Biomedical subjects

Guy A Rutter

Publications and source records attributed to Guy A Rutter.

At least 37 records · Page 2Linked to original sources

PubMed2004

Inhibition of mitochondrial Na+-Ca2+ exchange restores agonist-induced ATP production and Ca2+ handling in human complex I deficiency.

Human mitochondrial complex I (NADH:ubiquinone oxidoreductase) of the oxidative phosphorylation system is a multiprotein assembly comprising both nuclear and mitochondrially encoded subunits. Deficiency of this complex is associated with numerous clinical syndromes ranging from highly progressive, often early lethal encephalopathies, of which Leigh disease is the most frequent, to neurodegenerative disorders in adult life, including Leber's hereditary optic neuropathy and Parkinson disease. We show here that the cytosolic Ca2+ signal in response to hormonal stimulation with bradykinin was impaired in skin fibroblasts from children between the ages of 0 and 5 years with an isolated complex I deficiency caused by mutations in nuclear encoded structural subunits of the complex. Inhibition of mitochondrial Na+-Ca2+ exchange by the benzothiazepine CGP37157 completely restored the aberrant cytosolic Ca2+ signal. This effect of the inhibitor was paralleled by complete restoration of the bradykinin-induced increases in mitochondrial Ca2+ concentration and ensuing ATP production. Thus, impaired mitochondrial Ca2+ accumulation during agonist stimulation is a major consequence of human complex I deficiency, a finding that may provide the basis for the development of new therapeutic approaches to this disorder.

Adenosine Triphosphate↗

PubMed2004

Ca2+-induced Ca2+ release in pancreatic islet beta-cells: critical evaluation of the use of endoplasmic reticulum-targeted "cameleons".

Elevated glucose concentrations cause Ca2+ influx and the exocytotic release of insulin from pancreatic islet beta-cells. Whether increases in cytosolic free Ca2+ concentration also mobilize Ca2+ from intracellular stores (Ca2+-induced Ca2+ release) is unresolved. Endoplasmic reticulum-targeted cameleons have previously been used to explore the involvement of endoplasmic reticulum (ER) Ca2+ release in these cells, albeit with differing conclusions. Cameleons comprise two spectrally shifted green fluorescent proteins, enhanced cyan and yellow fluorescent protein, whose orientation is affected by Ca2+, changing intramolecular fluorescence resonance energy transfer. By measuring pH in the cytosol and ER lumen, we demonstrate that high K+ concentrations (>20 mm) acidify both compartments in clonal MIN6 beta-cells when external bicarbonate concentrations are low (<5 mm), interfering with measurements using Ycam-2 and Ycam-4ER. However, when intracellular pH is strongly buffered (24 mm HCO3-), glucose or cell depolarization increases ER [Ca2+] monitored with Ycam-4ER. KCl-induced increases in ER [Ca2+] were diminished when intracellular stores were sensitized with 1 mm caffeine and inhibited by pretreatment with ryanodine. Furthermore, preincubation with ryanodine tended to slow the falling phase of the ER Ca2+ transient after cell depolarization with KCl and reduced the peak cytosolic [Ca2+]. By contrast, stimulation with glucose increased ER [Ca2+] both in the absence and presence of caffeine or ryanodine. These observations suggest that Ca2+-induced ER Ca2+ release can occur in beta-cells under some conditions but may not be essential for glucose-stimulated insulin secretion.

Animals↗

PubMed2004

Involvement of Per-Arnt-Sim (PAS) kinase in the stimulation of preproinsulin and pancreatic duodenum homeobox 1 gene expression by glucose.

Per-Arnt-Sim (PAS) domain-containing kinases are common in prokaryotes, but a mammalian counterpart has only recently been described. Although the PAS domain of the mammalian PAS kinase (PASK) is closely related to the bacterial oxygen sensor FixL, it is unclear whether PASK activity is changed in mammalian cells in response to nutrients and might therefore contribute to signal transduction by these or other stimuli. Here, we show that elevated glucose concentrations rapidly increase PASK activity in pancreatic islet beta cells, an event followed by the accumulation of both PASK mRNA and protein. Demonstrating a physiological role for PASK activation, comicroinjection into clonal beta cells of cDNA encoding wild-type PASK, or PASK protein itself, mimics the induction of preproinsulin promoter activity by high glucose concentrations. Conversely, anti-PASK antibodies block promoter activation by the sugar, and the silencing of PASK expression by RNA interference suppresses the up-regulation by glucose of preproinsulin and pancreatic duodenum homeobox 1 gene expression, without affecting glucose-induced changes in the levels of mRNAs encoding glucokinase or uncoupling protein 2. We conclude that PASK is an important metabolic sensor in nutrient-sensitive mammalian cells and plays an unexpected role in the regulation of key genes involved in maintaining the differentiated phenotype of pancreatic beta cells.

