A Kir6.2 Mutation Causing Neonatal Diabetes Impairs Electrical Activity and Insulin Secretion From INS-1 β-Cells

Tarasov, Andrei I.; Welters, Hannah J.; Senkel, Sabine; Ryffel, Gerhart U.; Hattersley, Andrew T.; Morgan, Noel G.; Ashcroft, Frances M.

doi:10.2337/db06-0637

Cited by 41 publications

(31 citation statements)

References 37 publications

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“…Our data show that mutations at R1380 have modest (Ͻ2-fold) effects on the rate of ATP hydrolysis, the ability of ATP to block the K ATP channel, and the resting whole-cell K ATP current. However, because the K ATP channel dominates the membrane potential of the beta cell, even a small increase in K ATP current can cause a hyperpolarization sufficient to inhibit electrical activity and insulin secretion (21). Previous studies of Kir6.2 mutations have shown a good correlation between the magnitude of the K ATP current at physiological levels of MgATP and the severity of the clinical phenotype.…”

Section: Discussionmentioning

confidence: 99%

Increased ATPase activity produced by mutations at arginine-1380 in nucleotide-binding domain 2 of ABCC8 causes neonatal diabetes

Wet

Rees

Shimomura

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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Gain-of-function mutations in the genes encoding the ATPsensitive potassium (K ATP) channel subunits Kir6.2 (KCNJ11) and SUR1 (ABCC8) are a common cause of neonatal diabetes mellitus. Here we investigate the molecular mechanism by which two heterozygous mutations in the second nucleotide-binding domain (NBD2) of SUR1 (R1380L and R1380C) separately cause neonatal diabetes. SUR1 is a channel regulator that modulates the gating of the pore formed by Kir6.2. K ATP channel activity is inhibited by ATP binding to Kir6.2 but is stimulated by MgADP binding, or by MgATP binding and hydrolysis, at the NBDs of SUR1. Functional analysis of purified NBD2 showed that each mutation enhances MgATP hydrolysis by purified isolated fusion proteins of maltose-binding protein and NBD2. Inhibition of ATP hydrolysis by MgADP was unaffected by mutation of R1380, but inhibition by beryllium fluoride (which traps the ATPase cycle in the prehydrolytic state) was reduced. MgADP-dependent activation of K ATP channel activity was unaffected. These data suggest that the R1380L and R1380C mutations enhance the off-rate of P i, thereby enhancing the hydrolytic rate. Molecular modeling studies supported this idea. Because mutant channels were inhibited less strongly by MgATP, this would increase K ATP currents in pancreatic beta cells, thus reducing insulin secretion and producing diabetes.SUR1 ͉ KATP channel ͉ ATP hydrolysis ͉ sulfonylurea receptor A ctivating mutations in the ABCC8 and KCNJ11 genes are a common cause of neonatal diabetes (1-3). ABCC8 encodes the regulatory subunit of the ATP-sensitive potassium (K ATP ) channel. It coassembles with pore-forming Kir6.2 (KCNJ11) subunits to form an octameric channel, with a central tetrameric Kir6.2 pore being surrounded by four SUR1 subunits (4). Both Kir6.2 and SUR1 serve as metabolic sensors, enabling the K ATP channel to respond to changes in cellular metabolism. In addition, SUR1 endows the channel with sensitivity to the antidiabetic sulfonylurea drugs.In pancreatic beta cells, metabolic regulation of K ATP channels is crucial for glucose-stimulated insulin secretion (Fig. 1A) (3). In unstimulated cells, K ATP channels are open and hyperpolarize the membrane enough to keep voltage-gated Ca 2ϩ channels closed. When the plasma glucose concentration rises, glucose uptake and metabolism by the beta cell are enhanced, leading to K ATP channel closure. This causes a membrane depolarization that triggers opening of voltage-gated Ca 2ϩ channels and Ca 2ϩ -dependent electrical activity. The consequent Ca 2ϩ influx stimulates insulin release. Metabolic regulation of K ATP channel activity is mediated by changes in the concentration of adenine nucleotides, which interact with both Kir6.2 and SUR1 subunits. Binding of ATP to Kir6.2, in a Mg-independent fashion, shuts the channel (5). In contrast, MgADP binding to SUR1 stimulates channel opening (5, 6). Although MgATP can also stimulate K ATP channel activity by interaction with SUR1 (7) it must first be hydrolyzed to MgADP (8). K ATP channel activity...

