de Wet H , Shimomura K , Aittoniemi J , Ahmad N , Lafond M , Sansom MS , Ashcroft FM .

076436/Z/05/Z Wellcome Trust , 081188/A/06/Z Wellcome Trust , 084655 Wellcome Trust , WT084655 Wellcome Trust , Wellcome Trust

	Figure 1. Invariant residues in the NBDs of ABC proteinsA, alignment of the NBDs of various ABC proteins showing the invariant glycine residue that lies C terminal to the Walker A motif. Abbreviations for the eukaryotic proteins are: MRP, multidrug resistance related protein; CFTR, cystic fibrosis transmembrane conductance regulator; PgP, P-glycoprotein; Tap, transporter associated with antigen processing. MsbA, ModC, BtuCD and Sav1866 are bacterial ABC proteins. R, rat; h, human. B and C, homology model of the human SUR1-NBD1 and SUR1-NBD2 dimer (B) and SUR1-NBD2 alone (C). The Walker A motif is shown in red, the Walker B motif in purple, and the ABC signature sequence in orange. G1401 is shown as a sphere. NBD1 is shown in silver (right) and NBD2 in blue (left). The Mg2+ atom is shown as a white sphere and ATP (in ball-and-stick format) in green.
	Figure 2. ATPase activity of NBD2 is decreased by the G1401R mutationATPase activity of isolated MBP-NBD2. Wild-type: circles, n= 4. G1401R: triangles, n= 4. The lines are the best fit of the Michaelis–Menten equation to the mean data.
	Figure 3. ATP inhibition of wild-type and G1401R channels in the absence and presence of Mg2+A and B, KATP currents recorded in response to voltage ramps from −110 to +110 mV in an inside-out patch excised from oocytes expressing wild-type (A) or Kir6.2/SUR1-G1401R (B) channels. The dashed line indicates the zero current level. The bar indicates application of 10 μm ATP. C and D, mean relationships between [ATP] and KATP conductance (G), expressed relative to that in the absence of nucleotide (Gc), for wild-type (•, n= 10), and Kir6.2/SUR1-G1401R (o, n= 8) channels in the absence of Mg2+ (C); or for wild-type (•, n= 8) or Kir6.2/SUR1-G1401R (o, n= 21) currents in the presence of Mg2+ (D). The lines are drawn to eqn (1) with the following parameters. C: wild-type, IC50= 4.7 μm, h= 1.2; G1401R, IC50= 7.3 μm, h= 0.8, a= 0.011. D: wild-type, IC50= 12 μm, h= 1.0; G1401R, IC50= 27 μm, h= 1.1, a= 0.03.
	Figure 4. ADP regulation of wild-type and mutant KATP channelsA, mean wild-type (open bar, n= 8) and G1401R (filled bar, n= 7) currents recorded in the presence of 0.1 mm MgATP, 0.1 mm MgADP or 0.1 mm MgATP plus 0.1 mm MgADP. *P < 0.05. B, mean relationships between [ADP] and KATP conductance (G), expressed relative to that in the absence of nucleotide (Gc), for G1401R channels in the absence (o, n= 5) and presence (•, n= 9) of Mg2+. The lines are drawn to eqn (1) with the following parameters: Mg2+-free, IC50= 49 μm, h= 1.3, a= 0.05; with Mg2+, IC50= 105 μm, h= 1.6, a= 0.09.
	Figure 5. Mg-nucleotide activation of wild-type and mutant KATP channelsA and B, mean relationships between [MgADP] (A) or [MgATP] (B) and KATP conductance (G) expressed relative to that in the absence of nucleotide (Gc), for Kir6.2-G334D/SUR1 (o, n= 5) and Kir6.2-G334D/SUR1-G1401R (•, n= 5) channels. The lines through the G1401R data are drawn by eye. The lines through the wild-type data are drawn to eqn (2) with the following parameters: A: EC50= 38 μm, h= 2, a= 10; B: EC50= 196 μm, h= 1.6, a= 4.6.
	Figure 6. ATP inhibition of G1401R-KAKA channelsMean relationship between [ATP] and KATP conductance (G), expressed relative to that in the absence of nucleotide (Gc), for Kir6.2/SUR1-G1401R-KAKA channels (o, n= 12). The solution contained Mg2+. The line is drawn to eqn (1) with the following parameters: IC50= 9 μm, h= 1.1, a= 0.03. The lines through the data obtained for Kir6.2/SUR1-G1401R in the absence of Mg2+ (dotted line) and Kir6.2/SUR1-G1401R in the presence of Mg2+ (dashed line) are also shown.

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