Adenosine Triphosphate↗

PubMed2004

Impact of PPARgamma overexpression and activation on pancreatic islet gene expression profile analyzed with oligonucleotide microarrays.

Peroxisome proliferator-activated receptor-gamma (PPARgamma) serves as a target for the thiazolidinedione class of antidiabetic drugs and is an important regulator of adipose tissue differentiation. By contrast, the principal target genes for PPARgamma in the pancreatic islet and the impact of their induction on insulin secretion are largely undefined. Here, we show that mRNAs encoding both isoforms of rodent PPARgamma, gamma1 and gamma2, are expressed in primary rat islets and are upregulated by overexpresssion of the lipogenic transcription factor sterol response element-binding protein 1c. Unexpectedly, however, oligonucleotide microarray analysis demonstrates that graded activation of PPARgamma achieved with 1) the thiazolidinedione GW-347845, 2) transduction with adenoviral PPARgamma1, or 3) a combination of both treatments progressively enhances the expression of genes involved in fatty acid oxidation and transport. Moreover, maximal activation of PPARgamma1 reduces islet triglyceride levels and enhances the oxidation of exogenous palmitate while decreasing glucose oxidation, cellular ATP content, and glucose-, but not depolarization-stimulated, insulin secretion. We conclude that, in the context of the pancreatic islet, the principal response to PPARgamma expression and activation is the activation of genes involved in the disposal, rather than the synthesis, of fatty acids. Although fatty acid oxidation may have beneficial effects on beta-cell function in the longer term by countering beta-cell "lipotoxicity," the acute response to this metabolic shift is a marked inhibition of insulin secretion.

Animals↗

PubMed2004

Identification of a Ras GTPase-activating protein regulated by receptor-mediated Ca2+ oscillations.

Receptor-mediated increases in the concentration of intracellular free calcium ([Ca2+]i) are responsible for controlling a plethora of physiological processes including gene expression, secretion, contraction, proliferation, neural signalling, and learning. Increases in [Ca2+]i often occur as repetitive Ca2+ spikes or oscillations. Induced by electrical or receptor stimuli, these repetitive Ca2+ spikes increase their frequency with the amplitude of the receptor stimuli, a phenomenon that appears critical for the induction of selective cellular functions. Here we report the characterisation of RASAL, a Ras GTPase-activating protein that senses the frequency of repetitive Ca2+ spikes by undergoing synchronous oscillatory associations with the plasma membrane. Importantly, we show that only during periods of plasma membrane association does RASAL inactivate Ras signalling. Thus, RASAL senses the frequency of complex Ca2+ signals, decoding them through a regulation of the activation state of Ras. Our data provide a hitherto unrecognised link between complex Ca2+ signals and the regulation of Ras.

Amino Acid Sequence↗

PubMed2004

Over-expression of sterol-regulatory-element-binding protein-1c (SREBP1c) in rat pancreatic islets induces lipogenesis and decreases glucose-stimulated insulin release: modulation by 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR).

Accumulation of intracellular lipid by pancreatic islet beta-cells has been proposed to inhibit normal glucose-regulated insulin secretion ('glucolipotoxicity'). In the present study, we determine whether over-expression in rat islets of the lipogenic transcription factor SREBP1c (sterol-regulatory-element-binding protein-1c) affects insulin release, and whether changes in islet lipid content may be reversed by activation of AMPK (AMP-activated protein kinase). Infection with an adenovirus encoding the constitutively active nuclear fragment of SREBP1c resulted in expression of the protein in approx. 20% of islet cell nuclei, with a preference for beta-cells at the islet periphery. Real-time PCR (TaqMan) analysis showed that SREBP1c up-regulated the expression of FAS (fatty acid synthase; 6-fold), acetyl-CoA carboxylase-1 (2-fold), as well as peroxisomal-proliferator-activated receptor-gamma (7-fold), uncoupling protein-2 (1.4-fold) and Bcl2 (B-cell lymphocytic-leukaemia proto-oncogene 2; 1.3-fold). By contrast, levels of pre-proinsulin, pancreatic duodenal homeobox-1, glucokinase and GLUT2 (glucose transporter isoform-2) mRNAs were unaltered. SREBP1c-transduced islets displayed a 3-fold increase in triacylglycerol content, decreased glucose oxidation and ATP levels, and a profound inhibition of glucose-, but not depolarisation-, induced insulin secretion. Culture of islets with the AMPK activator 5-amino-4-imidazolecarboxamide riboside decreased the expression of the endogenous SREBP1c and FAS genes, and reversed the effect of over-expressing active SREBP1c on FAS mRNA levels and cellular triacylglycerol content. We conclude that SREBP1c over-expression, even when confined to a subset of beta-cells, leads to defective insulin secretion from islets and may contribute to some forms of Type II diabetes.

