Search

The newborn heart adapts to postnatal life by shifting from a fetal glycolytic metabolism to a mitochondrial oxidative metabolism. Abcc9, an ATP-binding cassette family member, increases expression concomitant with this metabolic shift. Abcc9 encodes a membrane-associated receptor that partners with a potassium channel to become the major potassium-sensitive ATP channel in the heart. Abcc9 also encodes a smaller protein enriched in the mitochondria. We now deleted exon 5 of Abcc9 to ablate expression of both plasma membrane and mitochondria-associated Abcc9-encoded proteins, and found that the myocardium failed to acquire normal mature metabolism, resulting in neonatal cardiomyopathy. Unlike wild-type neonatal cardiomyocytes, mitochondria from Ex5 cardiomyocytes were unresponsive to the KATP agonist diazoxide, consistent with loss of KATP activity. When exposed to hydrogen peroxide to induce cell stress, Ex5 neonatal cardiomyocytes displayed a rapid collapse of mitochondria membrane potential, distinct from wild-type cardiomyocytes. Ex5 cardiomyocytes had reduced fatty acid oxidation, reduced oxygen consumption and reserve. Morphologically, Ex5 cardiac mitochondria exhibited an immature pattern with reduced cross-sectional area and intermitochondrial contacts. In the absence of Abcc9, the newborn heart fails to transition normally from fetal to mature myocardial metabolism. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The ATP-sensitive potassium ( KATP) channel is a hetero-octameric complex that links cell metabolism to membrane electrical activity in many cells, thereby controlling physiological functions such as insulin release, muscle contraction and neuronal activity. It consists of four pore-forming Kir6.2 and four regulatory sulfonylurea receptor ( SUR) subunits. SUR2B serves as the regulatory subunit in smooth muscle and some neurones. An integrative approach, combining electron microscopy and homology modelling, has been used to obtain information on the structure of this large (megadalton) membrane protein complex. Single-particle electron microscopy of purified SUR2B tethered to a lipid monolayer revealed that it assembles as a tetramer of four SUR2B subunits surrounding a central hole. In the absence of an X-ray structure, a homology model for SUR2B based on the X-ray structure of the related ABC transporter Sav1866 was used to fit the experimental images. The model indicates that the central hole can readily accommodate the transmembrane domains of the Kir tetramer, suggests a location for the first transmembrane domains of SUR2B (which are absent in Sav1866) and suggests the relative orientation of the SUR and Kir6.2 subunits. Structured digital abstract SUR2B and SUR2B bind by electron microscopy (View interaction), SUR2B and SUR2B bind by molecular sieving (View interaction) [ABSTRACT FROM AUTHOR]

Being gated by high-energy nucleotides, cardiac ATP-sensitive potassium (KATP) channels are exquisitely sensitive to changes in cellular energy metabolism. An emerging view is that proteins associated with the KATP channel provide an additional layer of regulation. Using putative sulfonylurea receptor (SUR) coiled-coil domains as baits in a 2-hybrid screen against a rat cardiac cDNA library, we identified glycolytic enzymes (GAPDH and aldolase A) as putative interacting proteins. Interaction between aldolase and SUR was confirmed using GST pulldown assays and coimmunoprecipitation assays. Mass spectrometry of proteins from KATP channel immunoprecipitates of rat cardiac membranes identified glycolysis as the most enriched biological process. Coimmunoprecipitation assays confirmed interaction for several glycolytic enzymes throughout the glycolytic pathway. Immunocytochemistry colocalized many of these enzymes with KATP channel subunits in rat cardiac myocytes. The catalytic activities of aldolase and pyruvate kinase functionally modulate KATP channels in patch-clamp experiments, whereas D-glucose was without effect. Overall, our data demonstrate close physical association and functional interaction of the glycolytic process (particularly the distal ATP-generating steps) with cardiac KATP channels. [ABSTRACT FROM AUTHOR]

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 Km 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The ATP-sensitive potassium (KATP) channel couples glucose metabolism to insulin secretion in pancreatic β-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 ∼ 8 momomers. The ATPase activities expressed as maximal turnover rate [in nmol Pi·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 Km values (in mm) were 0.1, 0.6, 0.65 and 0.56, respectively. Thus sulfonylurea receptor 1 has a lower Km 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 Km 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 ( Ki 16 µm), 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. [ABSTRACT FROM AUTHOR]

