Proks P , de Wet H , Ashcroft FM .

089795/Z/09/Z Wellcome Trust , 084655 Wellcome Trust , WT084655 Wellcome Trust

	FIG. 1. The mechanism of sulphonylurea block of KATP channels. A: Schematic of the interaction of Mg-nucleotides with Kir6.2 and SUR1 subunits in the absence (left) and presence (right) of sulphonylureas for wild-type channels and channels carrying an ATP-insensitive mutation (G334) in Kir6.2. Nucleotides reduce the PO of wild-type channels by binding to Kir6.2 and increase PO by interacting with SUR1 (top left). Sulphonylureas inhibit channel activity by binding to SUR1. They also displace MgADP from SUR1, and consequently, ATP block at Kir6.2 is not balanced by MgATP activation at SUR1 (top right). Channels with a mutation that abolishes (G334D) ATP inhibition at Kir6.2 are activated but not blocked by ADP or MgADP (bottom left). Sulphonylureas still inhibit channel activity. They also displace MgADP from SUR1, abolishing activation; however, the total block is less because of the lack of ATP inhibition at Kir6.2 (bottom right). B: Concentration-response curves for gliclazide block of macroscopic KATP currents in excised patches in the absence (○; n = 4–8) and presence (●; n = 7–12) of 100 μmol/L MgADP. The lines are the best fit of Eq. 2 to the mean data, with IC50(1) = 50 nmol/L, IC50(2) = 3 mmol/L, h1 = h2 = 1, and a = 0.4 (0 µmol/L MgADP), or to Eq. 1, with IC50 = 204 nmol/L, h = 1.13, and a = 0.065 (100 µmol/L MgADP). Data are taken from Gribble and Ashcroft (23) and Proks et al. (33). SU, sulphonylurea.
	FIG. 2. Effect of gliclazide on ATP block of wild-type and R201C KATP channels. A–D: Representative Kir6.2/SUR1 (A and C) and Kir6.2-R201C/SUR1 (B and D) currents recorded at −60 mV in the absence (A and B) and presence (C and D) of Mg2+. Gliclazide and ATP were added as indicated by the bars. The dotted line indicates the zero current level. E and F: ATP concentration-inhibition curves for Kir6.2/SUR1 (E) and Kir6.2-R201C/SUR1 (F) channels measured in the absence of both gliclazide (SU) and Mg2+ (□) and in the absence of gliclazide but in the presence of 2 mmol/L Mg2+ (○), 30 μmol/L gliclazide without Mg2+ (■), and 30 μmol/L gliclazide plus 2 mmol/L Mg2+ (●). The lines are the best fit of Eq. 1 to the mean data with the following parameters: Kir6.2/SUR1 (E) IC50 = 6.8 μmol/L, h = 1.0 (□); IC50 = 4 μmol/L, h = 1.0 (■); IC50 = 16 μmol/L, h = 1.0 (○); and IC50 = 4 μmol/L, h = 0.97 (●) and Kir6.2-R201C/SUR1 (F): IC50 = 98 μmol/L, h = 1.4 (□); IC50 = 2.0 mmol/L, h = 1.1 (○); IC50 = 97 μmol/L, h = 0.87 (■); and IC50 = 107 μmol/L, h = 0.90 (●). The dotted line is the concentration-inhibition curve for Kir6.2/SUR1 channels in the absence of Mg2+ and gliclazide (Fig. 2E) (□). SU, sulfonylurea; WT, wild-type.
