
Figure 1. Nucleotide and sulfonylurea interactions with SUR. (A–D) Schematic showing interactions of nucleotides (A and B) and of nucleotides plus sulfonylureas (C and D) with SUR1 (A and C) and SUR2A (B and D). Minus signs indicate inhibitory effects; plus signs indicate interactions that stimulate channel activity.


Figure 2. Method of quantifying interactions between Mgnucleotides and gliclazide. Representative record showing activation of Kir6.2G334D/SUR1 by MgADP (1 or 3 mM) in the presence and absence of 30 µM gliclazide (as indicated). IMAX,1 and IMAX,2 are the steadystate KATP currents in the presence of a maximal stimulatory concentration of 1 mM MgADP before and after gliclazide application, respectively; I0,1 and I0,2 are the currents in the control (drug and nucleotide free) solution measured immediately before nucleotide application (I0,1) or after (I0,2) gliclazide application; IG is the current in the presence of gliclazide alone; and ING is the steadystate current in the presence of both drug and the test nucleotide concentration. The dotted line indicates the zero current level.


Figure 3. Sulfonylurea inhibition of Kir6.2/SUR1 and Kir6.2/SUR2Acontaining channels. (A and B) Representative KATP currents (top) and concentrationresponse relationships (bottom) for glibenclamide inhibition of Kir6.2/SUR1 (A; n = 6) and Kir6.2/SUR2A (B; n = 6) channels. The lines are the best fit of Eq. 1 to the mean data: (A) IC50 = 2.8 nM, h = 0.93, a = 0.32; (B) IC50 = 13 nM, h = 0.94, a = 0.32. (C and D) KATP currents (top) and concentrationresponse relationships (bottom) for gliclazide block of Kir6.2/SUR1 (C; n = 6) and Kir6.2/SUR2AYS (D; n = 5) channels. The lines are the best fit of Eq. 1 to the mean data: (C) IC50 = 72 nM, h = 1.2, a = 0.42; (D) IC50 = 1.3 µM, h = 1.1, a = 0.65. Mean ± SEM.


Figure 4. Effect of sulfonylureas on (Mg)ADP modulation of SUR1 and SUR2Acontaining channels. (A and B) Concentrationresponse relationships for ADP modulation of Kir6.2/SUR1 channels in the absence (A) or presence (B) of 2 mM Mg2+ and the absence (open symbols) or presence (closed symbols) of 30 µM gliclazide. (A) The lines are the best fit of Eq. 1 to the mean data: IC50 = 64 µM, h = 0.81 (open squares; n = 6); IC50 = 57 µM, h = 0.95 (closed squares; n = 6). (B) The lines are the best fit to the mean data of Eq. 4 with IC50 = 224 µM, h1 = 1.3 and EC50 = 11 µM, h2 = 1.3 and a = 2.7 (open circles; n = 12) or of Eq. 1 with IC50 = 64 µM, h = 0.91 (closed circles; n = 6). (C and D) Concentrationresponse relationships for ADP modulation of Kir6.2/SUR2AYS channels in the absence (C) and presence of 2 mM Mg2+ (D) and in the presence (closed symbols; n = 6) or absence (open symbols; n = 6) of 30 µM gliclazide. (C) The lines are the best fit of Eq. 1 to the mean data: IC50 = 94 µM, h = 1.1 (open squares; n = 6); IC50 = 76 µM, h = 1.2 (closed squares; n = 6). (D) The solid line is the best fit to the mean data of Eq. 4 with IC50 = 240 µM, h1 = 1.2 and EC50 = 19 µM, h2 = 1.5 and a = 1.3. The dotted line is the concentrationinhibition curve for Kir6.2/SUR2AYS channels in the absence of Mg2+ and gliclazide (C, open squares). (E and F) Concentrationresponse relationships for ADP modulation of Kir6.2/SUR2A channels in the absence (E) and presence of 2 mM Mg2+ (F) and in the absence (open symbols) or presence (closed symbols) of 1 µM glibenclamide. (E) The lines are the best fit of Eq. 1 to the mean data: IC50 = 90 µM, h = 0.86 (open squares; n = 6); IC50 = 70 µM, h = 0.95 (closed squares; n = 6). (F) Both open and closed circles are the mean of seven experiments. The solid line is the best fit to the mean data of Eq. 4 with IC50 = 270 µM, h1 = 1.4 and EC50 = 18 µM, h2 = 1.3 and a = 1.0. The dotted line is the concentrationinhibition curve for Kir6.2/SUR2A channels in the absence of Mg2+ and glibenclamide (E, open squares). Mean ± SEM.


Figure 5. Effect of MgADP and MgATP on sulfonylurea inhibition of SUR1 and SUR2Acontaining channels. (A–F) Currents in the presence of sulfonylurea (I) expressed as a fraction of that in drugfree solution (Ic). (A and B) Concentrationresponse relationships for gliclazide inhibition of Kir6.2/SUR1 (A) and Kir6.2/SUR2AYS (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) Concentrationresponse 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) Concentrationresponse relationships for gliclazide inhibition of Kir6.2G334D/SUR1 (E) and Kir6.2G334D/SUR2AYS (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.