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Section: Discussionmentioning

confidence: 99%

Increased ATPase activity produced by mutations at arginine-1380 in nucleotide-binding domain 2 of ABCC8 causes neonatal diabetes

Wet

Rees

Shimomura

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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“…Plasma membrane potential (V m ) and whole-cell K ATP channel conductance (G KATP ) of β-cells were recorded in perforated patch configuration (34). Electrical capacitance (C m ) and Ca 2+ current (I Ca ) through the plasma membrane of β-cells were recorded in standard whole-cell configuration (24).…”

Section: Methodsmentioning

confidence: 99%

Insulin Storage and Glucose Homeostasis in Mice Null for the Granule Zinc Transporter ZnT8 and Studies of the Type 2 Diabetes–Associated Variants

et al. 2009

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OBJECTIVEZinc ions are essential for the formation of hexameric insulin and hormone crystallization. A nonsynonymous single nucleotide polymorphism rs13266634 in the SLC30A8 gene, encoding the secretory granule zinc transporter ZnT8, is associated with type 2 diabetes. We describe the effects of deleting the ZnT8 gene in mice and explore the action of the at-risk allele.RESEARCH DESIGN AND METHODSSlc30a8 null mice were generated and backcrossed at least twice onto a C57BL/6J background. Glucose and insulin tolerance were measured by intraperitoneal injection or euglycemic clamp, respectively. Insulin secretion, electrophysiology, imaging, and the generation of adenoviruses encoding the low- (W325) or elevated- (R325) risk ZnT8 alleles were undertaken using standard protocols.RESULTSZnT8−/− mice displayed age-, sex-, and diet-dependent abnormalities in glucose tolerance, insulin secretion, and body weight. Islets isolated from null mice had reduced granule zinc content and showed age-dependent changes in granule morphology, with markedly fewer dense cores but more rod-like crystals. Glucose-stimulated insulin secretion, granule fusion, and insulin crystal dissolution, assessed by total internal reflection fluorescence microscopy, were unchanged or enhanced in ZnT8−/− islets. Insulin processing was normal. Molecular modeling revealed that residue-325 was located at the interface between ZnT8 monomers. Correspondingly, the R325 variant displayed lower apparent Zn2+ transport activity than W325 ZnT8 by fluorescence-based assay.CONCLUSIONSZnT8 is required for normal insulin crystallization and insulin release in vivo but not, remarkably, in vitro. Defects in the former processes in carriers of the R allele may increase type 2 diabetes risks.

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“…The parameters of the current injections were chosen to minimise their effect on the glucose-induced electrical activity. To control V m and impose electrical stimulations, the mode was periodically switched to voltage-clamp [49]. V m was held at the value of −70 mV, with 0.5 Hz +5/−10 mV pulses to monitor the K ATP conductance (see Suppl.…”

Section: Methodsmentioning

confidence: 99%

The Mitochondrial Ca2+ Uniporter MCU Is Essential for Glucose-Induced ATP Increases in Pancreatic β-Cells

et al. 2012

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Glucose induces insulin release from pancreatic β-cells by stimulating ATP synthesis, membrane depolarisation and Ca2+ influx. As well as activating ATP-consuming processes, cytosolic Ca2+ increases may also potentiate mitochondrial ATP synthesis. Until recently, the ability to study the role of mitochondrial Ca2+ transport in glucose-stimulated insulin secretion has been hindered by the absence of suitable approaches either to suppress Ca2+ uptake into these organelles, or to examine the impact on β-cell excitability. Here, we have combined patch-clamp electrophysiology with simultaneous real-time imaging of compartmentalised changes in Ca2+ and ATP/ADP ratio in single primary mouse β-cells, using recombinant targeted (Pericam or Perceval, respectively) as well as entrapped intracellular (Fura-Red), probes. Through shRNA-mediated silencing we show that the recently-identified mitochondrial Ca2+ uniporter, MCU, is required for depolarisation-induced mitochondrial Ca2+ increases, and for a sustained increase in cytosolic ATP/ADP ratio. By contrast, silencing of the mitochondrial Na+-Ca2+ exchanger NCLX affected the kinetics of glucose-induced changes in, but not steady state values of, cytosolic ATP/ADP. Exposure to gluco-lipotoxic conditions delayed both mitochondrial Ca2+ uptake and cytosolic ATP/ADP ratio increases without affecting the expression of either gene. Mitochondrial Ca2+ accumulation, mediated by MCU and modulated by NCLX, is thus required for normal glucose sensing by pancreatic β-cells, and becomes defective in conditions mimicking the diabetic milieu.

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A Kir6.2 Mutation Causing Neonatal Diabetes Impairs Electrical Activity and Insulin Secretion From INS-1 β-Cells

Cited by 41 publications

References 37 publications

Increased ATPase activity produced by mutations at arginine-1380 in nucleotide-binding domain 2 of ABCC8 causes neonatal diabetes

Increased ATPase activity produced by mutations at arginine-1380 in nucleotide-binding domain 2 of ABCC8 causes neonatal diabetes

Insulin Storage and Glucose Homeostasis in Mice Null for the Granule Zinc Transporter ZnT8 and Studies of the Type 2 Diabetes–Associated Variants

The Mitochondrial Ca2+ Uniporter MCU Is Essential for Glucose-Induced ATP Increases in Pancreatic β-Cells

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