Acetyltransferases↗

PubMed2004

Importin beta1 mediates the glucose-stimulated nuclear import of pancreatic and duodenal homeobox-1 in pancreatic islet beta-cells (MIN6).

The transcription factor PDX-1 (pancreatic and duodenal homeobox-1) is essential for pancreatic development and the maintainence of expression of islet beta-cell-specific genes. In an previous study [Rafiq, Kennedy and Rutter (1998) J. Biol. Chem. 273, 23241-23247] we demonstrated that PDX-1 may be activated at elevated glucose concentrations by translocation from undefined binding sites in the cytosol and nuclear membrane into the nucleoplasm. In the present study, we show that PDX-1 interacts directly and specifically in vitro with the nuclear import receptor family member, importin beta1, and that this interaction is mediated by the PDX-1 homeodomain (amino acids 146-206). Demonstrating the functional importance of the PDX-1-importin beta1 interaction, microinjection of MIN6 beta-cells with anti-(importin beta1) antibodies blocked both the nuclear translocation of PDX-1, and the activation by glucose (30 mM versus 3 mM) of the pre-proinsulin promoter. However, treatment with extracts from pancreatic islets incubated at either low or high glucose concentrations had no impact on the ability of PDX-1 to interact with importin beta1 in vitro. Furthermore, importin beta1 also interacted with SREBP1c (sterol-regulatory-element-binding protein 1c) in vitro, and microinjection of importin beta1 antibodies blocked the activation by glucose of SREBP1c target genes. Since the subcellular distribution of SREBP1c is unaffected by glucose, these findings suggest that a redistribution of importin beta1 is unlikely to explain the glucose-stimulated nuclear uptake of PDX-1. Instead, we conclude that the uptake of PDX-1 into the nucleoplasm, as glucose concentrations increase, may be mediated by release of the factor both from sites of retention in the cytosol and from non-productive complexes with importin beta1 at the nuclear membrane.

Active Transport, Cell Nucleus↗

PubMed2004

Metformin, but not leptin, regulates AMP-activated protein kinase in pancreatic islets: impact on glucose-stimulated insulin secretion.

Metformin, a drug widely used in the treatment of type 2 diabetes, has recently been shown to act on skeletal muscle and liver in part through the activation of AMP-activated protein kinase (AMPK). Whether metformin or the satiety factor leptin, which also stimulates AMPK in muscle, regulates this enzyme in pancreatic islets is unknown. We have recently shown that forced increases in AMPK activity inhibit insulin secretion from MIN6 cells (da Silva Xavier G, Leclerc I, Varadi A, Tsuboi T, Moule SK, and Rutter GA. Biochem J 371: 761-774, 2003). Here, we explore whether 1) glucose, metformin, or leptin regulates AMPK activity in isolated islets from rodent and human and 2) whether changes in AMPK activity modulate insulin secretion from human islets. Increases in glucose concentration from 0 to 3 and from 3 to 17 mM inhibited AMPK activity in primary islets from mouse, rat, and human, confirming previous findings in insulinoma cells. Incubation with metformin (0.2-1 mM) activated AMPK in both human islets and MIN6 beta-cells in parallel with an inhibition of insulin secretion, whereas leptin (10-100 nM) was without effect in MIN6 cells. These studies demonstrate that AMPK activity is subject to regulation by both glucose and metformin in pancreatic islets and clonal beta-cells. The inhibitory effects of metformin on insulin secretion may therefore need to be considered with respect to the use of this drug for the treatment of type 2 diabetes.

AMP-Activated Protein Kinases↗

PubMed2004

Kiss and run exocytosis of dense core secretory vesicles.