Figure 6. Effect of MgADP and MgATP on gliclazide inhibition of Kir6.2/SUR1 and Kir6.2/SUR2AYS. (A–F) Data are the same as in Fig. 5 (A–F), but currents in the presence of gliclazide (I) are expressed as a fraction of that in drug and nucleotidefree solution (Ic). (A and B) Concentrationresponse relationships for gliclazide inhibition of Kir6.2/SUR1 (A) and Kir6.2/SUR2AYS (B) channels in the presence and absence of 100 µM MgADP. The lines are the best fit of Eq. 1 to the mean data: IC50 = 72 nM, h = 1.1, a = 0.42 (A, open circles; n = 6); IC50 = 187 nM, h = 1.1, a = 0.18, L = 2.5 (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 = 1.61, L = 1.7 (B, closed circles; n = 5). (C and D) Concentrationresponse relationships for glibenclamide inhibition of Kir6.2/SUR1 (C) and Kir6.2/SUR2A (D) channels in the presence and absence 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 = 0.12, L = 2.8 (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 = 1.02, L = 1.5 (D, closed circles; n = 5). (E and F) Concentrationresponse relationships for gliclazide inhibition of Kir6.2G334D/SUR1 (E) and Kir6.2G334D/SUR2AYS (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 = 0.42, L = 2.0 (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 = 1.44, L = 1.8 (F, closed squares; n = 5). Mean ± SEM.


Figure 7. Gliclazide reduces MgATP and MgADP activation of Kir6.2G334D/SUR1. (A and C) Representative records showing activation of Kir6.2G334D/SUR1 currents by 1 mM MgADP (A) or 10 mM MgATP (C) in the presence and absence of 30 µM gliclazide (as indicated). The dotted lines indicate the zero current level. (B and D) Concentrationactivation relationships for MgADP (B) or MgATP (D) for Kir6.2G334D/SUR1 channels in the absence (open circles; n = 6) or presence (closed circles; n = 6) of 30 µM gliclazide. (B) The lines are the best fit of Eq. 2 to the mean data. Open circles: EC50 = 9 µM, h = 1.3; a was fixed at 1. Closed circles: EC50 = 560 µM, h = 1.5, a = 0.3. (D) The lines are the best fit to the mean data of Eq. 2 with a set to 1 (open circles: EC50 = 124 µM, h = 1.3) or Eq. 2 with a set to 0.3 (closed circles: EC50 = 8.1 mM, h = 1.3). Mean ± SEM.


Figure 8. Effects of mutations in the WA motif of SUR1 on Mgnucleotide activation. (A and B) Concentrationactivation relationships for MgADP (A) or MgATP (B) for channels composed of Kir6.2G334D and SUR1 (open circles; n = 6), SUR1KAKA (closed circles; n = 5), SUR1K1A (closed squares; n = 5), or SUR1K2A (open squares; n = 5) channels. The solid lines are the best fit of Eq. 3 to the mean data (A, open circles: a = 0.84, EC50 = 9 µM, h = 1.3; B, open circles: a = 0.88, EC50 = 124 µM, h = 1.3; closed squares: a = 0.52, EC50 = 150 µM, h = 1.3; open squares: a = 0.40, EC50 = 200 µM, h = 1.1; closed circles: a = 0.05, EC50 = 150 µM, h = 1.3) except for data for channels containing mutant SUR1 subunits in A, where the lines are drawn by hand. The dotted lines indicate the relationship obtained for Kir6.2G334D/SUR1 in the presence of 30 µM gliclazide (shown in Fig. 7). Mean ± SEM.


Figure 9. Simulations of the fractional occupancy of NBS2 by MgADP. (A and B) Simulations of the fractional occupancy of NBS2 by MgADP in the presence of either MgADP (A; Eq. 5) or MgATP (B; Eq. 6). The model and the values of the rate constants were taken from Bienengraeber et al. (2004). Calculated control curves for MgADP and MgATP predicted EC50 values for MgADP occupancy of NBS2 that were very similar to those measured experimentally for channel activation. (A) To simulate the effect of gliclazide or the K1A and K2A mutations (KA mutations) on MgADP activation, we assumed a threefold increase in the off rate for MgADP binding in the presence of gliclazide (as measured experimentally) or when K1A or K2A was mutated. A 30fold decrease in the on rate of MgADP binding in the presence of gliclazide (or the K1A or K2A mutation) predicted the measured EC50 for MgADP activation in the presence of gliclazide. (B) We used the same values for the rate constants for MgADP binding as in A and assumed the binding affinity of MgATP was reduced by gliclazide to the same extent as MgADP binding. This resulted in a predicted EC50 for the fractional occupancy of NBS2 by MgADP in the presence of gliclazide of 8 mM, which was the same as that measured experimentally for MgATP activation. To model the effect of the K1A and K2A mutations, we assumed the mutations had no effect on MgATP binding (as observed experimentally) and that they reduced the rate of ATP hydrolysis 100fold. This reduced the maximal fractional occupancy of NBS2 by MgADP, but had no effect on the EC50 (as observed experimentally).