	FIG. 3. Effect of gliclazide on ATP block of mutant KATP channels with impaired gating. ATP concentration-inhibition curves for Kir6.2-V59M/SUR1 (A) and Kir6.2-I296L/SUR1 (B) channels were measured in the absence of both gliclazide and Mg2+ (□) and in the absence of gliclazide but the presence of 2 mmol/L Mg2+ (○), 30 μmol/L gliclazide without Mg2+ (■), and 30 μmol/L gliclazide plus 2 mmol/L Mg2+ (●). The dotted line is the concentration-inhibition curve for Kir6.2/SUR1 channels in the absence of Mg2+ and gliclazide (Fig. 2E, □). The lines are the best fit of Eq. 1 to the mean data with the following parameters: Kir6.2-V59M/SUR1 (A): IC50 = 62 μmol/L, h = 1.0 (□); IC50 = 510 μmol/L, h = 0.7 (○); IC50 = 40 μmol/L, h = 1.1 (■); and IC50 = 46 μmol/L, h = 1.1 (●) and Kir6.2-I296L/SUR1 (B): IC50 = 2.8 mmol/L, h = 0.76 (□); IC50 = 2.3 mmol/L, h = 0.86 (■); and IC50 = 2.4 mmol/L, h = 1.0 (●). The line through the open circles was drawn by hand. SU, sulfonylurea; WT, wild-type.
	FIG. 4. ATP modulation of gliclazide block of wild-type KATP channels and mutant channels with impaired ATP binding to Kir6.2. A–C: Gliclazide concentration-inhibition relations for wild-type and mutant channels in the absence (○) and presence (●) of MgATP. Currents are expressed relative to those in the absence of gliclazide. The MgATP concentration was 15 μmol/L for Kir6.2/SUR1, 1 mmol/L for Kir6.2-G334D/SUR1, and 100 μmol/L for Kir6.2-R201C/SUR1, respectively. The lines are the best fit of Eq. 1 to the mean data with the following parameters: Kir6.2/SUR1 (A): IC50 = 67 nmol/L, h = 1.3, a = 0.45 (○) and IC50 = 71 nmol/L, h = 1.0, a = 0.20 (●); Kir6.2-G334D/SUR1 (B): IC50 = 67 nmol/L, h = 1.1, a = 0.39 (○) and IC50 = 213 nmol/L, h = 1.0, a = 0.21 (●); and Kir6.2-R201C/SUR1 (C): IC50 = 49 nmol/L, h = 1.2, a = 0.48 (○) and IC50 = 190 nmol/L, h = 1.2, a = 0.27 (●). n = 6 in all experiments.
	FIG. 5. ATP modulation of gliclazide block of mutant KATP channels with impaired gating. A and B: Gliclazide concentration-inhibition relations for Kir6.2-V59M/SUR1 (n = 6) and Kir6.2-I296L/SUR1 (n = 6) channels in the absence (○) and presence (●) of MgATP. The MgATP concentration was 100 μmol/L for Kir6.2-V59M/SUR1 and 3 mmol/L for Kir6.2-I296L/SUR1. The lines are the best fit of Eq. 1 to the mean data with the following parameters: Kir6.2-V59M/SUR1 (A): IC50 = 200 nmol/L, h = 0.92, a = 0.79 (○) and IC50 = 140 nmol/L, h = 0.88, a = 0.39 (●) and Kir6.2-I296L/SUR1 (B): IC50 = 930 nmol/L, h = 1.5, a = 0.95 (○) and IC50 = 1,200 nmol/L, h = 0.98, a = 0.41 (●). C and D: Simulation of the dependence of high-affinity gliclazide block on PO in nucleotide-free solutions with a Monod-Wyman-Changeux model. C: Fractional block of KATP current, as a function of gliclazide concentration, for different values of PO. Currents are expressed relative to those in the absence of gliclazide. The lines are drawn to the following: (1 + F + E0) × (1 + [S]/Kd,O)4/ [(1 + F) × (1 + [S]/Kd,O)4 + E0 × (1 + [S]/Kd,C)4], where F = 0.16 is the equilibrium gating constant for the fast intraburst transitions (from maximal PO of 0.86 when E0 = 0), E0 = [1 − PO × (1 + F)] / PO is the equilibrium gating constant for the slow interburst transitions, [S] is the gliclazide concentration, and Kd,O = 70 nmol/L and Kd,C = 50 nmol/L are the dissociation constants for gliclazide binding to the open and closed states, respectively. D: Monod-Wyman-Changeux model for gliclazide-dependent gating of KATP channels used to derive the current traces in C. This model is the simplest mechanism of concerted gating, which has previously been shown to occur in KATP channels (48–50). KC, sulphonylurea binding constant for the closed state; KO, sulphonylurea binding constant for the open state; S, sulphonylurea; t, proportionality factor reflecting the change in the equilibrium gating constant E0 when sulphonylurea is bound to the channel (t = KO/KC).
