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Background: Diazoxide maintains myocyte volume and contractility during stress via an unknown mechanism. The mechanism of action may involve an undefined (genotype unknown) mitochondrial ATP-sensitive potassium channel and is dependent on the ATP-sensitive potassium channel subunit sulfonylurea type 1 receptor (SUR1). The ATP-sensitive potassium channel openers have been shown to inhibit succinate dehydrogenase (SDH) and a gene for a portion of SDH has been found in the SUR intron. Diazoxide may be cardioprotective via inhibition of SDH, which can form part of an ATP-sensitive potassium channel or share its genetic material. This study investigated the role of inhibition of SDH by diazoxide and its relationship to the SUR1 subunit., Study Design: Mitochondria were isolated from wild-type and SUR1 knockout mice. Succinate dehydrogenase activity was measured by spectrophotometric analysis of 2,6-dichloroindophenol reduction for 20 minutes as the relative change in absorbance over time. Mitochondria were treated with succinate (20 mM), succinate + 1% dimethylsulfoxide, succinate + malonate (8 mM) (competitive inhibitor of SDH), or succinate + diazoxide (100 μM)., Results: Both malonate and diazoxide inhibit SDH activity in mitochondria of wild-type mice and in mice lacking the SUR1 subunit (p < 0.05 vs control)., Conclusions: The ability of DZX to inhibit SDH persists even after deletion of the SUR1 gene. Therefore, the enzyme complex SDH is not dependent on the SUR1 gene. The inhibition of SDH by DZX can play a role in the cardioprotection afforded by DZX; however, this role is independent of the ATP-sensitive potassium channel subunit SUR1., (Copyright © 2013 American College of Surgeons. Published by Elsevier Inc. All rights reserved.)

K(ATP) channels link cell metabolism to excitability in many cells. They are formed as tetramers of Kir6.2 subunits, each associated with a SUR1 subunit. We used mutant GFP-based FRET to assess domain organization in channel complexes. Full-length Kir6.2 subunits were linked to YFP or cyan fluorescent protein (CFP) at N or C termini, and all such constructs, including double-tagged YFP-Kir6.2-CFP (Y6.2C), formed functional K(ATP) channels. In intact COSm6 cells, background emission of YFP excited by 430-nm light was ∼6%, but the Y6.2C construct expressed alone exhibited an apparent FRET efficiency of ∼25%, confirmed by trypsin digestion, with or without SUR1 co-expression. Similar FRET efficiency was detected in mixtures of CFP- and YFP-tagged full-length Kir6.2 subunits and transmembrane domain only constructs, when tagged at the C termini but not at the N termini. The FRET-reported Kir6.2 tetramer domain organization was qualitatively consistent with Kir channel crystal structures: C termini and M2 domains are centrally located relative to N termini and M1 domains, respectively. Additional FRET analyses were performed on cells in which tagged full-length Kir6.2 and tagged SUR1 constructs were co-expressed. These analyses further revealed that 1) NBD1 of SUR1 is closer to the C terminus of Kir6.2 than to the N terminus; 2) the Kir6.2 cytoplasmic domain is not essential for complexation with SUR1; and 3) the N-terminal half of SUR1 can complex with itself in the absence of either the C-terminal half or Kir6.2.

Chloroquine and related compounds can inhibit inwardly rectifying potassium channels by multiple potential mechanisms, including pore block and allosteric effects on channel gating. Motivated by reports that chloroquine inhibition of cardiac ATP-sensitive inward rectifier K(+) current (I(KATP)) is antifibrillatory in rabbit ventricle, we investigated the mechanism of chloroquine inhibition of ATP-sensitive potassium (K(ATP)) channels (Kir6.2/SUR2A) expressed in human embryonic kidney 293 cells, using inside-out patch-clamp recordings. We found that chloroquine inhibits the Kir6.2/SUR2A channel by interacting with at least two different sites and by two mechanisms of action. A fast-onset effect is observed at depolarized membrane voltages and enhanced by the N160D mutation in the central cavity, probably reflecting direct channel block resulting from the drug entering the channel pore from the cytoplasmic side. Conversely, a slow-onset, voltage-independent inhibition of I(KATP) is regulated by chloroquine interaction with a different site and probably involves disruption of interactions between Kir6.2/SUR2A and phosphatidylinositol 4,5-bisphosphate. Our findings reveal multiple mechanisms of K(ATP) channel inhibition by chloroquine, highlighting the numerous convergent regulatory mechanisms of these ligand-dependent ion channels.

