Institute of Biomedical and Clinical Research

Exeter, United Kingdom

Institute of Biomedical and Clinical Research

Exeter, United Kingdom
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Mannikko R.,University of Oxford | Stansfeld P.J.,University of Oxford | Ashcroft A.S.,University of Oxford | Hattersley A.T.,Institute of Biomedical and Clinical Research | And 3 more authors.
Journal of Physiology | Year: 2011

We identified a novel heterozygous mutation, W68R, in the Kir6.2 subunit of the ATP-sensitive potassium (K ATP) channel, in a patient with transient neonatal diabetes. This tryptophan is absolutely conserved in mammalian Kir channels. The functional effects of mutations at residue 68 of Kir6.2 were studied by heterologous expression in Xenopus oocytes, and by homology modelling. We found the Kir6.2-W68R mutation causes a small reduction in ATP inhibition in the heterozygous state and an increase in the whole-cell K ATP current. This can explain the clinical phenotype of the patient. The effect of the mutation was not charge or size dependent, the order of potency for ATP inhibition being W


Mannikko R.,University of Oxford | Jefferies C.,Starship Childrens Health | Flanagan S.E.,Institute of Biomedical and Clinical Research | Hattersley A.,Institute of Biomedical and Clinical Research | And 2 more authors.
Human Molecular Genetics | Year: 2010

ATP-sensitive potassium (KATP) channels regulate insulin secretion from pancreatic beta-cells. Gain-of-function mutations in the genes encoding the Kir6.2 and SUR1 subunits of this channel cause neonatal diabetes. We report two novel mutations on the same haplotype (cis), F60Y and V64L, in the slide helix of Kir6.2 in a patient with neonatal diabetes, developmental delay and epilepsy. Functional analysis revealed the F60Y mutation increases the intrinsic channel open probability (Po(0)), thereby indirectly producing a marked decrease in channel inhibition by ATP and an increase in whole-cell KATP currents. When expressed alone, the V64L mutation caused a small reduction in apparent ATP inhibition, by enhancing the ability of MgATP to stimulate channel activity. The V64L mutation also ameliorated the deleterious effects on the F60Y mutation when it was expressed on the same (but not a different) subunit. These data indicate that F60Y is the pathogenic mutation and reveal that interactions between slide helix residues can influence KATP channel gating. © The Author 2009. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org.


Mannikko R.,University of Oxford | Flanagan S.E.,Institute of Biomedical and Clinical Research | Sim X.,University of Oxford | Segal D.,Center for Diabetes and Endocrinology | And 4 more authors.
Diabetes | Year: 2011

OBJECTIVE - Two novel mutations (E1506D, E1506G) in the nucleotide-binding domain 2 (NBD2) of the ATP-sensitive K+ channel (KATP channel) sulfonylurea receptor 1 (SUR1) subunit were detected heterozygously in patients with neonatal diabetes. A mutation at the same residue (E1506K) was previously shown to cause congenital hyperinsulinemia. We sought to understand why mutations at the same residue can cause either neonatal diabetes or hyperinsulinemia. RESEARCH DESIGN AND METHODS - Neonatal diabetic patients were sequenced for mutations in ABCC8 (SUR1) and KCNJ11 (Kir6.2). Wild-type and mutant KATP channels were expressed in Xenopus laevis oocytes and studied with electrophysiological methods. RESULTS - Oocytes expressing neonatal diabetes mutant channels had larger resting whole-cell KATP currents than wild-type, consistent with the patients' diabetes. Conversely, no E1506K currents were recorded at rest or after metabolic inhibition, as expected for a mutation causing hyperinsulinemia. KATP channels are activated by Mg-nucleotides (via SUR1) and blocked by ATP (via Kir6.2). All mutations decreased channel activation by MgADP but had little effect on MgATP activation, as assessed using an ATP-insensitive Kir6.2 subunit. Importantly, using wild-type Kir6.2, a 30-s preconditioning exposure to physiological MgATP concentrations (>300 μmol/L) caused a marked reduction in the ATP sensitivity of neonatal diabetic channels, a small decrease in that of wild-type channels, and no change for E1506K channels. This difference in MgATP inhibition may explain the difference in resting whole-cell currents found for the neonatal diabetes and hyperinsulinemia mutations. CONCLUSIONS - Mutations in the same residue can cause either hyperinsulinemia or neonatal diabetes. Differentially altered nucleotide regulation by NBD2 of SUR1 can explain the respective clinical phenotypes. © 2011 by the American Diabetes Association.

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