Männikkö R et al. (2011), Mutations of the same conserved glutamate resid...

XB-ART-43300

Diabetes 2011 Jun 01;606:1813-22. doi: 10.2337/db10-1583.

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Mutations of the same conserved glutamate residue in NBD2 of the sulfonylurea receptor 1 subunit of the KATP channel can result in either hyperinsulinism or neonatal diabetes.

Männikkö R , Flanagan SE , Sim X , Segal D , Hussain K , Ellard S , Hattersley AT , Ashcroft FM .

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OBJECTIVE: Two novel mutations (E1506D, E1506G) in the nucleotide-binding domain 2 (NBD2) of the ATP-sensitive K(+) channel (K(ATP) 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 K(ATP) channels were expressed in Xenopus laevis oocytes and studied with electrophysiological methods. RESULTS: Oocytes expressing neonatal diabetes mutant channels had larger resting whole-cell K(ATP) 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. K(ATP) 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.

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Species referenced: Xenopus laevis
Genes referenced: abcc8 kcnj11 kcnj21

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	FIG. 1. Whole-cell KATP currents for wild-type and mutant channels. A–C: Representative whole-cell current amplitudes evoked by repeated voltage steps from −10 to −30 mV for wild-type (A, WT), and homomeric (B, homE1506D) or heterozygous (C, hetE1506D) Kir6.2/SUR1-E1506D channels. The bars indicate application of 3 mmol/L azide, 0.34 mmol/L diazoxide (Dz), and 0.5 mmol/L tolbutamide (Tb). The dashed line (- - -) indicates the zero current level. Scale bars are 0.5 μA (y-axis) and 400 s (x-axis). D and E: Mean steady-state whole-cell KATP current amplitudes for wild-type homomeric (D) and heterozygous (E) mutant channels. Currents were evoked by a voltage step from –10 to –30 mV before (control, ■) and after (▨) application of 3 mmol/L azide, in the presence of 3 mmol/L azide plus 0.34 mmol/L Dz (□), and 3 azide + 0.5 mmol/L Tb (▧) for WT and SUR1 mutant channels, as indicated. The number of oocytes tested is given below the bars. P ≤ 0.05, P ≤ 0.01, **P ≤ 0.001 against WT (Student t test). The error bars show the SEM.
	FIG. 2. ATP sensitivity of wild-type (WT) and mutant channels. A: Representative inside-out patch currents recorded in response to MgATP (100 μmol/L) for channels composed of WT Kir6.2 and WT or mutant SUR1, as indicated. The dashed lines (- - -) indicate the zero-current level. The solid lines (–––) indicate nucleotide application. B: Mean relationship between KATP current (I), expressed relative to that in the absence of nucleotide (Ic), and ATP concentration in the presence (B–D) or absence (E–G) of 2 mmol/L Mg2+ for WT (○, - - - ) and mutant (●, –––) channels. The lines are drawn to eq. 1, with the following parameters (in μmol/L for IC50): B–D: wild-type (n = 23), IC50 = 12.2, h = 1.05; E–G: WT (n = 7), IC50 = 8.3, h = 1.09. B: Kir6.2/SUR1-E1506D (n = 11), IC50 = 20.1, h = 1.05. C: Kir6.2/SUR1-E1506G (n = 10), IC50 = 18.8, h = 0.93. D: Kir6.2/SUR1-E1506K (n = 11), IC50 = 11.0, h = 1.14. E: Kir6.2/SUR1-E1506D (n = 7), IC50 = 8.2, h = 1.00. F: Kir6.2/SUR1-E1506G (n = 7), IC50 = 6.9, h = 0.86. G: SUR1-E1506K (n = 7), IC50 = 6.4, h = 1.00.
	FIG. 3. MgADP activation of wild-type (WT) and mutant channels. A: Representative inside-out patch currents recorded in response to application of MgADP (100 μmol/L) for channels composed of WT Kir6.2 and WT or mutant SUR1, as indicated. The dashed lines (- - -) indicate the zero-current level. The solid bars (■) indicate the application of nucleotide. Note that 100 μmol/L MgADP activates WT-SUR1 channels but blocks all the mutant E1506 channels. B and C: MgADP activation of WT or mutant Kir6.2/SUR1-E1506 channels, as indicated. The number of patches is indicated below the bars. B: KATP current (I) in the presence of 100 μmol/L MgADP is expressed relative to the current in the absence of nucleotides (IC). C: KATP current (I) in the presence of 100 μmol/L MgADP and 100 μmol/L MgATP is expressed relative to the current in the presence of 100 μmol/L MgATP alone (IATP). P ≤ 0.05, P ≤ 0.01, **P ≤ 0.001 against wild type (Student t test). Statistical significance between mutant E1506 channels is reported in Supplementary Table 1. The error bars show the SEM.