The sulfonylurea receptor SUR1 associates with Kir6.2 or Kir6.1 to form K(ATP) channels, which link metabolism to excitability in multiple cell types. The strong physical coupling of SUR1 with Kir6 subunits appears exclusive, but recent studies argue that SUR1 also modulates TRPM4, a member of the transient receptor potential family of non-selective cation channels. It has been reported that, following stroke, brain, or spinal cord injury, SUR1 is increased in neurovascular cells at the site of injury. This is accompanied by up-regulation of a non-selective cation conductance with TRPM4-like properties and apparently sensitive to sulfonylureas, leading to the postulation that post-traumatic non-selective cation currents are determined by TRPM4/SUR1 channels. To investigate the mechanistic hypothesis for the coupling between TRPM4 and SUR1, we performed electrophysiological and FRET studies in COSm6 cells expressing TRPM4 channels with or without SUR1. TRPM4-mediated currents were Ca(2+)-activated, voltage-dependent, underwent desensitization, and were inhibited by ATP but were insensitive to glibenclamide and tolbutamide. These properties were not affected by cotransfection with SUR1. When the same SUR1 was cotransfected with Kir6.2, functional K(ATP) channels were formed. In cells cotransfected with Kir6.2, SUR1, and TRPM4, we measured K(ATP)-mediated K(+) currents and Ca(2+)-activated, sulfonylurea-insensitive Na(+) currents in the same patch, further showing that SUR1 controls K(ATP) channel activity but not TRPM4 channels. FRET signal between fluorophore-tagged TRPM4 subunits was similar to that between Kir6.2 and SUR1, whereas there was no detectable FRET efficiency between TRPM4 and SUR1. Our data suggest that functional or structural association of TRPM4 and SUR1 is unlikely.

KATP channels regulate insulin secretion by coupling β-cell metabolism to membrane excitability. These channels are comprised of a pore-forming Kir6.2 tetramer which is enveloped by four regulatory SUR1 subunits. ATP acts on Kir6.2 to stabilize the channel closed state while ADP (coordinated with Mg(2+)) activates channels via the SUR1 domains. Aberrations in nucleotide-binding or in coupling binding to gating can lead to hyperinsulinism or diabetes. Here, we report a case of diabetes in a 7-mo old child with compound heterozygous mutations in ABCC8 (SUR1[A30V] and SUR1[G296R]). In unison, these mutations lead to a gain of KATP channel function, which will attenuate the β-cell response to increased metabolism and will thereby decrease insulin secretion. (86)Rb(+) flux assays on COSm6 cells coexpressing the mutant subunits (to recapitulate the compound heterozygous state) show a 2-fold increase in basal rate of (86)Rb(+) efflux relative to WT channels. Experiments on excised inside-out patches also reveal a slight increase in activity, manifested as an enhancement in stimulation by MgADP in channels expressing the compound heterozygous mutations or homozygous G296R mutation. In addition, the IC 50 for ATP inhibition of homomeric A30V channels was increased ~6-fold, and was increased ~3-fold for both heteromeric A30V+WT channels or compound heterozygous (A30V +G296R) channels. Thus, each mutation makes a mechanistically distinct contribution to the channel gain-of-function that results in neonatal diabetes, and which we predict may contribute to diabetes in related carrier individuals.