Abstract: 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) β-cells by total internal reflection fluorescence microscopy. Sur1KO β-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 β-cells. [Copyright &y& Elsevier]

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. [ABSTRACT FROM AUTHOR]

Cardiac ATP-sensitive K+ (KATP) channels are proposed to contribute to cardio-protection and ischemic preconditioning. Although mRNAs for all subunits of KATP channels (Kir6.0 and sulfonylurea receptors SURs) were detected in hearts, subcellular localization of their proteins and the subunit combination are not well elucidated. We address these questions in rat hearts, using anti-peptide antibodies raised against each subunit. By immunoblot analysis, all of the subunits were detected in microsomal fractions including sarcolemmal membranes, while they were not detected in mitochondrial fractions at all. Immunoprecipitation and sucrose gradient sedimentation of the digitonin-solubilized microsomes indicated that Kir6.2 exclusively assembled with SUR2A. The molecular mass of the Kir6.2–SUR2A complex estimated by sucrose sedimentation was 1150 kDa, significantly larger than the calculated value for (Kir6.2)4–(SUR2A)4, suggesting a potential formation of micellar complex with digitonin but no indication of hybrid channel formation under the conditions. These findings provide additional information on the structural and functional relationships of cardiac KATP channel proteins involving subcellular localization and roles for cardioprotection and ischemic preconditioning. [Copyright &y& Elsevier]

The sulfonylurea receptor (SUR), an ATP-binding cassette (ABC) protein, assembles with a potassium channel subunit (Kir6) to form the ATP-sensitive potassium channel (KATP) complex. Although SUR is an important regulator of Kir6, the specific SUR domain that associates with Kir6 is stilt 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 electrophysiotogical 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 KATP 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. [ABSTRACT FROM AUTHOR]

ATP-sensitive K+ channels are an octameric assembly of two proteins, a sulfonylurea receptor (SUR1) and an ion conducting subunit (Kir 6.0). We have examined the role of the C-terminus of SUR1 by expressing a series of truncation mutants together with Kir6.2 stably in HEK293 cells. Biochemical analyses using coimmunoprecipitation indicate that SUR1 deletion mutants and Kir6.2 assemble and that a SUR1 deletion mutant binds glibenclamide with high affinity. Electrophysiological recordings indicate that ATP sensitivity is normal but the response of the mutant channel complexes to tolbutamide, MgADP and diazoxide is disturbed. Quantitative immunofluorescence and cell surface biotinylation supports the idea that there is little disturbance in the efficiency of trafficking. Our data show that deletions of the C-terminal most cytoplasmic domain of SUR1, can result in functional channels at the plasma membrane in mammalian cells that have an abnormal response to physiological and pharmacological agents. [ABSTRACT FROM AUTHOR]

ATP-sensitive potassium (KATP) channels are composed of an ATP-binding cassette (ABC) protein (SUR1, SUR2A or SUR2B) and an inwardly rectifying K+ channel (Kir6.1 or Kir6.2). Like other ABC proteins, the nucleotide binding domains (NBDs) of SUR contain a highly conserved 'signature sequence' (the tinker, LSGGQ) whose function is unclear. Mutation of the conserved serine to arginine in the tinker of NBD1 (S1R) or NBD2 (S2R) did not alter the ability of ATP or ADP (100 μM) to displace 8-azido-[32P]ATP binding to SUR1, or abolish ATP hydrolysis at NBD2. We co-expressed Kir6.2 with wild-type or mutant SUR in Xenopus oocytes and recorded the resulting currents in inside-out macropatches. The S1R mutation in SUR1, SUR2A or SUR2B reduced KATP current activation by 100 μM MgADP, whereas the S2R mutation in SUR1 or SUR2B (but not SUR2A) abolished MgADP activation completely. The tinker mutations also reduced (S1R) or abolished (S2R) MgATP-dependent activation of Kir6.2-RSOG co-expressed with SUR1 or SUR2B. These results suggest that the tinker serines are not required for nucteotide binding but may be involved in transducing nucleotide binding into channel activation. [ABSTRACT FROM AUTHOR]

