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Sulfonylureas suppress the stimulatory action of Mg-nucleotides on Kir6.2/SUR1 but not Kir6.2/SUR2A KATP channels: a mechanistic study.

Proks P, de Wet H, Ashcroft FM - J. Gen. Physiol. (2014)

Bottom Line: We found that both MgATP and MgADP increased gliclazide inhibition of Kir6.2/SUR1 channels and reduced inhibition of Kir6.2/SUR2A-Y1206S.Mutation of one (or both) of the Walker A lysines in the catalytic site of the nucleotide-binding domains of SUR1 may have a similar effect to gliclazide on MgADP binding and transduction, but it does not appear to impair MgATP binding.Our results have implications for the therapeutic use of sulfonylureas.

View Article: PubMed Central - HTML - PubMed

Affiliation: Oxford Centre for Gene Function and Department of Physiology, Anatomy, and Genetics, University of Oxford, Oxford OX1 3PT, England, UK Oxford Centre for Gene Function and Department of Physiology, Anatomy, and Genetics, University of Oxford, Oxford OX1 3PT, England, UK.

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Effect of MgADP and MgATP on sulfonylurea inhibition of SUR1- and SUR2A-containing channels. (A–F) Currents in the presence of sulfonylurea (I) expressed as a fraction of that in drug-free solution (Ic). (A and B) Concentration-response relationships for gliclazide inhibition of Kir6.2/SUR1 (A) and Kir6.2/SUR2A-YS (B) channels in the presence and absence (same data as in Fig. 3) of 100 µM MgADP. The lines are the best fit of Eq. 1 to the mean data: IC50 = 72 nM, h = 1.2, a = 0.42 (A, open circles; n = 6); IC50 = 187 nM, h = 1.1, a = 0.07 (A, closed circles; n = 6); IC50 = 1.3 µM, h = 1.1, a = 0.65 (B, open circles; n = 5); IC50 = 1.6 µM, h = 1.2, a = 0.85 (B, closed circles; n = 5). (C and D) Concentration-response relationships for glibenclamide inhibition of Kir6.2/SUR1 (C) and Kir6.2/SUR2A (D) channels in the presence and absence (data from Fig. 3) of 100 µM MgADP. The lines are the best fit of Eq. 1 to the mean data: IC50 = 2.8 nM, h = 0.93, a = 0.32 (C, open circles; n = 6); IC50 = 3.7 nM, h = 1.2, a = 0.04 (C, closed circles; n = 6); IC50 = 13 nM, h = 0.94, a = 0.32 (D, open circles; n = 5); IC50 = 30 nM, h = 0.75, a = 0.68 (D, closed circles; n = 5). (E and F) Concentration-response relationships for gliclazide inhibition of Kir6.2-G334D/SUR1 (E) and Kir6.2-G334D/SUR2A-YS (F) channels in the presence and absence of 1 mM MgATP. The lines are the best fit of Eq. 1 to the mean data: IC50 = 70 nM, h = 1.0, a = 0.39 (E, open squares; n = 6); IC50 = 210 nM, h = 1.0, a = 0.21 (E, closed squares; n = 6); IC50 = 1.3 µM, h = 1.2, a = 0.64 (F, open squares; n = 5); IC50 = 2.5 µM, h = 1.0, a = 0.91 (F, closed squares; n = 5). Mean ± SEM.
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fig5: Effect of MgADP and MgATP on sulfonylurea inhibition of SUR1- and SUR2A-containing channels. (A–F) Currents in the presence of sulfonylurea (I) expressed as a fraction of that in drug-free solution (Ic). (A and B) Concentration-response relationships for gliclazide inhibition of Kir6.2/SUR1 (A) and Kir6.2/SUR2A-YS (B) channels in the presence and absence (same data as in Fig. 3) of 100 µM MgADP. The lines are the best fit of Eq. 1 to the mean data: IC50 = 72 nM, h = 1.2, a = 0.42 (A, open circles; n = 6); IC50 = 187 nM, h = 1.1, a = 0.07 (A, closed circles; n = 6); IC50 = 1.3 µM, h = 1.1, a = 0.65 (B, open circles; n = 5); IC50 = 1.6 µM, h = 1.2, a = 0.85 (B, closed circles; n = 5). (C and D) Concentration-response relationships for glibenclamide inhibition of Kir6.2/SUR1 (C) and Kir6.2/SUR2A (D) channels in the presence and absence (data from Fig. 3) of 100 µM MgADP. The lines are the best fit of Eq. 1 to the mean data: IC50 = 2.8 nM, h = 0.93, a = 0.32 (C, open circles; n = 6); IC50 = 3.7 nM, h = 1.2, a = 0.04 (C, closed circles; n = 6); IC50 = 13 nM, h = 0.94, a = 0.32 (D, open circles; n = 5); IC50 = 30 nM, h = 0.75, a = 0.68 (D, closed circles; n = 5). (E and F) Concentration-response relationships for gliclazide inhibition of Kir6.2-G334D/SUR1 (E) and Kir6.2-G334D/SUR2A-YS (F) channels in the presence and absence of 1 mM MgATP. The lines are the best fit of Eq. 1 to the mean data: IC50 = 70 nM, h = 1.0, a = 0.39 (E, open squares; n = 6); IC50 = 210 nM, h = 1.0, a = 0.21 (E, closed squares; n = 6); IC50 = 1.3 µM, h = 1.2, a = 0.64 (F, open squares; n = 5); IC50 = 2.5 µM, h = 1.0, a = 0.91 (F, closed squares; n = 5). Mean ± SEM.

