Sulfonylurea Receptor 2 Research Articles

Deletion of Sulfonylurea Receptor 2 in the Adult Myocardium Enhances Cardiac Glucose Uptake and Is Cardioprotective

The adult myocardium relies on oxidative metabolism. In ischemic myocardium, such as the embryonic heart, glycolysis contributes more prominently as a fuel source. The sulfonylurea receptor 2 (SUR2) was previously implicated in the normal myocardial transition from glycolytic to oxidative metabolism that occurs during adaptation to postnatal life. This receptor was now selectively deleted in adult mouse myocardium resulting in protection from ischemia reperfusion injury. SUR2-deleted cardiomyocytes had enhanced glucose uptake, and SUR2 forms a complex with the major glucose transporter. These data identify the SUR2 receptor as a target to shift cardiac metabolism to protect against myocardial injury.

Conserved functional consequences of disease-associated mutations in the slide helix of Kir6.1 and Kir6.2 subunits of the ATP-sensitive potassium channel

Cantu syndrome (CS) is a condition characterized by a range of anatomical defects, including cardiomegaly, hyperflexibility of the joints, hypertrichosis, and craniofacial dysmorphology. CS is associated with multiple missense mutations in the genes encoding the regulatory sulfonylurea receptor 2 (SUR2) subunits of the ATP-sensitive K+ (KATP) channel as well as two mutations (V65M and C176S) in the Kir6.1 (KCNJ8) subunit. Previous analysis of leucine and alanine substitutions at the Val-65-equivalent site (Val-64) in Kir6.2 indicated no major effects on channel function. In this study, we characterized the effects of both valine-to-methionine and valine-to-leucine substitutions at this position in both Kir6.1 and Kir6.2 using ion flux and patch clamp techniques. We report that methionine substitution, but not leucine substitution, results in increased open state stability and hence significantly reduced ATP sensitivity and a marked increase of channel activity in the intact cell irrespective of the identity of the coassembled SUR subunit. Sulfonylurea inhibitors, such as glibenclamide, are potential therapies for CS. However, as a consequence of the increased open state stability, both Kir6.1(V65M) and Kir6.2(V64M) mutations essentially abolish high-affinity sensitivity to the KATP blocker glibenclamide in both intact cells and excised patches. This raises the possibility that, at least for some CS mutations, sulfonylurea therapy may not prove to be successful and highlights the need for detailed pharmacogenomic analyses of CS mutations.

Effect of tobacco smoke on hydrogen sulfide-induced rat thoracic aorta relaxation.

Levels of hydrogen sulfide (H2S), a gaseous signaling molecule, are reduced in the serum of individuals who smoke. We hypothesized that tobacco smoke influenced smooth muscle relaxation by decreasing H2S levels and this effect could also influence expression of cystathionine γ-lyase (CSE) and sulfonylurea receptor-2 (SUR-2). The aim of this study was to explore the effect of tobacco smoke on H2S-mediated rat thoracic aorta relaxation and its possible mechanism. Thirty-two Sprague-Dawley rats were divided into four groups: control (C) group, short-term smoker (SS) group, mid-term smoker (MS) group, and long-term smoker (LS) group. H2S concentrations in serum, action of H2S on rat aortic vascular relaxation, and expression of CSE and SUR-2 in thoracic aortic smooth muscle were measured. Although there was no significant difference in H2S between the C and the SS groups, concentration of H2S was significantly reduced in both the LS and MS groups compared to control (P<0.01). Furthermore, H2S was significantly lower in the LS than in the MS group (P<0.05). Rat aortic vascular relaxation was lower in all three treatment groups compared to the control, with the most significant decrease observed in the LS group (P<0.05 compared to the MS group). Expression of CSE and SUR-2 was reduced in the LS and MS groups compared to control (P<0.05), with the lowest levels observed in the LS group (P<0.05). Therefore, tobacco smoke reduced expression of CSE and SUR-2 in rat thoracic aorta, which may inhibit H2S production and vascular dilation.

Nicorandil, a Katp channel opener, alleviates chronic renal injury by targeting podocytes and macrophages