The mechanisms by which large, dense core, peptide-containing vesicles release their cargo from neuroendocrine cells has been the subject of intense debate during the past few years. Thanks to recent studies of the vesicle membrane post fusion, the view that exocytosis is obligatorily linked to the complete collapse of these vesicles at the cell surface is gradually being revised. We discuss here the evidence supporting a model in which (1) exocytosis and endocytosis of dense core vesicles are closely coupled, and (2) the release of peptides occurs from largely intact vesicles via the formation of a large, but transient, fusion pore.

Animals↗

PubMed2004

Distinct roles for insulin and insulin-like growth factor-1 receptors in pancreatic beta-cell glucose sensing revealed by RNA silencing.

The importance of the insulin receptor (IR) and the insulin-like growth factor-1 receptor (IGF-1R) for glucose-regulated insulin secretion and gene expression in pancreatic islet beta-cells is at present unresolved. Here, we have used small interfering RNAs (siRNAs) to silence the expression of each receptor selectively in clonal MIN6 beta-cells. Reduction of IR levels by >90% completely inhibited glucose (30 mM compared with 3 mM)-induced insulin secretion, but had no effect on depolarization-stimulated secretion. IR depletion also blocked the accumulation of preproinsulin (PPI), pancreatic duodenum homoeobox-1 (PDX-1) and glucokinase (GK) mRNAs at elevated glucose concentrations, as assessed by quantitative real-time PCR analysis (TaqMan). Similarly, depletion of IGF-1R inhibited glucose-induced insulin secretion but, in contrast with the effects of IR silencing, had little impact on the regulation of gene expression by glucose. Moreover, loss of IGF-1R, but not IR, markedly inhibited glucose-stimulated increases in cytosolic and mitochondrial ATP, suggesting a role for IGF-1R in the maintenance of oxidative metabolism and in the generation of mitochondrial coupling factors. RNA silencing thus represents a useful tool for the efficient and selective inactivation of receptor tyrosine kinases in isolated beta-cells. By inhibiting glucose-stimulated insulin secretion through the inactivation of IGF-1R, this approach also demonstrates the existence of insulin-independent mechanisms whereby elevated glucose concentrations regulate PPI, PDX-1 and GK gene expression in beta-cells.

Adenosine Triphosphate↗

PubMed2004

Mitochondrial localization as a determinant of capacitative Ca2+ entry in HeLa cells.

Whether different subsets of mitochondria play distinct roles in shaping intracellular Ca2+ signals is presently unresolved. Here, we determine the role of mitochondria located beneath the plasma membrane in controlling (a) Ca2+ release from the endoplasmic reticulum (ER) and (b) capacitative Ca2+ entry. By over-expression of the dynactin subunit dynamitin, and consequent inhibition of the fission factor, dynamin-related protein (Drp-1), mitochondria were relocalised from the plasma membrane towards the nuclear periphery in HeLa cells. The impact of these changes on free calcium concentration in the cytosol ([Ca2+]c), mitochondria ([Ca2+]m) and ER ([Ca2+]ER) was then monitored with specifically-targeted aequorins. Whilst dynamitin over-expression increased the number of close contacts between the ER and mitochondria by >2.5-fold, assessed using organelle-targeted GFP variants, histamine-induced changes in organellar [Ca2+] were unaffected. By contrast, Ca2+ influx elicited significantly smaller increases in [Ca2+]c and [Ca2+]m in dynamitin-expressing than in control cells. These data suggest that the strategic localisation of a subset of mitochondria beneath the plasma membrane is required for normal Ca2+ influx, but that the transfer of Ca2+ ions between the ER and mitochondria is relatively insensitive to gross changes in the spatial relationship between these two organelles.

Calcium↗

PubMed2004

Temperature-sensitive random insulin granule diffusion is a prerequisite for recruiting granules for release.

Glucose-evoked insulin secretion exhibits a biphasic time course and is associated with accelerated intracellular granule movement. We combined live confocal imaging of EGFP-labelled insulin granules with capacitance measurements of exocytosis in clonal INS-1 cells to explore the relation between distinct random and directed modes of insulin granule movement, as well as exocytotic capacity. Reducing the temperature from 34 degrees C to 24 degrees C caused a dramatic 81% drop in the frequency of directed events, but reduced directed velocities by a mere 25%. The much stronger temperature sensitivity of the frequency of directed events (estimated energy of activation approximately 135 kJ/mol) than that of the granule velocities (approximately 22 kJ/mol) suggests that cooling-induced suppression of insulin granule movement is attributable to factors other than reduced motor protein adenosine 5'-triphosphatase activity. Indeed, cooling suppresses random granule diffusion by approximately 50%. In the single cell, the number of directed events depends on the extent of granule diffusion. Finally, single-cell exocytosis exhibits a biphasic pattern corresponding to that observed in vivo, and only the component reflecting 2nd phase insulin secretion is affected by cooling. We conclude that random diffusive movement is a prerequisite for directed insulin granule transport and for the recruitment of insulin granules released during 2nd phase insulin secretion.

