switch functional properties from K(ATP) channel activation to inhibition.