	FIG. 4. MgADP and MgATP activation of SUR1-E1506 mutations on a Kir6.2-G334D background. MgADP and MgATP activation of KATP channels composed of Kir6.2-G334D and WT or mutant SUR1, as indicated. The number of patches is indicated below the bars. A: The maximal KATP current after patch excision is expressed relative to that in the cell-attached patch. KATP current (I) in the presence of 100 μmol/L MgADP (B) or 100 μmol/L MgATP (C) is expressed relative to the current in the absence of nucleotides (IC). P ≤ 0.05, P ≤ 0.01, **P ≤ 0.001 against wild type (Student t test). Statistical significance between mutant E1506 channels is reported in Supplementary Table 1. The error bars show the SEM.
	FIG. 5. Time course of MgADP and MgATP activation of KATP channels. Channels were composed of Kir6.2-G334D and wild-type (WT) or mutant SUR1 subunits, as indicated. Current activation and deactivation were fit with a single exponential function. The number of patches is indicated below the bars. Time constant of current activation (τon) by 100 μmol/L MgADP (A) or 100 μmol/L MgATP (B) is shown. Time constant of current deactivation (τoff) after removal of 100 μmol/L MgADP (C) or 100 μmol/L MgATP (D) is shown. P ≤ 0.05 P ≤ 0.01, **P ≤ 0.001 against wild-type, (Student t test). Statistical significance between mutant E1506 channels is reported in Supplementary Table 1. The error bars show the SEM. E: Representative current traces for WT and Kir6.2/SUR1-E1506D (E1506D) channels. The horizontal bar indicates the duration of application of 100 μmol/L MgATP. The WT trace is interrupted to align the time point of ATP removal with that of the E1506D trace. Current amplitudes are normalized.
	FIG. 6. ATP sensitivity of wild-type and mutant channels after preconditioning with 10 mmol/L MgATP. A: Representative homE1506D currents recorded in response to different MgATP concentrations (as indicated), preceded by a preconditioning pulse in 10 mmol/L MgATP solution (indicated by the unlabeled solid line [–––]) and followed by control (nucleotide-free) solution. The dashed lines (- - -) indicate the zero current. B–D: Mean relationship between MgATP concentration and KATP current (I), expressed relative to that in the absence of nucleotide (Ic), after 30-s preincubation in 10 mmol/L MgATP for WT (○, n = 9), heterozygous (●), and homomeric mutant (▲) channels. Channels were composed of Kir6.2 and wild-type or mutant SUR1 subunits, as indicated. B: homE1506D (n = 8), hetE1506D (n = 9). C: homE1506G (n = 7), hetE1506G (n = 7). D: homE1506K (n = 7), hetE1506K (n = 5). The lines are drawn to eq. 1, with the following parameters (in μmol/L for IC50): wild-type, IC50 = 28, h = 1.2; homE1506D, IC50 = 78, h = 1.2; hetE1506D, IC50 = 45, h = 1.2; homE1506G, IC50 = 105, h = 1.3; hetE1506G, IC50 = 45, h = 1.3; homE1506K, IC50 = 18, h = 1.6; hetE1506K, IC50 = 26, h = 1.4.
	FIG. 7. Concentration dependence of MgATP preconditioning on KATP channel ATP sensitivity. A: Representative homE1506G currents recorded in response to test pulses of 100 μmol/L MgATP (unlabeled solid line [–––]), preceded by a preconditioning pulse to a variable MgATP concentration (10 μmol/L to 10 mmol/L, as indicated) and followed by control (nucleotide-free) solution. B: MgATP concentration during the conditioning prepulse plotted against the current during a 100 μmol/L MgATP test pulse. Current is expressed as a fraction of that in control (nucleotide-free) solution after the test pulse for wild-type (○, n = 14), homE1506D (●, n = 15), homE1506G (▲, n = 10), and homE1506K (■, n = 8) channels. The lines are drawn through the points by eye. The error bars show the SEM. C: The SUR1 ATPase catalytic cycle.