	FIG. 6. ATP modulation of gliclazide block of wild-type and mutant KATP channels. A–E: Gliclazide concentration-inhibition relations for wild-type and mutant channels in the absence (○) and presence (●) of MgATP. Currents are expressed relative to those in the absence of both MgATP and gliclazide. MgATP concentrations were 15 μmol/L (A, Kir6.2/SUR1), 100 μmol/L (B, Kir6.2-R201C/SUR1), 1 mmol/L (C, Kir6.2-G334D/SUR1), 100 μmol/L (D, Kir6.2-V59M/SUR1), and 3 mmol/L (E, Kir6.2-I296L/SUR1). The lines are the best fit of Eq. 1 to the mean data with the following parameters: Kir6.2/SUR1 (A): IC50 = 67 nmol/L, h = 1.3, a = 0.45 (○) and IC50 = 71 nmol/L, h = 1.0, a = 0.10 (●); Kir6.2-R201C/SUR1 (B): IC50 = 67 nmol/L, h = 1.1, a = 0.39 (○) and IC50 = 190 nmol/L, h = 1.2, a = 0.25 (●); Kir6.2-G334D/SUR1 (C): IC50 = 140 nmol/L, h = 0.88, a = 0.31 (○) and IC50 = 213 nmol/L, h = 1.0, a = 0.39 (●); Kir6.2-V59M/SUR1 (D): IC50 = 200 nmol/L, h = 0.92, a = 0.79 (○) and IC50 = 49 nmol/L, h = 1.2, a = 0.48 (●); and Kir6.2-I296L/SUR1 (E): IC50 = 930 nmol/L, h = 1.5, a = 0.95 (○) and IC50 = 1,200 nmol/L, h = 0.98, a = 0.38 (●). n = 6 in all experiments. Note that in the absence of gliclazide Kir6.2-G334D/SUR1 currents are greater in the presence of ATP than in the absence of ATP because the G334D mutation abolishes the inhibitory effect of ATP at Kir6.2, leaving only the stimulatory effect at SUR1 (C). In contrast, currents are smaller in the presence of MgATP for all other channels because both inhibition and activation are present. F and G: Current remaining in the presence of 100 μmol/L MgATP or 1 mmol/L MgATP in the absence (open bars) and presence (solid bars) of 30 μmol/L gliclazide for wild-type channels and KATP channels carrying ND mutations. The current is expressed as a fraction of that in drug- and nucleotide-free solution. n = 6 in all experiments. WT, wild-type.
