Tammaro P et al. (2008), A Kir6.2 mutation causing severe functional eff...

XB-ART-37365

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 .

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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.

???displayArticle.pubmedLink??? 18335204
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???displayArticle.link??? Diabetologia
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Species referenced: Xenopus laevis
Genes referenced: abcc8 ins kcnj11

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	Fig. 1. Whole-cell KATP current. Mean steady state whole-cell currents evoked by voltage steps from −10 to −30 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 −110 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, n = 9), hetL164P (white/black circles, n = 6) or homL164P (black circles, n = 4) 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 −60 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 −60 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 means ± SEM. 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]

References [+] :

Antcliff, Functional analysis of a structural model of the ATP-binding site of the KATP channel Kir6.2 subunit. 2005, Pubmed