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Diabetologia
2008 May 01;515:802-10. doi: 10.1007/s00125-008-0923-1.
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A Kir6.2 mutation causing severe functional effects in vitro produces neonatal diabetes without the expected neurological complications.
Tammaro P
,
Flanagan SE
,
Zadek B
,
Srinivasan S
,
Woodhead H
,
Hameed S
,
Klimes I
,
Hattersley AT
,
Ellard S
,
Ashcroft FM
.
Abstract
AIMS/HYPOTHESIS: Heterozygous activating mutations in the pancreatic ATP-sensitive K+ channel cause permanent neonatal diabetes mellitus (PNDM). This results from a decrease in the ability of ATP to close the channel, which thereby suppresses insulin secretion. PNDM mutations that cause a severe reduction in ATP inhibition may produce additional symptoms such as developmental delay and epilepsy. We identified a heterozygous mutation (L164P) in the pore-forming (Kir6.2) subunit of the channel in three unrelated patients and examined its functional effects.
METHODS: The patients (currently aged 2, 8 and 20 years) developed diabetes shortly after birth. The two younger patients attempted transfer to sulfonylurea therapy but were unsuccessful (up to 1.1 mg kg(-1) day(-1)). They remain insulin dependent. None of the patients displayed neurological symptoms. Functional properties of wild-type and mutant channels were examined by electrophysiology in Xenopus oocytes.
RESULTS: Heterozygous (het) and homozygous L164P K(ATP) channels showed a marked reduction in channel inhibition by ATP. Consistent with its predicted location within the pore, L164P enhanced the channel open state, which explains the reduction in ATP sensitivity. HetL164P currents exhibited greatly increased whole-cell currents that were unaffected by sulfonylureas. This explains the inability of sulfonylureas to ameliorate the diabetes of affected patients.
CONCLUSIONS/INTERPRETATION: Our results provide the first demonstration that mutations such as L164P, which produce a severe reduction in ATP sensitivity, do not inevitably cause developmental delay or neurological problems. However, the neonatal diabetes of these patients is unresponsive to sulfonylurea therapy. Functional analysis of PNDM mutations can predict the sulfonylurea response.
Fig. 1. Whole-cell KATP current. Mean steady state whole-cell currents evoked by voltage steps from â10 to â30 mV before (control, white bars) and after application of 3 mmol/l azide (grey bars) and in the presence of 3 mmol/l azide plus 0.5 mmol/l tolbutamide (black bars). The number of oocytes was five to seven in each case. G, KATP conductance; Gc, KATP conductance expressed relative to the conductance in the absence of the nucleotide. WT, wild-type; hetL164P and homL164P channels as indicated
Fig. 2. ATP-inhibition of L164P channels is less that that of wild-type channels. a, c Currents recorded in inside-out patches excised from Xenopus oocytes expressing hetKir6.2-L164P/SUR1 (hetL164P) or homKir6.2-L164P/SUR1 (homL164P) channels, as indicated, in response to 3 s voltage ramps from â110 to +100 mV. ATP (10 mmol/l) was applied as indicated by the horizontal bars in the absence (a) or presence (c) of 2 mmol/l Mg2+. b, d, mean relationship between [ATP] and KATP conductance (G), expressed relative to the conductance in the absence of the nucleotide (Gc), for wild-type (white circles, nâ=â9), hetL164P (white/black circles, nâ=â6) or homL164P (black circles, nâ=â4) channels. Experiments were carried out in the absence (b) or presence (d) of 2 mmol/l Mg2+. The continuous lines through the black circles were drawn by eye. The smooth curves are the best fit to the Hill equation with IC50 of 11 μmol/l (wild-type) and 100 μmol/l (hetL164P) (b, 0 mmol/l Mg2+) or IC50 of 16 μmol/l (wild-type) and 122 μmol/l (hetL164P) (d, 2 mmol/l Mg2+)
Fig. 3. Homology model of Kir6.2 [32]. For clarity, only two subunits are shown. ATP (yellow) is shown in its binding site. Residue L164 is shown in red
Fig. 4. The L164P mutation enhances single-channel activity. Representative single KATP channel currents recorded at â60 mV in inside-out patches from oocytes expressing wild-type or homL164P channels, as indicated. Currents were recorded in the absence of Mg2+ and nucleotides
Fig. 5. Sensitivity to MgADP of wild-type and hetL164P channels. a Representative currents (I) recorded at â60 mV from inside-out excised membrane patches from Xenopus oocytes expressing wild-type or hetL164P channels, as indicated. Patches were exposed to 100 μmol/l ADP in the continuous presence of 100 μmol/l ATP: 2 mmol/l Mg2+ was present throughout. b Mean current in the presence of 100 μmol/l MgATP or 30 or 100 μmol/l MgADP plus 100 μmol/l MgATP, normalised to the current in the absence of nucleotides for wild-type (grey bars) and hetL164P (black bars) channels. Bars indicate meansâ±âSEM. The number of patches was three to four in each case
Fig. 6. Mean KATP current (expressed as a % of maximum) measured in the presence of 3 mmol/l MgATP from inside-out patches expressing heterozygous mutant channels as indicated. The dashed line indicates the maximal current amplitude normally associated with PNDM. iDEND, intermediate DEND syndrome (i.e. neonatal diabetes with developmental delay [21]). Data for wild-type (WT) and Kir6.2-R201H channels are from [14], for Y330C and F333I from [26], for Q52R, V59G and R201C from [15], for V59M from [24], for I296L from [16] and for G334D from [34]
Antcliff,
Functional analysis of a structural model of the ATP-binding site of the KATP channel Kir6.2 subunit.
2005, Pubmed
Antcliff,
Functional analysis of a structural model of the ATP-binding site of the KATP channel Kir6.2 subunit.