Animals↗

PubMed2004

Impaired glucose homeostasis in transgenic mice expressing the human transient neonatal diabetes mellitus locus, TNDM.

Transient neonatal diabetes mellitus (TNDM) is a rare inherited diabetic syndrome apparent in the first weeks of life and again during early adulthood. The relative contributions of reduced islet beta cell number and impaired beta cell function to the observed hypoinsulinemia are unclear. The inheritance pattern of this imprinted disorder implicates overexpression of one or both genes within the TNDM locus: ZAC, which encodes a proapoptotic zinc finger protein, and HYMAI, which encodes an untranslated mRNA. To investigate the consequences for pancreatic function, we have developed a high-copy transgenic mouse line, TNDM29, carrying the human TNDM locus. TNDM29 neonates display hyperglycemia, and older adults, impaired glucose tolerance. Neonatal hyperglycemia occurs only on paternal transmission, analogous to paternal dependence of TNDM in humans. Embryonic pancreata of TNDM29 mice showed reductions in expression of endocrine differentiation factors and numbers of insulin-staining structures. By contrast, beta cell mass was normal or elevated at all postnatal stages, whereas pancreatic insulin content in neonates and peak serum insulin levels after glucose infusion in adults were reduced. Expression of human ZAC and HYMAI in these transgenic mice thus recapitulates key features of TNDM and implicates impaired development of the endocrine pancreas and beta cell function in disease pathogenesis.

Aging↗

PubMed2004

Imaging glucose-regulated insulin secretion and gene expression in single islet beta-cells: control by AMP-activated protein kinase.

The mechanisms by which changes in glucose concentration regulate gene expression and insulin secretion in pancreatic islet beta-cells are only partly understood. Here we describe the development of new technologies for examining these processes at the level of single living beta-cells. We also present recent findings, made using these and other techniques, which implicate a role for adenosine 5'-monophosphate-activated protein kinase in glucose signaling in these cells.

AMP-Activated Protein Kinases↗

PubMed2004

Role for plasma membrane-related Ca2+-ATPase-1 (ATP2C1) in pancreatic beta-cell Ca2+ homeostasis revealed by RNA silencing.

Changes in intracellular Ca(2+) concentration play a key role in the regulation of insulin secretion by glucose and other secretagogues. Here, we explore the importance of the secretory pathway Ca(2+)-ATPase, plasma membrane-related Ca(2+)-ATPase-1 (PMR1; human orthologue ATP2C1) in intracellular Ca(2+) homeostasis in pancreatic islet beta-cells. Endogenous PMR1 mRNA and protein were detected in both isolated rat islets and beta-cell-derived lines (MIN6 and INS1). Subcellular fractionation of the cell lines revealed PMR1 immunoreactivity in both microsomal and dense-core secretory vesicle-enriched fractions. Correspondingly, depletion of cellular PMR1 with small interfering RNAs inhibited Ca(2+) uptake into the endoplasmic reticulum and secretory vesicles by approximately 20%, as assessed using organelle-targeted aequorins in permeabilized INS1 cells. In intact cells, PMR1 depletion markedly enhanced flux though L-type Ca(2+) channels and augmented glucose-stimulated, but not basal, insulin secretion. Whereas average cytosolic [Ca(2+)] increases in response to 30.0 mmol/l glucose were unaffected by PMR1 depletion, [Ca(2+)] oscillation shape, duration, and decay rate in response to glucose plus tetraethylammonium were modified in PMR1-depleted single cells, imaged using fluo-3-acetoxymethylester. PMR1 thus plays an important role, which is at least partially nonoverlapping with that of sarco(endo-)plasmic reticulum Ca(2+)-ATPases, in the control of beta-cell Ca(2+) homeostasis and insulin secretion.

ATP-Binding Cassette Transporters↗

PubMed2004

AMP-activated protein kinase: a new beta-cell glucose sensor?: Regulation by amino acids and calcium ions.

