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 [●]).

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