Nicorandil exhibits a protective effect in the vascular system, which is thought to be due to vasodilatation from opening ATP-dependent potassium channels and donation of nitric oxide. Recently, nicorandil was shown to be renoprotective in models of acute kidney injury and glomerulonephritis. However, the specific mechanisms of renoprotection are unclear. We evaluated the effect of nicorandil on the rat remnant kidney model of chronic kidney disease. Blood pressure was unchanged by a 10-wk course of nicorandil, while albuminuria was significantly reduced. Glomerular injury and tubulointerstitial injury were also ameliorated by nicorandil. Oxidative stress, as noted by renal nitrotyrosine level and urine 8-hydroxy-2'-deoxyguanosine, were elevated in this model and was significantly reduced by nicorandil treatment. Treatment was associated with maintenance of the mitochondrial antioxidant, manganese SOD, in podocytes and with suppression of xanthine oxidase expression in infiltrating macrophages. Interestingly, these two cell types express sulfonylurea receptor 2 (SUR2), a binding site of nicorandil in the ATP-dependent K channel. Consistently, we found that stimulating SUR2 with nicorandil prevented angiotensin II-mediated upregulation of xanthine oxidase in the cultured macrophage, while xanthine oxidase expression was rather induced by blocking SUR2 with glibenclamide. In conclusion, nicorandil reduces albuminuria and ameliorates renal injury by blocking oxidative stress in chronic kidney disease.

Abstract 16645: Abcc9 Contributes to Mitochondrial Katp Function and Protection From Cardiomyopathy

In response to intracellular energy supply, ATP-sensitive potassium (KATP) channels alter membrane potential and mediate cell stress response. The Abcc9 gene encodes the major regulatory subunit in the heart, sulfonylurea receptor 2 (SUR2), as well as smaller mitochondria-enriched proteins that contribute to sulfonylurea-insensitive KATP channels. Pharmacological studies suggest mitochondrial KATP channels are critical regulators of cell stress, however the molecular composition of this channel has been unclear. We now studied the role of KATP channels by deleting exon 5 of Abcc9 . This strategy ablated expression of both full length SUR2 protein as well as the smaller mitochondrial 55 KDa protein. Homozygous exon 5 (Ex5) mice died within 14 days of birth with progressive cardiac contractile dysfunction. Diazoxide was found to depolarize mitochondria from wildtype cardiomyocytes but not Ex5 mitochondria, consistent with disrupted mitochondrial KATP channels. Ex5 mitochondria had a reduced cross sectional area (WT: 1.27±0.11 vs. Ex5: 0.67±0.04µm 2 , p&lt;0.05), and Ex5 cardiomyocytes also exhibited reduced oxygen consumption(WT: 1.86±0.41 vs. Ex5: 0.61±0.14nmO 2 /s, p&lt;0.05). Deletion of exons 14 to 18 in Abcc9 disrupts full length SUR2 but leaves intact the 55 KDa protein. To determine the contribution of the mitochondrial 55 KDa protein, we compared the ability of Ex14/18 and Ex5 cardiomyocytes to withstand H2O2-induced stress. H 2 O 2 application resulted in a more rapid collapse of mitochondria membrane potential in Ex5 mitochondria (5.2±0.3s) compared to wildtype (15.1±0.6s) and Ex14/18 (13.1±0.6s) mitochondria. We conclude the Abcc9 -encoded 55 KDa protein contributes to mitochondrial KATP channels, and that mitochondrial KATP channels mediate the acute stress associated with cardiac function in the neonatal window.

Differential roles for SUR subunits in KATP channel membrane targeting and regulation

since their discovery over 25 years ago, ATP-sensitive K+ (KATP) channels have been studied extensively in many tissues, including heart, pancreas, brain, skeletal and smooth muscle, pituitary, and kidney ([6][1], [10][2], [18][3], [19][4]). KATP channels are critical sensors that couple cellular

The mitochondrial bioenergetic phenotype for protection from cardiac ischemia in SUR2 mutant mice

The sulfonylurea receptor-2 (SUR2) is a subunit of ATP-sensitive potassium channels (K(ATP)) in heart. Mice with the SUR2 gene disrupted (SUR2m) are constitutively protected from ischemia-reperfusion (I/R) cardiac injury. This was surprising because K(ATP), either sarcolemmal or mitochondrial or both, are thought to be important for cardioprotection. We hypothesized that SUR2m mice have an altered mitochondrial phenotype that protects against I/R. Mitochondrial membrane potential (ΔΨ(m)), tolerance to Ca(2+) load, and reactive oxygen species (ROS) generation were studied by fluorescence-based assays, and volumetric changes in response to K(+) were measured by light scattering in isolated mitochondria. For resting SUR2m mitochondria compared with wild type, the ΔΨ(m) was less polarized (46.1 ± 0.4 vs. 51.9 ± 0.6%), tolerance to Ca(2+) loading was increased (163 ± 2 vs. 116 ± 2 μM), and ROS generation was enhanced with complex I [8.5 ± 1.2 vs. 4.9 ± 0.2 arbitrary fluorescence units (afu)/s] or complex II (351 ± 51.3 vs. 166 ± 36.2 afu/s) substrates. SUR2m mitochondria had greater swelling in K(+) medium (30.2 ± 3.1%) compared with wild type (14.5 ± 0.6%), indicating greater K(+) influx. Additionally, ΔΨ(m) decreased and swelling increased in the absence of ATP in SUR2m, but the sensitivity to ATP was less compared with wild type. When the mitochondria were subjected to hypoxia-reoxygenation, the decrease in respiration rates and respiratory control index was less in SUR2m. ΔΨ(m) maintenance in the SUR2m intact myocytes was also more tolerant to metabolic inhibition. In conclusion, the cardioprotection observed in the SUR2m mice is associated with a protected mitochondrial phenotype resulting from enhanced K(+) conductance that partially dissipated ΔΨ(m). These results have implications for possible SUR2 participation in mitochondrial K(ATP).