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	FIG. 1. Whole-cell KATP currents for wild-type and mutant channels. A–C: Representative whole-cell current amplitudes evoked by repeated voltage steps from −10 to −30 mV for wild-type (A, WT), and homomeric (B, homE1506D) or heterozygous (C, hetE1506D) Kir6.2/SUR1-E1506D channels. The bars indicate application of 3 mmol/L azide, 0.34 mmol/L diazoxide (Dz), and 0.5 mmol/L tolbutamide (Tb). The dashed line (- - -) indicates the zero current level. Scale bars are 0.5 μA (y-axis) and 400 s (x-axis). D and E: Mean steady-state whole-cell KATP current amplitudes for wild-type homomeric (D) and heterozygous (E) mutant channels. Currents were evoked by a voltage step from –10 to –30 mV before (control, ■) and after (▨) application of 3 mmol/L azide, in the presence of 3 mmol/L azide plus 0.34 mmol/L Dz (□), and 3 azide + 0.5 mmol/L Tb (▧) for WT and SUR1 mutant channels, as indicated. The number of oocytes tested is given below the bars. P ≤ 0.05, P ≤ 0.01, **P ≤ 0.001 against WT (Student t test). The error bars show the SEM.
	FIG. 2. ATP sensitivity of wild-type (WT) and mutant channels. A: Representative inside-out patch currents recorded in response to MgATP (100 μmol/L) for channels composed of WT Kir6.2 and WT or mutant SUR1, as indicated. The dashed lines (- - -) indicate the zero-current level. The solid lines (–––) indicate nucleotide application. B: Mean relationship between KATP current (I), expressed relative to that in the absence of nucleotide (Ic), and ATP concentration in the presence (B–D) or absence (E–G) of 2 mmol/L Mg2+ for WT (○, - - - ) and mutant (●, –––) channels. The lines are drawn to eq. 1, with the following parameters (in μmol/L for IC50): B–D: wild-type (n = 23), IC50 = 12.2, h = 1.05; E–G: WT (n = 7), IC50 = 8.3, h = 1.09. B: Kir6.2/SUR1-E1506D (n = 11), IC50 = 20.1, h = 1.05. C: Kir6.2/SUR1-E1506G (n = 10), IC50 = 18.8, h = 0.93. D: Kir6.2/SUR1-E1506K (n = 11), IC50 = 11.0, h = 1.14. E: Kir6.2/SUR1-E1506D (n = 7), IC50 = 8.2, h = 1.00. F: Kir6.2/SUR1-E1506G (n = 7), IC50 = 6.9, h = 0.86. G: SUR1-E1506K (n = 7), IC50 = 6.4, h = 1.00.
	FIG. 3. MgADP activation of wild-type (WT) and mutant channels. A: Representative inside-out patch currents recorded in response to application of MgADP (100 μmol/L) for channels composed of WT Kir6.2 and WT or mutant SUR1, as indicated. The dashed lines (- - -) indicate the zero-current level. The solid bars (■) indicate the application of nucleotide. Note that 100 μmol/L MgADP activates WT-SUR1 channels but blocks all the mutant E1506 channels. B and C: MgADP activation of WT or mutant Kir6.2/SUR1-E1506 channels, as indicated. The number of patches is indicated below the bars. B: KATP current (I) in the presence of 100 μmol/L MgADP is expressed relative to the current in the absence of nucleotides (IC). C: KATP current (I) in the presence of 100 μmol/L MgADP and 100 μmol/L MgATP is expressed relative to the current in the presence of 100 μmol/L MgATP alone (IATP). P ≤ 0.05, P ≤ 0.01, **P ≤ 0.001 against wild type (Student t test). Statistical significance between mutant E1506 channels is reported in Supplementary Table 1. The error bars show the SEM.