Murine ventricular and atrial ATP-sensitive potassium (K(ATP)) channels contain different sulfonylurea receptors (ventricular K(ATP) channels are Kir6.2/SUR2A complexes, while atrial K(ATP) channels are Kir6.2/SUR1 complexes). HMR 1098, the sodium salt of HMR 1883 {1-[[5-[2-(5-chloro-o-anisamido)ethyl]-2-methoxyphenyl]sulfonyl]-3-methylthiourea}, has been considered as a selective sarcolemmal (i.e. SUR2A-dependent) K(ATP) channel inhibitor. However, it is not clear whether HMR 1098 would preferentially inhibit ventricular K(ATP) channels over atrial K(ATP) channels. To test this, we used whole-cell patch clamp techniques on mouse atrial and ventricular myocytes as well as (86)Rb(+) efflux assays and excised inside-out patch clamp techniques on Kir6.2/SUR1 and Kir6.2/SUR2A channels heterologously expressed in COSm6 cells. In mouse atrial myocytes, both spontaneously activated and diazoxide-activated K(ATP) currents were effectively inhibited by 10 μM HMR 1098. By contrast, in ventricular myocytes, pinacidil-activated K(ATP) currents were inhibited by HMR 1098 at a high concentration (100 μM) but not at a low concentration (10 μM). Consistent with this finding, HMR 1098 inhibits (86)Rb(+) effluxes through Kir6.2/SUR1 more effectively than Kir6.2/SUR2A channels in COSm6 cells. In excised inside-out patches, HMR 1098 inhibited Kir6.2/SUR1 channels more effectively, particularly in the presence of MgADP and MgATP (mimicking physiological stimulation). Finally, dose-dependent enhancement of insulin secretion from pancreatic islets and decrease of blood glucose level confirm that HMR 1098 is an inhibitor of Kir6.2/SUR1-composed K(ATP) channels., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Objective: Animal and human myocytes demonstrate significant swelling and reduced contractility during exposure to stress (metabolic inhibition, hyposmotic stress, or hyperkalemic cardioplegia), and these detrimental consequences may be inhibited by the addition of diazoxide (adenosine triphosphate-sensitive potassium channel opener) via an unknown mechanism. Both SUR1 and SUR2A subunits have been localized to the heart, and mouse sarcolemmal adenosine triphosphate-sensitive potassium channels are composed of SUR2A/Kir6.2 subunits in the ventricle and SUR1/Kir6.2 subunits in the atria. This study was performed to localize the mechanism of diazoxide by direct probing of sarcolemmal adenosine triphosphate-sensitive potassium channel current and by genetic deletion of channel subunits., Methods: Sarcolemmal adenosine triphosphate-sensitive potassium channel current was recorded in isolated wild-type ventricular mouse myocytes during exposure to Tyrode's solution, Tyrode's + 100 μmol/L diazoxide, hyperkalemic cardioplegia, cardioplegia + diazoxide, cardioplegia + 100 μmol/L pinacidil, or metabolic inhibition using whole-cell voltage clamp (N = 7-12 cells per group). Ventricular myocyte volume was measured from SUR1(-/-) and wild-type mice during exposure to control solution, hyperkalemic cardioplegia, or cardioplegia + 100 μmol/L diazoxide (N = 7-10 cells per group)., Results: Diazoxide did not increase sarcolemmal adenosine triphosphate-sensitive potassium current in wild-type myocytes, although they demonstrated significant swelling during exposure to cardioplegia that was prevented by diazoxide. SUR1(-/-) myocytes also demonstrated significant swelling during exposure to cardioplegia, but this was not altered by diazoxide., Conclusions: Diazoxide does not open the ventricular sarcolemmal adenosine triphosphate-sensitive potassium channel but provides volume homeostasis via an SUR1-dependent pathway in mouse ventricular myocytes, supporting a mechanism of action distinct from sarcolemmal adenosine triphosphate-sensitive potassium channel activation., (Copyright © 2010 The American Association for Thoracic Surgery. Published by Mosby, Inc. All rights reserved.)

Buffering of blood pressure during change of posture such as standing is controlled largely by the baroreflex. In our population-based Victorian Family Heart Study (VFHS), we previously demonstrated that, on average, systolic blood pressure (SBP) changes very little on standing; however, interindividual variation is substantial and shows familial aggregation, with approximately 25% of the variance attributable to genetic factors. Our genomewide linkage analysis suggests a region on chromosome 12p that harbors two strong candidate genes, KCNJ8 and ABCC9, encoding the channel-forming inward rectifier subunit Kir6.1 and the ATP-sensitive binding cassette SUR2B, respectively. These are key components of smooth muscle ATP-sensitive potassium (K(ATP)) channels, important regulators of arterial tone and blood flow and central to autonomic baroreceptor control of changes in total peripheral resistance. We performed a comprehensive association analysis of 47 tag single nucleotide polymorphisms (SNPs) spanning the KCNJ8 and ABCC9 gene regions with postural change in SBP (DeltaSBP). To augment power, we took a selective genotyping approach in which we compared allele and genotype frequencies between 150 unrelated individuals with high (positive) DeltaSBP (> or = 7 mmHg) and 150 unrelated individuals with low (negative) DeltaSBP (< or = -7 mmHg) drawn from the offspring generation (18-30 yr) of the VFHS. Association analyses showed that no SNPs demonstrated statistically significant differences in genotype frequencies between groups, particularly after adjustments for multiple testing. We conclude that sequence variants in KCNJ8 and ABCC9 are unlikely to contribute to variation in DeltaSBP. Other genes in the identified chromosome 12p region warrant investigation.