Cardiomyocyte sulfonylurea receptor 2-KATP channel mediates cardioprotection and ST segment elevation.

Sulfonylurea receptor-containing ATP-sensitive potassium (K(ATP)) channels have been implicated in cardioprotection, but the cell type and constitution of channels responsible for this protection have not been clear. Mice deleted for the first nucleotide binding region of sulfonylurea receptor 2 (SUR2) are referred to as SUR2 null since they lack full-length SUR2 and glibenclamide-responsive K(ATP) channels in cardiac, skeletal, and smooth muscle. As previously reported, SUR2 null mice develop electrocardiographic changes of ST segment elevation that were shown to correlate with coronary artery vasospasm. Here we restored expression of the cardiomyocyte SUR2-K(ATP) channel in SUR2 null mice by generating transgenic mice with ventricular cardiomyocyte-restricted expression of SUR2A. Introduction of the cardiomyocyte SUR2A transgene into the SUR2 null background restored functional cardiac K(ATP) channels. Hearts isolated from rescued mice, referred to as MLC2A, had significantly reduced infarct size (27 ± 3% of area at risk) compared with SUR2 null mice (36 ± 3% of area at risk). Compared with SUR2 null hearts, MLC2A hearts exhibited significantly improved cardiac function during the postischemia reperfusion period primarily because of preservation of low diastolic pressures. Additionally, restoration of cardiac SUR2-K(ATP) channels significantly reduced the degree and frequency of ST segment elevation episodes in MLC2A mice. Therefore, cardioprotective mechanisms both dependent and independent of SUR2-K(ATP) channels contribute to cardiac function.

Expression of sulfonylurea receptors in rat taste buds

To test the possibility that a fast-onset promoting agent repaglinide may initiate prandial insulin secretion through the mechanism of cephalic-phase insulin release, we explored the expression and distribution character of sulfonylurea receptors in rat taste buds. Twenty male Wistar rats aged 10 weeks old were killed after general anesthesia. The circumvallate papillae, fungiform papillae and pancreas tissues were separately collected. Immunohistochemical staining was used to detect the expression and distribution of sulfonylurea receptor 1 (SUR1) or sulfonylurea receptor 2 (SUR2) in rat taste buds. Reverse transcriptase polymerase chain reaction (RT-PCR) was used to analyze the expression of SUR1 or SUR2 mRNA. The pancreatic tissues from the same rat were used as positive control. This is the first study to report that SUR1 is uniquely expressed in the taste buds of fungiform papillae of each rat tongue, while the expression of SUR1 or SUR2 was not detected in the taste buds of circumvallate papillae. SUR1 is selectively expressed in rat taste buds, and its distribution pattern may be functionally relevant, suggesting that the rapid insulin secretion-promoting effect of repaglinide may be exerted through the cephalic-phase secretion pathway mediated by taste buds.

The Mitochondrial Bioenergetic Phenotype for Protection from Ischemia in Sur2-Mutant Mice

ATP-sensitive potassium channels (KATP) in mitochondria are postulated to play a key role to protect mitochondria and myocytes from cardiac ischemic insult. The sulfonylurea receptor-2 (SUR2) is a subunit of KATP in sarcolemma, although its role in mitochondrial physiology is unclear. Mice where the SUR2 gene was disrupted (SUR2 mutant) have been shown to be constitutively protected from ischemic injury.

Mice lacking sulfonylurea receptor 2 (SUR2) ATP-sensitive potassium channels are resistant to acute cardiovascular stress

Adenosine triphosphate-sensitive potassium (K ATP) channels are thought to mediate the stress response by sensing intracellular ATP concentration. Cardiomyocyte K ATP channels are composed of the pore-forming Kir6.2 subunit and the regulatory sulfonylurea receptor 2 (SUR2). We studied the response to acute isoproterenol in SUR2 null mice as a model of acute adrenergic stress and found that the episodic coronary vasospasm observed at baseline in SUR2 null mice was alleviated. Similar results were observed following administration of a nitric oxide donor consistent with a vasodilatory role. Langendorff-perfused hearts were subjected to global ischemia, and hearts from SUR2 null mice exhibited significantly reduced infarct size (54 ± 4 versus 30 ± 3%) and improved cardiac function compared to control mice. SUR2 null mice have hypertension and develop cardiac hypertrophy. However, despite longstanding hypertension, fibrosis was absent in SUR2 null mice. SUR2 null mice were administered nifedipine to block baseline coronary vasospasm, and hearts from nifedipine-treated SUR2 null mice exhibited increased infarct size compared to untreated SUR2 null mice (42 ± 3% versus 54 ± 3%). We conclude that conventional sarcolemmal cardiomyocyte K ATP channels containing full-length SUR2 are not required for mediating the response to acute cardiovascular stress.