KATP channels incorporate a regulatory subunit of the ATP-binding cassette (ABC) transporter family, the sulfonylurea receptor (SUR), which defines their pharmacology. The therapeutically important K+ channel openers (e.g. pinacidil, cromakalim, nicorandil) act specifically on the SUR2 muscle isoforms but, except for diazoxide, remain ineffective on the SUR1 neuronal/pancreatic isoform. This SUR1/2 dichotomy underpinned a chimeric strategy designed to identify the structural determinants of opener action, which led to a minimal set of two residues within the last transmembrane helix of SUR. Transfer of either residue from SUR2A to SUR1 conferred opener sensitivity to SUR1, while the reverse operation abolished SUR2A sensitivity. It is therefore likely that these residues form part of the site of interaction of openers with the channel Thus, openers would target a region that, in other ABC transporters, is known to be tightly involved with the binding of substrates and other ligands. This first glimpse of the site of action of pharmacological openers should permit rapid progress towards understanding the structural determinants of their affinity and specificity. [ABSTRACT FROM AUTHOR]

The KATP channel is a heterooctamer composed of two different subunits, four inwardly rectifying K+ channel subunits, either Kir 6.1 or Kir 6.2, and four sulfonylurea receptors (SUR), which belong to the family of ABC transporters. This unusual molecular architecture is related to the complex gating behaviour of these channels. Intracellular ATP inhibits KATP channels by binding to the Kir 6.x subunits, whereas Mg-ADP increases channel activity by a hydrolysis reaction at the SUR. This ATP/ADP dependence allows KATP channels to link metabolism to excitability, which is important for many physiological functions, such as insulin secretion and cell protection during periods of ischemic stress. Recent work has uncovered a new class of regulatory molecules for KATP channel gating. Membrane phospholipids such as phosphoinositol 4,5-bisphosphate and phosphatidylinositiol 4-monophosphate were found to interact with KATP channels resulting in increased open probability and markedly reduced ATP sensitivity. The membrane concentration of these phospholipids is regulated by a set of enzymes comprising phospholipases, phospholipid phosphatases and phospholipid kinases providing a possible mechanism for control of cell excitability through signal transduction pathways that modulate activity of these enzymes. This review discusses the mechanisms and molecular determinants that underlie gating of KATP channel by nucleotides and phospholipids and their physiological implications. [ABSTRACT FROM AUTHOR]

To determine the interaction site(s) of ATP-sensitive K+ (KATP) channels for G-proteins, sulfonylurea receptor (SUR2A or SUR1) and pore-forming (Kir6.2) subunits were reconstituted in the mammalian cell line, COS-7. Intracellular application of the G-protein βγ2-subunits (Gβγ2) caused a reduction of ATP-induced inhibition of Kir6.2/SUR channel activities by lessening the ATP sensitivity of the channels. Gβγ2 bound in vitro to both intracellular (loop-NBD) and C-terminal segments of SUR2A, each containing a nucleotide-binding domain (NBD). Furthermore, a single amino acid substitution in the loop-NBD of SUR (Arg656Ala in SUR2A or Arg665Ala in SUR1) abolished the Gβγ2-dependent alteration of the channel activities. These findings provide evidence that Gβγ modulates KATP channels through a direct interaction with the loop-NBD of SUR. [ABSTRACT FROM AUTHOR]

ATP-sensitive K+ (KATP) channels are inhibited by intracellular ATP and activated by intracellular nucleoside diphosphates, and thus provide a link between cellular metabolism and excitability. KATP channels are widely distributed in various tissues and may be associated with diverse cellular functions. In the heart, the KATP channel appears to be activated during ischemic or hypoxic conditions and may be responsible for the increase of K+ efflux and shortening of the action potential duration. Therefore, opening of this channel may result in cardioprotective as well as proarrhythmic effects. In the vascular smooth muscle, the KATP channel is believed to mediate the relaxation of vascular tone. Thus, KATP channels play important regulatory roles in the cardiovascular system. Furthermore, KATP channels are the targets of two important classes of drugs, i.e., the antidiabetic sulfonylureas, which block the channels, and a series of vasorelaxants called "K+ channel openers," ' which tend to maintain the channels in an open conformation. Recently, the molecular structure of KATP channels bas been clarified. The KATP channel in pancreatic β-cells is a complex composed of at least two subunits, a member of inwardly rectifying K+ channels and a sulfonylurea receptor. Subsequently, two additional homologs of the sulfonylurea receptor, which form cardiac and smooth muscle type KATP channels, respectively, have been reported. Further works are now in progress to understand the molecular mechanisms of KATP channel function. [ABSTRACT FROM AUTHOR]