K(ATP) channels are generally cardioprotective under conditions of metabolic impairment, consisting of pore-forming (Kir6.1 and/or Kir6.2) and sulphonylurea-binding, modulatory subunits [sulfonylurea receptor (SUR) 1, 2A, or 2B]. Cardiovascular K(ATP) channels are generally thought to consist of Kir6.2/SUR2A subunits (in the case of heart muscle) or Kir6.1/SUR2B subunits (smooth muscle), whereas SUR1-containing channels have well-documented roles in pancreatic insulin release. Recent data, however, demonstrated the presence of SUR1 subunits in mouse cardiac tissue (particularly in atria) and a surprising protection from myocardial ischemia/reperfusion in SUR1-null mice. Here, we review some of the extra-pancreatic roles assigned to SUR1 subunits and consider whether these might be involved in the sequelae of ischemia/reperfusion.

The isoform-specific structure of the ATP-sensitive potassium (K(ATP)) channel endows it with differential fundamental properties, including physiological activation and pharmacology. Numerous studies have convincingly demonstrated that the pore-forming Kir6.2 (KCNJ11) and regulatory SUR2A (ABCC9) subunits are essential elements of the sarcolemmal K(ATP) channel in cardiac ventricular myocytes. Using a novel antibody directed against the COOH terminus of SUR1 (ABCC8), we show that this K(ATP) subunit is also expressed in mouse myocardium and is the dominant SUR isoform in the atrium. This suggests differential sarcolemmal K(ATP) composition in atria and ventricles, and, to test this, K(ATP) currents were measured in isolated atrial and ventricular myocytes from wild-type and SUR1(-/-) animals. K(ATP) conductance is essentially abolished in SUR1(-/-) atrial myocytes but is normal in SUR1(-/-) ventricular myocytes. Furthermore, pharmacological properties of wild-type atrial K(ATP) match closely the properties of heterologously expressed SUR1/Kir6.2 channels, whereas ventricular K(ATP) properties match those of heterologously expressed SUR2A/Kir6.2 channels. Collectively, the data demonstrate a previously unappreciated K(ATP) channel heterogeneity: SUR1 is an essential component of atrial, but not ventricular, K(ATP) channels. Differential molecular make-up of the 2 channels underlies differential pharmacology, with important implications when considering sulfonylurea therapy or dissecting the role of cardiac K(ATP) pharmacologically, as well as for understanding of the role of diazoxide in preconditioning.

ATP-sensitive K(+) (K(ATP)) channels modulate their activity as a function of inhibitory ATP and stimulatory Mg-nucleotides. They are constituted by two proteins: a pore-forming K(+) channel subunit (Kir6.1, Kir6.2) and a regulatory sulfonylurea receptor (SUR) subunit, an ATP-binding cassette (ABC) transporter that confers MgADP stimulation to the channel. Channel regulation by MgADP is dependent on nucleotide interaction with the cytoplasmic nucleotide binding folds (NBF1 and NBF2) of the SUR subunit. Crystal structures of bacterial ABC proteins indicate that NBFs form as dimers, suggesting that NBF1-NBF2 heterodimers may form in SUR and other eukaryotic ABC proteins. We have modeled SUR1 NBF1 and NBF2 as a heterodimer, and tested the validity of the predicted dimer interface by systematic mutagenesis. Engineered cysteine mutations in this region have significant effects, both positive and negative, on MgADP stimulation of K(ATP) channels in excised patches and on macroscopic channel activity in intact cells. Additionally, the mutations cluster in the model structure according to their functional effect, such that patterns of alteration emerge. Of note, three gain-of-function mutations, leading to MgADP hyperstimulation of the channel, are located in the D-loop region at the center of the predicted dimer interface. Overall, the data support the idea that SUR1 NBFs assemble as heterodimers and that this interaction is functionally critical.