2005,
Pubmed
Ashcroft,
Glucose induces closure of single potassium channels in isolated rat pancreatic beta-cells.
,
Pubmed
Ashcroft,
Type 2 diabetes mellitus: not quite exciting enough?
2004,
Pubmed
Ashcroft,
ATP-sensitive potassium channelopathies: focus on insulin secretion.
2005,
Pubmed
Clement,
Association and stoichiometry of K(ATP) channel subunits.
1997,
Pubmed
Cordes,
Proline-induced distortions of transmembrane helices.
2002,
Pubmed
Drain,
KATP channel inhibition by ATP requires distinct functional domains of the cytoplasmic C terminus of the pore-forming subunit.
1998,
Pubmed
,
Xenbase
Enkvetchakul,
The kinetic and physical basis of K(ATP) channel gating: toward a unified molecular understanding.
2000,
Pubmed
Enkvetchakul,
ATP interaction with the open state of the K(ATP) channel.
2001,
Pubmed
Flanagan,
Mutations in KCNJ11, which encodes Kir6.2, are a common cause of diabetes diagnosed in the first 6 months of life, with the phenotype determined by genotype.
2006,
Pubmed
Flanagan,
Mutations in ATP-sensitive K+ channel genes cause transient neonatal diabetes and permanent diabetes in childhood or adulthood.
2007,
Pubmed
Gloyn,
Activating mutations in the gene encoding the ATP-sensitive potassium-channel subunit Kir6.2 and permanent neonatal diabetes.
2004,
Pubmed
,
Xenbase
Gloyn,
Relapsing diabetes can result from moderately activating mutations in KCNJ11.
2005,
Pubmed
,
Xenbase
Gribble,
Properties of cloned ATP-sensitive K+ currents expressed in Xenopus oocytes.
1997,
Pubmed
,
Xenbase
Gribble,
The essential role of the Walker A motifs of SUR1 in K-ATP channel activation by Mg-ADP and diazoxide.
1997,
Pubmed
,
Xenbase
Gribble,
Sulphonylurea action revisited: the post-cloning era.
2003,
Pubmed
Haider,
Identification of the PIP2-binding site on Kir6.2 by molecular modelling and functional analysis.
2007,
Pubmed
Haider,
Focus on Kir6.2: a key component of the ATP-sensitive potassium channel.
2005,
Pubmed
Inagaki,
Reconstitution of IKATP: an inward rectifier subunit plus the sulfonylurea receptor.
1995,
Pubmed
Loussouarn,
Flexibility of the Kir6.2 inward rectifier K(+) channel pore.
2001,
Pubmed
Loussouarn,
Structure and dynamics of the pore of inwardly rectifying K(ATP) channels.
2000,
Pubmed
Masia,
An ATP-binding mutation (G334D) in KCNJ11 is associated with a sulfonylurea-insensitive form of developmental delay, epilepsy, and neonatal diabetes.
2007,
Pubmed
Massa,
KCNJ11 activating mutations in Italian patients with permanent neonatal diabetes.
2005,
Pubmed
Nichols,
Adenosine diphosphate as an intracellular regulator of insulin secretion.
1996,
Pubmed
Pearson,
Switching from insulin to oral sulfonylureas in patients with diabetes due to Kir6.2 mutations.
2006,
Pubmed
,
Xenbase
Proks,
Functional effects of KCNJ11 mutations causing neonatal diabetes: enhanced activation by MgATP.
2005,
Pubmed
,
Xenbase
Proks,
A gating mutation at the internal mouth of the Kir6.2 pore is associated with DEND syndrome.
2005,
Pubmed
,
Xenbase
Proks,
Molecular basis of Kir6.2 mutations associated with neonatal diabetes or neonatal diabetes plus neurological features.
2004,
Pubmed
,
Xenbase
Reimann,
Involvement of the n-terminus of Kir6.2 in coupling to the sulphonylurea receptor.
1999,
Pubmed
,
Xenbase
Sagen,
Permanent neonatal diabetes due to mutations in KCNJ11 encoding Kir6.2: patient characteristics and initial response to sulfonylurea therapy.
2004,
Pubmed
Seino,
Physiological and pathophysiological roles of ATP-sensitive K+ channels.
2003,
Pubmed
Shimomura,
Mutations at the same residue (R50) of Kir6.2 (KCNJ11) that cause neonatal diabetes produce different functional effects.
2006,
Pubmed
,
Xenbase
Shyng,
Octameric stoichiometry of the KATP channel complex.
1997,
Pubmed
Shyng,
Regulation of KATP channel activity by diazoxide and MgADP. Distinct functions of the two nucleotide binding folds of the sulfonylurea receptor.
1997,
Pubmed
Stanik,
Prevalence of permanent neonatal diabetes in Slovakia and successful replacement of insulin with sulfonylurea therapy in KCNJ11 and ABCC8 mutation carriers.
2007,
Pubmed
Tammaro,
Kir6.2 mutations causing neonatal diabetes provide new insights into Kir6.2-SUR1 interactions.
2005,
Pubmed
,
Xenbase
Trapp,
Molecular analysis of ATP-sensitive K channel gating and implications for channel inhibition by ATP.
1998,
Pubmed
,
Xenbase
Tucker,
Truncation of Kir6.2 produces ATP-sensitive K+ channels in the absence of the sulphonylurea receptor.
1997,
Pubmed
,
Xenbase
Zingman,
Signaling in channel/enzyme multimers: ATPase transitions in SUR module gate ATP-sensitive K+ conductance.
2001,
Pubmed
Zung,
Glibenclamide treatment in permanent neonatal diabetes mellitus due to an activating mutation in Kir6.2.
2004,
Pubmed