Mentions: The relationship between ADP concentration and KATP current inhibition in Fig. 4 and between sulfonylurea concentration and KATP current inhibition in Figs. 3, 5, and 6, was fit with(1)IXIC=a+L−a1+([X]IC50)h,where IX is the steady-state KATP current in the presence of the test nucleotide or drug concentration [X], IC is the current in nucleotide (or drug)-free solution obtained by averaging the current before and after application, IC50 is the nucleotide (drug) concentration at which the inhibition is half maximal, h is the Hill coefficient, and a is the fraction of KATP current remaining at gliclazide concentrations that saturate the high-affinity binding site (for ADP, a = 0). The factor L equals 1 except for data in Fig. 6 (closed symbols), where it reflects the extent of channel activation by Mg-nucleotides in drug-free solution.


Sulfonylureas suppress the stimulatory action of Mg-nucleotides on Kir6.2/SUR1 but not Kir6.2/SUR2A KATP channels: a mechanistic study.

Proks P, de Wet H, Ashcroft FM - J. Gen. Physiol. (2014)

Effect of MgADP and MgATP on sulfonylurea inhibition of SUR1- and SUR2A-containing channels. (A–F) Currents in the presence of sulfonylurea (I) expressed as a fraction of that in drug-free solution (Ic). (A and B) Concentration-response relationships for gliclazide inhibition of Kir6.2/SUR1 (A) and Kir6.2/SUR2A-YS (B) channels in the presence and absence (same data as in Fig. 3) of 100 µM MgADP. The lines are the best fit of Eq. 1 to the mean data: IC50 = 72 nM, h = 1.2, a = 0.42 (A, open circles; n = 6); IC50 = 187 nM, h = 1.1, a = 0.07 (A, closed circles; n = 6); IC50 = 1.3 µM, h = 1.1, a = 0.65 (B, open circles; n = 5); IC50 = 1.6 µM, h = 1.2, a = 0.85 (B, closed circles; n = 5). (C and D) Concentration-response relationships for glibenclamide inhibition of Kir6.2/SUR1 (C) and Kir6.2/SUR2A (D) channels in the presence and absence (data from Fig. 3) of 100 µM MgADP. The lines are the best fit of Eq. 1 to the mean data: IC50 = 2.8 nM, h = 0.93, a = 0.32 (C, open circles; n = 6); IC50 = 3.7 nM, h = 1.2, a = 0.04 (C, closed circles; n = 6); IC50 = 13 nM, h = 0.94, a = 0.32 (D, open circles; n = 5); IC50 = 30 nM, h = 0.75, a = 0.68 (D, closed circles; n = 5). (E and F) Concentration-response relationships for gliclazide inhibition of Kir6.2-G334D/SUR1 (E) and Kir6.2-G334D/SUR2A-YS (F) channels in the presence and absence of 1 mM MgATP. The lines are the best fit of Eq. 1 to the mean data: IC50 = 70 nM, h = 1.0, a = 0.39 (E, open squares; n = 6); IC50 = 210 nM, h = 1.0, a = 0.21 (E, closed squares; n = 6); IC50 = 1.3 µM, h = 1.2, a = 0.64 (F, open squares; n = 5); IC50 = 2.5 µM, h = 1.0, a = 0.91 (F, closed squares; n = 5). Mean ± SEM.