Sarcolemmal ATP-sensitive potassium channels (K(ATP)) act as metabolic sensors that facilitate adaptation of the left ventricle to changes in energy requirements. This study examined the mechanism by which K(ATP) dysfunction impairs the left ventricular response to stress using transgenic mouse strains with cardiac-specific disruption of K(ATP) activity (SUR1-tg mice) or Kir6.2 gene deficiency (Kir6.2 KO). Both SUR1-tg and Kir6.2 KO mice had normal left ventricular mass and function under unstressed conditions. Following chronic transverse aortic constriction, both SUR1-tg and Kir6.2 KO mice developed more severe left ventricular hypertrophy and dysfunction as compared with their corresponding WT controls. Both SUR1-tg and Kir6.2 KO mice had significantly decreased expression of peroxisome proliferator-activated receptor gamma coactivator (PGC)-1alpha and a group of energy metabolism related genes at both protein and mRNA levels. Furthermore, disruption of K(ATP) repressed expression and promoter activity of PGC-1alpha in cultured rat neonatal cardiac myocytes in response to hypoxia, indicating that K(ATP) activity is required to maintain PGC-1alpha expression under stress conditions. PGC-1alpha gene deficiency also exacerbated chronic transverse aortic constriction-induced ventricular hypertrophy and dysfunction, suggesting that depletion of PGC-1alpha can worsen systolic overload induced ventricular dysfunction. Both SUR1-tg and Kir6.2 KO mice had decreased FOXO1 after transverse aortic constriction, in agreement with the reports that a decrease of FOXO1 can repress PGC-1alpha expression. Furthermore, inhibition of K(ATP) caused a decrease of FOXO1 associated with PGC-1alpha promoter. These data indicate that K(ATP) channels facilitate the cardiac response to stress by regulating PGC-1alpha and its target genes, at least partially through the FOXO1 pathway.

Sulfonylurea receptors (SURs) associate with Kir6.x subunits to form tetradimeric K(ATP) channel complexes. SUR1 and SUR2 confer differential channel sensitivities to nucleotides and pharmacological agents, and are expressed in specific, but overlapping, tissues. This raises the question of whether these different SUR subtypes can assemble in the same channel complex and generate channels with hybrid properties. To test this, we engineered dimeric constructs of wild type or N160D mutant Kir6.2 fused to SUR1 or SUR2A. Dimeric fusions formed functional, ATP-sensitive, channels. Coexpression of weakly rectifying SUR1-Kir6.2 (WTF-1) with strongly rectifying SUR1-Kir6.2[N160D] (NDF-1) in COSm6 cells results in mixed subunit complexes that exhibit unique rectification properties. Coexpression of NDF-1 and SUR2A-Kir6.2 (WTF-2) results in similar complex rectification, reflecting the presence of SUR1- and SUR2A-containing dimers in the same channel. The data demonstrate clearly that SUR1 and SUR2A subunits associate randomly, and suggest that heteromeric channels will occur in native tissues.

ATP-sensitive K(+) (K(ATP)) channels are oligomeric complexes of pore-forming Kir6 subunits and regulatory Sulfonylurea Receptor (SUR) subunits. SUR, an ATP-Binding Cassette (ABC) transporter, confers Mg-nucleotide stimulation to the channel via nucleotide interactions with its two cytoplasmic domains (Nucleotide Binding Folds 1 and 2; NBF1 and NBF2). Regulation of K(ATP) channel expression is a complex process involving subunit assembly in the ER, SUR glycosylation in the Golgi, and trafficking to the plasma membrane. Dysregulation can occur at different steps of the pathway, as revealed by disease-causing mutations. Here, we have addressed the role of SUR1 NBF1 in gating and expression of reconstituted channels. Deletion of NBF1 severely impairs channel expression and abolishes MgADP stimulation. Total SUR1 protein levels are decreased, suggestive of increased protein degradation, but they are not rescued by treatment with sulfonylureas or the proteasomal inhibitor MG-132. Similar effects of NBF1 deletion are observed in recombinant K(ATP) channels obtained by "splitting" SUR1 into two separate polypeptides (a N-terminal "half" and a C-terminal "half"). Interestingly, the location of the "splitting point" in the vicinity of NBF1 has marked effects on the MgADP stimulation of resulting channels. Finally, ablation of the ER retention motif upstream of NBF1 (in either "split" or full-length SUR1) does not rescue expression of channels lacking NBF1. These results indicate that, in addition to NBF1 being required for MgADP stimulation of the channel, it plays an important role in the regulation of channel expression that is independent of the ER retention checkpoint and the proteasomal degradation pathway.