Stimulation of AMP-activated protein kinase (AMPK) in skeletal muscle and liver is seen as an exciting prospect for the treatment of type 2 diabetes. However, we have recently demonstrated that changes in AMPK activity accompany the exposure of pancreatic islet beta-cells to elevated glucose concentrations and may be involved in the activation of insulin secretion. Here, we discuss this hypothesis and explore the potential role of changes in AMPK activity in the actions of other secretagogues. Amino acids decreased AMPK activity in MIN6 beta-cells with an order of potency for inhibition: arg=leu < gln= leu + glu < glucose, which was closely correlated with the stimulation of insulin release (r2=0.76). By contrast, increases in intracellular Ca2+ concentration provoked by cell depolarization with KCl activated AMPK in the face of increased free intracellular ATP concentrations. Elevation of intracellular cAMP levels with isobutylmethylxanthine or forskolin had no effect on AMPK activity. We conclude that metabolizable amino acids regulate AMPK in the beta-cell via increases in the cytosolic ATP/AMP ratio and via phosphorylation by the upstream kinase LKB1. Intracellular Ca2+ ions may activate AMPK by calmodulin kinase 1 kinase-mediated phosphorylation. The latter may act as a novel feedback mechanism to inhibit excessive insulin secretion under some circumstances.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed2004

Impact of adenoviral transduction with SREBP1c or AMPK on pancreatic islet gene expression profile: analysis with oligonucleotide microarrays.

Accumulation of triglyceride in islets may contribute to the loss of glucose-stimulated insulin secretion (GSIS) in some forms of type 2 diabetes (Diraison et al., Biochem J 373:769-778, 2004). Here, we use adenoviral vectors and oligonucleotide microarrays to determine the effects of the forced expression of SREBP1c on the gene expression profile of rat islets. Sterol regulatory element binding protein-1c (SREBP1c) overexpression led to highly significant (P <0.1 with respect to null adenovirus) changes in the expression of 1,238 genes or expressed sequence tags, of which 1,180 (95.3%) were upregulated. By contrast, overexpression of constitutively active AMP-activated protein kinase (AMPK), expected to promote lipolysis, altered the expression of 752 genes, of which 702 (93%) were upregulated. To identify specific targets for SREBP1c or AMPK, we eliminated messages that were 1) affected in the same direction by the expression of either protein, 2) changed by less than twofold, or 3) failed a positive false discovery test; 206 SREBP1c-regulated genes (195; 95% upregulated) and 48 AMPK-regulated genes (33; 69% upregulated) remained. As expected, SREBP1c-induced genes included those involved in cholesterol (6), fatty acid (3), and eicosanoid synthesis. Interestingly, somatostatin receptor (sstr1) expression was increased by SREBP1c, whereas AMPK induced the expression of peptide YY, the early endocrine pancreas marker.

Adenoviridae↗

PubMed2003

ATP-dependent interaction of the cytosolic domains of the inwardly rectifying K+ channel Kir6.2 revealed by fluorescence resonance energy transfer.

ATP-sensitive K(+) (K(ATP)) channels play important roles in the regulation of membrane excitability in many cell types. ATP inhibits channel activity by binding to a specific site formed by the N and C termini of the pore-forming subunit, Kir6.2, but the structural changes associated with this interaction remain unclear. Here, we use fluorescence resonance energy transfer (FRET) to study the ATP-dependent interaction between the N and C termini of Kir6.2 using a construct bearing fused cyan and yellow fluorescent proteins (ECFP-Kir6.2-EYFP). When expressed in human embryonic kidney cells, ECFP-Kir6.2-EYFP/SUR1 channels displayed FRET that was augmented by agonist stimulation and diminished by metabolic poisoning. Addition of ATP to permeabilized cells or isolated plasma membrane sheets increased FRET. FRET changes were abolished by Kir6.2 mutations that altered ATP-dependent channel closure and channel gating. In the wild-type channel, the ATP concentrations, which increased FRET (EC(50) = 1.36 mM), were significantly higher than those causing channel inhibition (IC(50) = 0.29 mM). Demonstrating the existence of intermolecular interactions, a dimeric construct comprising two molecules of Kir6.2 linked head-to-tail (ECFP-Kir6.2-Kir6.2-EYFP) displayed less FRET than the monomer in the absence of nucleotide but still exhibited ATP-dependent FRET increases (EC(50) = 1.52 mM) and channel inhibition. We conclude that binding of ATP to Kir6.2, (i). alters the interaction between the N- and C-terminal domains, (ii). probably involves both intrasubunit and intersubunit interactions, (iii). reflects ligand binding not channel gating, and (iv). occurs in intact cells when subplasmalemmal [ATP] changes in the millimolar range.

Adenosine Diphosphate↗