	FIG. 7. Reduction of gliclazide block by nucleotides. A: Mean ± SEM current remaining in the presence of 100 nmol/L gliclazide and nucleotides (n = 6) for Kir6.2-G334D/SUR1 or Kir6.2-G334D/SUR1-KAKA (KAKA) channels. B: Fractional block of Kir6.2-G334D/SUR1-KAKA currents as a function of gliclazide concentration in the absence (○; n = 6) and presence (●; n = 6) of 1 mmol/L MgATP or 1 mmol/L MgADP (▲; n = 6) and in the absence (□; n = 6) and presence (■; n = 6) of 1 mmol/L ATP (Mg-free solution). The lines are the best fit of Eq. 1 to the mean data with the following parameters: IC50 = 63 nmol/L, h = 1.1, a = 0.42 (○); IC50 = 15 nmol/L, h = 1.2, a = 0.46 (●); IC50 = 75 nmol/L, h = 1.2, a = 0.42 (▲); IC50 = 64 nmol/L, h = 1.2, a = 0.29 (□); and IC50 = 67 nmol/L, h = 1.2, a = 0.29 (■). A and B: Experiments were performed in the inside-out patch configuration. C: Dependence of single-channel PO on gliclazide concentration for Kir6.2-G334D/SUR1 channels (control [●]; n = 6) and Kir6.2-G334D/SUR1-KAKA channels (KAKA [○]; n = 6) measured in cell-attached patches. The lines are the best fit of the following to the mean data: , where PO(0) is the single-channel PO in the absence of gliclazide, [X] is the gliclazide concentration, IC50 is the gliclazide concentration at which the inhibition is half maximal, h is the Hill coefficient, and a is the fractional PO at gliclazide concentrations that saturate the high-affinity SUR1 site. PO(0) = 0.76, IC50 = 440 nmol/L, h = 0.92, a = 0.47 (●) and PO(0) = 0.59, IC50 = 30 nmol/L, h = 1.0, a = 0.60 (●). D: Mean ± SEM relationship between glibenclamide and the whole-cell KATP current measured in β-cells from control mice (○; n = 6 cells, three mice) and mice in which the Kir6.2-V59M mutation was induced (V59M [●]; n = 6 cells, three mice). Current (I) is expressed relative to that in drug-free solution (IC). The curves are the best fit of Eq. 2 to the mean data with the following parameters: IC50(1) = 2 nmol/L, h1 = 1.3, IC50(2) = 45 μmol/L, h2 = 1, a = 0.03 (control [○]) and IC50(1) = 6 nmol/L, h1 = 0.74, IC50(2) = 80 μmol/L, h2 = 1, a = 0.1 (V59M [●]).

Abdelmoneim, Variations in tissue selectivity amongst insulin secretagogues: a systematic review. 2012, Pubmed

Abdelmoneim, Variations in tissue selectivity amongst insulin secretagogues: a systematic review. 2012, Pubmed
Abraham, Coupling of cell energetics with membrane metabolic sensing. Integrative signaling through creatine kinase phosphotransfer disrupted by M-CK gene knock-out. 2002, Pubmed
Ashcroft, Properties of single potassium channels modulated by glucose in rat pancreatic beta-cells. 1988, Pubmed
Ashcroft, Electrophysiology of the pancreatic beta-cell. 1989, Pubmed
Ashcroft, New uses for old drugs: neonatal diabetes and sulphonylureas. 2010, Pubmed
Babenko, A novel ABCC8 (SUR1)-dependent mechanism of metabolism-excitation uncoupling. 2008, Pubmed
Babenko, Activating mutations in the ABCC8 gene in neonatal diabetes mellitus. 2006, Pubmed
Barrett-Jolley, Kinetic analysis of the inhibitory effect of glibenclamide on KATP channels of mammalian skeletal muscle. 1997, Pubmed
Codner, Sulfonylurea treatment in young children with neonatal diabetes: dealing with hyperglycemia, hypoglycemia, and sick days. 2007, Pubmed
Craig, How ATP inhibits the open K(ATP) channel. 2008, Pubmed , Xenbase
de Wet, Studies of the ATPase activity of the ABC protein SUR1. 2007, Pubmed
Dor, Adult pancreatic beta-cells are formed by self-duplication rather than stem-cell differentiation. 2004, Pubmed
Drain, Concerted gating mechanism underlying KATP channel inhibition by ATP. 2004, Pubmed , Xenbase
Drain, ATP and sulfonylurea linkage in the K(ATP) channel solves a diabetes puzzler. 2013, Pubmed
Drain, KATP channel inhibition by ATP requires distinct functional domains of the cytoplasmic C terminus of the pore-forming subunit. 1998, Pubmed , Xenbase
Gillis, Effects of sulfonamides on a metabolite-regulated ATPi-sensitive K+ channel in rat pancreatic B-cells. 1989, Pubmed