Objective: We sought to examine the molecular mechanisms underlying permanenent neonatal diabetes mellitus (PNDM) in a patient with a heterozygous de novo L225P mutation in the L0 region of the sulfonylurea receptor (SUR)1, the regulatory subunit of the pancreatic ATP-sensitive K(+) channel (K(ATP) channel)., Research Design and Methods: The effects of L225P on the properties of recombinant K(ATP) channels in transfected COS cells were assessed by patch-clamp experiments on excised membrane patches and by macroscopic Rb-flux experiments in intact cells., Results: L225P-containing K(ATP) channels were significantly more active in the intact cell than in wild-type channels. In excised membrane patches, L225P increased channel sensitivity to stimulatory Mg nucleotides without altering intrinsic gating or channel inhibition by ATP in the absence of Mg(2+). The effects of L225P were abolished by SUR1 mutations that prevent nucleotide hydrolysis at the nucleotide binding folds. L225P did not alter channel inhibition by sulfonylurea drugs, and, consistent with this, the patient responded to treatment with oral sulfonylureas., Conclusions: L225P underlies K(ATP) channel overactivity and PNDM by specifically increasing Mg-nucleotide stimulation of the channel, consistent with recent reports of mechanistically similar PNDM-causing mutations in SUR1. The mutation does not affect sulfonylurea sensitivity, and the patient is successfully treated with sulfonylureas.

Mutations in the pancreatic ATP-sensitive K(+) channel (K(ATP) channel) cause permanent neonatal diabetes mellitus (PNDM) in humans. All of the K(ATP) channel mutations examined result in decreased ATP inhibition, which in turn is predicted to suppress insulin secretion. Here we describe a patient with severe PNDM, which includes developmental delay and epilepsy, in addition to neonatal diabetes (developmental delay, epilepsy, and neonatal diabetes [DEND]), due to a G334D mutation in the Kir6.2 subunit of K(ATP) channel. The patient was wholly unresponsive to sulfonylurea therapy (up to 1.14 mg . kg(-1) . day(-1)) and remained insulin dependent. Consistent with the putative role of G334 as an ATP-binding residue, reconstituted homomeric and mixed WT+G334D channels exhibit absent or reduced ATP sensitivity but normal gating behavior in the absence of ATP. In disagreement with the sulfonylurea insensitivity of the affected patient, the G334D mutation has no effect on the sulfonylurea inhibition of reconstituted channels in excised patches. However, in macroscopic rubidium-efflux assays in intact cells, reconstituted mutant channels do exhibit a decreased, but still present, sulfonylurea response. The results demonstrate that ATP-binding site mutations can indeed cause DEND and suggest the possibility that sulfonylurea insensitivity of such patients may be a secondary reflection of the presence of DEND rather than a simple reflection of the underlying molecular basis.

The lack of pathological consequences of cardiac ATP-sensitive potassium channel (K(ATP)) channel gene manipulation is in stark contrast to the effect of similar perturbations in the pancreatic beta-cell. Because the pancreatic and cardiac channel share the same pore-forming subunit (Kir6.2), the different effects of genetic manipulation likely reflect, at least in part, the tissue-specific expression of the regulatory subunit (SUR1 in pancreas vs. SUR2A in heart) of the bipartite channel complex. To examine this, we have generated transgenic (TG) mice that overexpress epitope-tagged SUR1 or SUR2A under the transcriptional control of the alpha-myosin heavy chain promoter. Western blot and real time RT-PCR analysis confirm transgene expression in the heart, and variable levels of SUR1 RNA and protein, in 16 viable founder lines. Surprisingly, activation of channels by either pharmacological agents (diazoxide and pinacidil) or metabolic inhibitors (oligomycin and 2-deoxyglucose) reveals a suppression of total K(ATP) conductance in high expressing TG mice. Moreover, K(ATP) channel activity was significantly reduced in excised cardiac patches from TG myocytes that overexpress either SUR1 or SUR2A. Using a recombinant cell system, we show that overexpression of either SUR1 or Kir6.2 suppresses the functional expression of K(ATP) from optimized dimeric SUR1-Kir6.2. Thus, the graded effect of SUR1 expression in the intact heart appears to demonstrate an in vivo requirement for 1:1 expression ratio of Kir6.2 and SURx.