Spontaneous Coronary Vasospasm in K ATP Mutant Mice Arises From a Smooth Muscle–Extrinsic Process

In the vasculature, ATP-sensitive potassium channels (KATP) channels regulate vascular tone. Mice with targeted gene disruptions of KATP subunits expressed in vascular smooth muscle develop spontaneous coronary vascular spasm and sudden death. From these models, it was hypothesized that the loss of KATP channel activity in arterial vascular smooth muscle was responsible for coronary artery spasm. We now tested this hypothesis using a transgenic strategy where the full-length sulfonylurea receptor containing exon 40 was expressed under the control of a smooth muscle-specific SM22alpha promoter. Two transgenic founder lines were generated and independently bred to sulfonylurea receptor 2 (SUR2) null mice to generate mice that restored expression of KATP channels in vascular smooth muscle. Transgenic expression of the sulfonylurea receptor in vascular smooth muscle cells was confirmed by detecting mRNA and protein from the transgene. Functional restoration was determined by recording pinacidil-based KATP current by whole cell voltage clamping of isolated aortic vascular smooth muscle cells isolated from the transgenic restored mice. Despite successful restoration of KATP channels in vascular smooth muscle, transgene-restored SUR2 null mice continued to display frequent episodes of spontaneous ST segment elevation, identical to the phenotype seen in SUR2 null mice. As in SUR2 null mice, ST segment elevation was frequently followed by atrioventricular heart block. ST segment elevation and coronary perfusion pressure in the restored mice did not differ significantly between transgene-negative and transgene-positive SUR2 null mice. We conclude that spontaneous coronary vasospasm and sudden death in SUR2 null mice arises from a coronary artery vascular smooth muscle-extrinsic process.

Characterization of human urinary bladder K ATP channels containing SUR2B splice variants expressed in L-cells

The molecular properties of the sulfonylurea receptor 2 (SUR2) subunits of K ATP channels expressed in urinary bladder were assessed by polymerase chain reaction (PCR). This showed that SUR2B exon 17− mRNA (72%) was predominant over the SUR2B exon 17+ splice variant (28%). The pharmacological properties of both of these isoforms stably expressed in mouse Ltk −cells (L-cells) with K IR 6.2 were determined by measuring changes in membrane potential responses evoked by K + channel openers using bis-(1,3-dibutylbarbituric acid) trimethine oxonol (DiBAC 4(3)) fluorescence. The rank order potency of a variety of structurally distinct K + channel openers was found to be the same in both stable cell lines and compared well with guinea pig bladder cells. The potency of these compounds in the SUR2B exon 17− cells more closely resembled the potency measured in guinea pig bladder unlike the cell line containing the SUR2B exon 17+ subtype. Analysis of the displacement of [ 125I]A-312110 binding with the same K + channel openers to the SUR2B exon 17- cells showed excellent correlation to those measured in guinea pig bladder. This study supports the notion that K ATP channels containing SUR2B exon 17− represent a major splice variant expressed in urinary bladder smooth muscle.

Molecular characterization of human SUR2-containing K ATP channels

The distribution of human sulfonylurea receptor-2 (SUR2)-containing K ATP channels was investigated using reverse transcriptase-polymerase chain reaction (RT-PCR). mRNA for SUR2B was detected in a variety of tissues including brain, skeletal, cardiac and smooth muscle, whereas SUR2A message was restricted to cardiac and skeletal muscle. An additional splice variant of SUR2 that lacked exon 17 was also identified by RT-PCR in tissues expressing both SUR2A and SUR2B or SUR2B alone. Quantification of RNA for SUR2 exon 17+ and SUR2 exon 17− splice variants using real-time Taqman PCR indicated differential levels of expression in brain, kidney, skeletal muscle, heart and small intestine. Interestingly, the SUR2 exon 17+ variant is the major species expressed in all tissues examined in this study. Each of the SUR2 splice variants transiently expressed with the inward rectifier Kir 6.2 formed functional K ATP channels in HEK 293 cells as assessed either by changes in DiBAC 4(3) fluorescence responses or glyburide-sensitive whole cell currents. Collectively, our findings demonstrate that various SUR2 splice variants have distinct expression patterns and can form functional K ATP channels.