	FIG. 4. MgADP and MgATP activation of SUR1-E1506 mutations on a Kir6.2-G334D background. MgADP and MgATP activation of KATP channels composed of Kir6.2-G334D and WT or mutant SUR1, as indicated. The number of patches is indicated below the bars. A: The maximal KATP current after patch excision is expressed relative to that in the cell-attached patch. KATP current (I) in the presence of 100 μmol/L MgADP (B) or 100 μmol/L MgATP (C) is expressed relative to the current in the absence of nucleotides (IC). P ≤ 0.05, P ≤ 0.01, **P ≤ 0.001 against wild type (Student t test). Statistical significance between mutant E1506 channels is reported in Supplementary Table 1. The error bars show the SEM.
	FIG. 5. Time course of MgADP and MgATP activation of KATP channels. Channels were composed of Kir6.2-G334D and wild-type (WT) or mutant SUR1 subunits, as indicated. Current activation and deactivation were fit with a single exponential function. The number of patches is indicated below the bars. Time constant of current activation (τon) by 100 μmol/L MgADP (A) or 100 μmol/L MgATP (B) is shown. Time constant of current deactivation (τoff) after removal of 100 μmol/L MgADP (C) or 100 μmol/L MgATP (D) is shown. P ≤ 0.05 P ≤ 0.01, **P ≤ 0.001 against wild-type, (Student t test). Statistical significance between mutant E1506 channels is reported in Supplementary Table 1. The error bars show the SEM. E: Representative current traces for WT and Kir6.2/SUR1-E1506D (E1506D) channels. The horizontal bar indicates the duration of application of 100 μmol/L MgATP. The WT trace is interrupted to align the time point of ATP removal with that of the E1506D trace. Current amplitudes are normalized.
	FIG. 6. ATP sensitivity of wild-type and mutant channels after preconditioning with 10 mmol/L MgATP. A: Representative homE1506D currents recorded in response to different MgATP concentrations (as indicated), preceded by a preconditioning pulse in 10 mmol/L MgATP solution (indicated by the unlabeled solid line [–––]) and followed by control (nucleotide-free) solution. The dashed lines (- - -) indicate the zero current. B–D: Mean relationship between MgATP concentration and KATP current (I), expressed relative to that in the absence of nucleotide (Ic), after 30-s preincubation in 10 mmol/L MgATP for WT (○, n = 9), heterozygous (●), and homomeric mutant (▲) channels. Channels were composed of Kir6.2 and wild-type or mutant SUR1 subunits, as indicated. B: homE1506D (n = 8), hetE1506D (n = 9). C: homE1506G (n = 7), hetE1506G (n = 7). D: homE1506K (n = 7), hetE1506K (n = 5). The lines are drawn to eq. 1, with the following parameters (in μmol/L for IC50): wild-type, IC50 = 28, h = 1.2; homE1506D, IC50 = 78, h = 1.2; hetE1506D, IC50 = 45, h = 1.2; homE1506G, IC50 = 105, h = 1.3; hetE1506G, IC50 = 45, h = 1.3; homE1506K, IC50 = 18, h = 1.6; hetE1506K, IC50 = 26, h = 1.4.
	FIG. 7. Concentration dependence of MgATP preconditioning on KATP channel ATP sensitivity. A: Representative homE1506G currents recorded in response to test pulses of 100 μmol/L MgATP (unlabeled solid line [–––]), preceded by a preconditioning pulse to a variable MgATP concentration (10 μmol/L to 10 mmol/L, as indicated) and followed by control (nucleotide-free) solution. B: MgATP concentration during the conditioning prepulse plotted against the current during a 100 μmol/L MgATP test pulse. Current is expressed as a fraction of that in control (nucleotide-free) solution after the test pulse for wild-type (○, n = 14), homE1506D (●, n = 15), homE1506G (▲, n = 10), and homE1506K (■, n = 8) channels. The lines are drawn through the points by eye. The error bars show the SEM. C: The SUR1 ATPase catalytic cycle.