ATP-sensitive potassium (K(ATP)) channels are heterooctamers of an inward rectifier potassium channel (Kir6) and a sulfonylurea receptor (SUR, a member of the ATP-binding cassette (ABC) transporter family). In the pancreatic beta-cell K(ATP) channels are dynamically active, and transgenic expression of overactive Kir6.2 mutants leads to severe neonatal diabetes and death, while in the ventricular cardiomyocyte they are closed except under conditions of severe metabolic inhibition, and similarly overactive transgenes are without gross phenotypic consequence. This discrepancy may arise in part from differences at the molecular level between the two SUR isotypes that constitute the regulatory subunit of the K(ATP) channel in those tissues: SUR1 in the pancreas, SUR2A in the heart. K(ATP) channels generated from coexpression of Kir6.2 with SUR1 exhibit greater MgADP stimulation than channels generated from coexpression of Kir6.2 with SUR2A. This difference persists when the open state stability of the channel is enhanced by application of PIP(2), consistent with each isotype transducing an intrinsically different energetic contribution to the channel pore. When expressed as isolated, affinity-purified protein constructs, NBF2 of SUR1 exhibits increased in vitro ATP hydrolysis compared to NBF2 of SUR2A. This biochemical difference may underlie the increased MgADP stimulation exhibited by SUR1-containing channels vs. SUR2A-containing channels, which may in turn contribute to physiological differences, observed at the tissue level, between pancreatic and cardiac K(ATP) channels.

The ATP-sensitive potassium channel, K(ATP) channel, a functional complex of the sulfonylurea receptor 1, SUR1, and an inward rectifier potassium channel subunit, Kir6.2, regulates insulin secretion in the pancreas. Mutations in both the Kir6.2 and SUR1 genes are associated with persistent hyperinsulinemic hypoglycemia of infancy (PHHI), a disorder of pancreatic beta-cell function characterized by excess insulin secretion and hypoglycemia. We have studied the functional properties of novel SUR1 mutations identified in PHHI patients, including H125Q, N188S, F591L, T1139M, R1215Q, G1382S, and R1394H. R1394H and deltaF1388 SUR1, a previously identified PHHI mutation, resulted in no functional channels when coexpressed with Kir6.2 in COS cells, while H125Q, N188S, F591L, T1139M, R1215Q, and G1382S SUR1 generated functional channels in the absence of ATP. With the exception of N188S and H125Q, all mutants had reduced response to stimulation by MgADP. These results indicate that lack of, or reduction of, K(ATP) channel sensitivity to MgADP is a common molecular defect associated with the disease. The mutant channels also showed varied response to activation by the potassium channel opener diazoxide. Because these mutations are distributed throughout the molecule, our data have new implications for structure-function relationships of the K(ATP) channel, suggesting that structural elements in SUR1 outside of the two nucleotide-binding folds are also important in regulating channel activity.

ATP-sensitive potassium (KATP) channels link cellular metabolism to electrical activity in nerve, muscle, and endocrine tissues. They are formed as a functional complex of two unrelated subunits-a member of the Kir inward rectifier potassium channel family, and a sulfonylurea receptor (SUR), a member of the ATP-binding cassette transporter family, which includes cystic fibrosis transmembrane conductance regulators and multidrug resistance protein, regulators of chloride channel activity. This recent discovery has brought together proteins from two very distinct superfamilies in a novel functional complex. The pancreatic KATP channel is probably formed specifically of Kir6.2 and SUR1 isoforms. The relationship between SUR1 and Kir6.2 must be determined to understand how SUR1 and Kir6.2 interact to form this unique channel. We have used mutant Kir6.2 subunits and dimeric (SUR1-Kir6.2) constructs to examine the functional stoichiometry of the KATP channel. The data indicate that the KATP channel pore is lined by four Kir6.2 subunits, and that each Kir6.2 subunit requires one SUR1 subunit to generate a functional channel in an octameric or tetradimeric structure.

KATP channels were reconstituted in COSm6 cells by coexpression of the sulfonylurea receptor SUR1 and the inward rectifier potassium channel Kir6.2. The role of the two nucleotide binding folds of SUR1 in regulation of KATP channel activity by nucleotides and diazoxide was investigated. Mutations in the linker region and the Walker B motif (Walker, J.E., M.J. Saraste, M.J. Runswick, and N.J. Gay. 1982. EMBO [Eur. Mol. Biol. Organ.] J. 1:945-951) of the second nucleotide binding fold, including G1479D, G1479R, G1485D, G1485R, Q1486H, and D1506A, all abolished stimulation by MgADP and diazoxide, with the exception of G1479R, which showed a small stimulatory response to diazoxide. Analogous mutations in the first nucleotide binding fold, including G827D, G827R, and Q834H, were still stimulated by diazoxide and MgADP, but with altered kinetics compared with the wild-type channel. None of the mutations altered the sensitivity of the channel to inhibition by ATP4-. We propose a model in which SUR1 sensitizes the KATP channel to ATP inhibition, and nucleotide hydrolysis at the nucleotide binding folds blocks this effect. MgADP and diazoxide are proposed to stabilize this desensitized state of the channel, and mutations at the nucleotide binding folds alter the response of channels to MgADP and diazoxide by altering nucleotide hydrolysis rates or the coupling of hydrolysis to channel activation.