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fig5: Effect of MgADP and MgATP on sulfonylurea inhibition of SUR1- and SUR2A-containing channels. (A–F) Currents in the presence of sulfonylurea (I) expressed as a fraction of that in drug-free solution (Ic). (A and B) Concentration-response relationships for gliclazide inhibition of Kir6.2/SUR1 (A) and Kir6.2/SUR2A-YS (B) channels in the presence and absence (same data as in Fig. 3) of 100 µM MgADP. The lines are the best fit of Eq. 1 to the mean data: IC50 = 72 nM, h = 1.2, a = 0.42 (A, open circles; n = 6); IC50 = 187 nM, h = 1.1, a = 0.07 (A, closed circles; n = 6); IC50 = 1.3 µM, h = 1.1, a = 0.65 (B, open circles; n = 5); IC50 = 1.6 µM, h = 1.2, a = 0.85 (B, closed circles; n = 5). (C and D) Concentration-response relationships for glibenclamide inhibition of Kir6.2/SUR1 (C) and Kir6.2/SUR2A (D) channels in the presence and absence (data from Fig. 3) of 100 µM MgADP. The lines are the best fit of Eq. 1 to the mean data: IC50 = 2.8 nM, h = 0.93, a = 0.32 (C, open circles; n = 6); IC50 = 3.7 nM, h = 1.2, a = 0.04 (C, closed circles; n = 6); IC50 = 13 nM, h = 0.94, a = 0.32 (D, open circles; n = 5); IC50 = 30 nM, h = 0.75, a = 0.68 (D, closed circles; n = 5). (E and F) Concentration-response relationships for gliclazide inhibition of Kir6.2-G334D/SUR1 (E) and Kir6.2-G334D/SUR2A-YS (F) channels in the presence and absence of 1 mM MgATP. The lines are the best fit of Eq. 1 to the mean data: IC50 = 70 nM, h = 1.0, a = 0.39 (E, open squares; n = 6); IC50 = 210 nM, h = 1.0, a = 0.21 (E, closed squares; n = 6); IC50 = 1.3 µM, h = 1.2, a = 0.64 (F, open squares; n = 5); IC50 = 2.5 µM, h = 1.0, a = 0.91 (F, closed squares; n = 5). Mean ± SEM.
Mentions: The relationship between ADP concentration and KATP current inhibition in Fig. 4 and between sulfonylurea concentration and KATP current inhibition in Figs. 3, 5, and 6, was fit with(1)IXIC=a+L−a1+([X]IC50)h,where IX is the steady-state KATP current in the presence of the test nucleotide or drug concentration [X], IC is the current in nucleotide (or drug)-free solution obtained by averaging the current before and after application, IC50 is the nucleotide (drug) concentration at which the inhibition is half maximal, h is the Hill coefficient, and a is the fraction of KATP current remaining at gliclazide concentrations that saturate the high-affinity binding site (for ADP, a = 0). The factor L equals 1 except for data in Fig. 6 (closed symbols), where it reflects the extent of channel activation by Mg-nucleotides in drug-free solution.

Bottom Line: We found that both MgATP and MgADP increased gliclazide inhibition of Kir6.2/SUR1 channels and reduced inhibition of Kir6.2/SUR2A-Y1206S.Mutation of one (or both) of the Walker A lysines in the catalytic site of the nucleotide-binding domains of SUR1 may have a similar effect to gliclazide on MgADP binding and transduction, but it does not appear to impair MgATP binding.Our results have implications for the therapeutic use of sulfonylureas.

View Article: PubMed Central - HTML - PubMed

Affiliation: Oxford Centre for Gene Function and Department of Physiology, Anatomy, and Genetics, University of Oxford, Oxford OX1 3PT, England, UK Oxford Centre for Gene Function and Department of Physiology, Anatomy, and Genetics, University of Oxford, Oxford OX1 3PT, England, UK.

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