Girard, Expression of an activating mutation in the gene encoding the KATP channel subunit Kir6.2 in mouse pancreatic beta cells recapitulates neonatal diabetes. 2009, Pubmed
Gloyn, Activating mutations in the gene encoding the ATP-sensitive potassium-channel subunit Kir6.2 and permanent neonatal diabetes. 2004, Pubmed , Xenbase
Göpel, Voltage-gated and resting membrane currents recorded from B-cells in intact mouse pancreatic islets. 1999, Pubmed
Gribble, Differential sensitivity of beta-cell and extrapancreatic K(ATP) channels to gliclazide. 1999, Pubmed , Xenbase
Gribble, Sulphonylurea action revisited: the post-cloning era. 2003, Pubmed
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, The interaction of nucleotides with the tolbutamide block of cloned ATP-sensitive K+ channel currents expressed in Xenopus oocytes: a reinterpretation. 1997, Pubmed , Xenbase
Gribble, MgATP activates the beta cell KATP channel by interaction with its SUR1 subunit. 1998, Pubmed , Xenbase
Hambrock, Glibenclamide binding to sulphonylurea receptor subtypes: dependence on adenine nucleotides. 2002, Pubmed
Hattersley, Activating mutations in Kir6.2 and neonatal diabetes: new clinical syndromes, new scientific insights, and new therapy. 2005, Pubmed
Henquin, Regulation of insulin secretion: a matter of phase control and amplitude modulation. 2009, Pubmed
Jones, Reevaluation of a case of type 1 diabetes mellitus diagnosed before 6 months of age. 2010, Pubmed
Koster, ATP and sulfonylurea sensitivity of mutant ATP-sensitive K+ channels in neonatal diabetes: implications for pharmacogenomic therapy. 2005, Pubmed
Koster, The G53D mutation in Kir6.2 (KCNJ11) is associated with neonatal diabetes and motor dysfunction in adulthood that is improved with sulfonylurea therapy. 2008, Pubmed
Krauter, Phospholipids as modulators of K(ATP) channels: distinct mechanisms for control of sensitivity to sulphonylureas, K(+) channel openers, and ATP. 2001, Pubmed , Xenbase
Masia, An ATP-binding mutation (G334D) in KCNJ11 is associated with a sulfonylurea-insensitive form of developmental delay, epilepsy, and neonatal diabetes. 2007, Pubmed
Matsuo, KATP channel interaction with adenine nucleotides. 2005, Pubmed
McTaggart, The role of the KATP channel in glucose homeostasis in health and disease: more than meets the islet. 2010, Pubmed
Misler, A metabolite-regulated potassium channel in rat pancreatic B cells. 1986, Pubmed
Nakanishi, A wortmannin-sensitive phosphatidylinositol 4-kinase that regulates hormone-sensitive pools of inositolphospholipids. 1995, Pubmed
Nichols, Adenosine diphosphate as an intracellular regulator of insulin secretion. 1996, Pubmed
Ortiz, Two neonatal diabetes mutations on transmembrane helix 15 of SUR1 increase affinity for ATP and ADP at nucleotide binding domain 2. 2012, Pubmed
Pearson, Switching from insulin to oral sulfonylureas in patients with diabetes due to Kir6.2 mutations. 2006, Pubmed , Xenbase
Proks, A gating mutation at the internal mouth of the Kir6.2 pore is associated with DEND syndrome. 2005, Pubmed , Xenbase
Proks, Functional effects of KCNJ11 mutations causing neonatal diabetes: enhanced activation by MgATP. 2005, Pubmed , Xenbase
Proks, Activation of the K(ATP) channel by Mg-nucleotide interaction with SUR1. 2010, Pubmed , Xenbase
Proks, Sulfonylurea stimulation of insulin secretion. 2002, Pubmed
Proks, Molecular basis of Kir6.2 mutations associated with neonatal diabetes or neonatal diabetes plus neurological features. 2004, Pubmed , Xenbase
Rafiq, Effective treatment with oral sulfonylureas in patients with diabetes due to sulfonylurea receptor 1 (SUR1) mutations. 2008, Pubmed
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
Sakura, Glucose modulation of ATP-sensitive K-currents in wild-type, homozygous and heterozygous glucokinase knock-out mice. 1998, 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
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