Adenosine triphosphate (ATP)-sensitive potassium (KATP) channels couple the cellular metabolic state to electrical activity and are a critical link between blood glucose concentration and pancreatic insulin secretion. A mutation in the second nucleotide-binding fold (NBF2) of the sulfonylurea receptor (SUR) of an individual diagnosed with persistent hyperinsulinemic hypoglycemia of infancy generated KATP channels that could be opened by diazoxide but not in response to metabolic inhibition. The hamster SUR, containing the analogous mutation, had normal ATP sensitivity, but unlike wild-type channels, inhibition by ATP was not antagonized by adenosine diphosphate (ADP). Additional mutations in NBF2 resulted in the same phenotype, whereas an equivalent mutation in NBF1 showed normal sensitivity to MgADP. Thus, by binding to SUR NBF2 and antagonizing ATP inhibition of KATP++ channels, intracellular MgADP may regulate insulin secretion.

Sulfonylureas are a class of drugs widely used to promote insulin secretion in the treatment of non-insulin-dependent diabetes mellitus. These drugs interact with the sulfonylurea receptor of pancreatic beta cells and inhibit the conductance of adenosine triphosphate (ATP)-dependent potassium (KATP) channels. Cloning of complementary DNAs for the high-affinity sulfonylurea receptor indicates that it is a member of the ATP-binding cassette or traffic ATPase superfamily with multiple membrane-spanning domains and two nucleotide binding folds. The results suggest that the sulfonylurea receptor may sense changes in ATP and ADP concentration, affect KATP channel activity, and thereby modulate insulin release.

We tested for the presence of sulfonylurea receptors in pancreatic alpha cells. Two high affinity sulfonylurea receptors were identified in clonal pancreatic alpha cells (alpha TC-6): a 140-kDa species observed previously in clonal pancreatic beta cells (HIT) and a second 150-kDa protein. The dissociation constant (Kd) for both receptors is approximately 3.5 nM for an iodinated glyburide analog, 5-iodo-2-hydroxyglyburide. The estimated number of receptors (Bmax) increases approximately 2-fold, from 3.1 to 6.8 pmol/mg of membrane protein as the pH of the binding buffer is reduced from 7.5 to 6. Consistent with the notion that high affinity sulfonylurea receptors are integral components of the ATP-sensitive K+ channel, we demonstrated the presence of ATP-sensitive K+ channels in inside-out patches of alpha TC-6 cells. Whole cell K+ currents that activated with time showed inward rectification at positive potentials (above 0 mV) and were almost completely suppressed by 5 nM glyburide. Likewise, glyburide blocked 86Rb+ efflux from ATP-depleted alpha TC-6 cells, an effect that was reversed by 400 microM diazoxide. The presence of sulfonylurea receptors provides a mechanism by which sulfonylureas can directly modulate alpha cell function. The properties of the 150-kDa receptor and the role of ATP-sensitive K+ channels in alpha cells remain to be elucidated, but as in beta cells, ATP-sensitive K+ channels may be involved in metabolic regulation of alpha cells by glucose.

Cell membranes isolated from hamster insulinoma (HIT T15) cells at passages 65-74 contain high and low affinity receptors for a sulfonylurea derivative, 5-[125I]iodo,2-hydroxyglyburide (KD values of approximately 7 nM and 16 microM). Between passages 75 and 85, the estimated B(max) for the high affinity receptor decreases approximately 10-fold from approximately 1.6 to 0.16 pmol/mg membrane protein. By contrast, the density of low affinity binding sites, 800-1000 pmol/mg, is unaltered. The drop in high affinity receptors is paralleled by a decrease in the density of KATP channels assessed using patch-clamp and 86Rb(+)-efflux techniques. These results strongly support the idea that the high affinity sulfonylurea receptor is an integral part of the KATP channel.