Craig TJ , Ashcroft FM , Proks P .

Wellcome Trust

	Figure 1. Schematic representation of connected tetramers. Schematic representation of the six tetrameric constructs employed. The distribution of wild-type (WT) and mutant K185E subunits is shown, together with the connecting amino acids. The arginine-serine (RS) in the connecting regions is supplied by the fused BglII and BamHI sites used in cloning. Open circles, wild-type Kir6.2 subunits; filled circles, Kir6.2-K185E subunits.
	Figure 2. Three types of models for KATP channel gating. Schematic representation of the different types of models. (A) Four independent gating units (4GU1), each formed by one Kir6.2 subunit. (B) One gating unit formed by all four Kir6.2 subunits (GU4). (C) Two independent gating units, each formed by two Kir6.2 subunits (2GU2). In each case, the transitions between open and closed states are shown for a single gating unit. Each Kir6.2 subunit can be either in an “open” (circle) or “closed” (square) conformation. It is assumed that ATP binds independently to all subunits and A indicates the presence of bound ATP. KO and KC are the binding constants to the open and closed states, respectively. EO is the equilibrium gating constant in the absence of ATP. The term t indicates the change in the equilibrium gating constant due to bound ATP. It is assumed that ATP binding has an additive effect on the energy of the gating unit in both open and closed conformations.
	Figure 3. Predictions for the ATP dependence of the burst ratio for tetrameric constructs based on different models. Relationship between the burst ratio (BR) and ATP concentration for connected KATP tetramers containing between one and four WT ATP-binding sites for channels containing four (A), one (B) or two (C) gating units. These correspond to the HH, MWC, and D models respectively. D depicts the relationship when the first ATP bound has a dominant effect on channel closure. The number of WT ATP-binding sites is indicated to the right of each trace. 2 SD, tetrameric construct with 2WT ATP-binding sites located on the same dimer subunit; 2 DD, tetrameric construct with two WT ATP-binding sites each located on a different dimer subunit. The lines are fit assuming mutant ATP-binding sites are nonfunctional (i.e., m = 1 or KO = 0). The lines are drawn using Eqs. 16–23 given in the Appendix. The KO and m parameters for the wild-type subunits of each model were obtained by fitting the data for unconnected four WT KATP channels (Fig. S12 B, open circles).
	Figure 4. Single-channel recordings. Single-channel currents recorded from inside-out patches at −60 mV in the presence or absence of 1 mM ATP. The composition of each tetramer is shown schematically on the right. Open circles, WT subunits; filled circles, Kir6.2-K185E subunits. The dotted line indicates the zero current level.
	Figure 5. ATP dependence of burst ratio. Relationship between burst ratio and ATP concentration measured experimentally for connected KATP channel tetramers containing different numbers of wild-type and mutant subunits (as indicated to the right of each trace). Each data point is the average of three to five experiments. (A) Linear scale. The lines were generated by fitting Eq. 14 to the data for each tetramer separately. The best fit of Eq. 11 to the data for 4M channels gave values of KO,M = 3.3 mM−1 and mM = 1.13, and these were used for the mutant subunits in all other fits. The best fit of Eq. 14 to KATP channels containing one to four WT subunits was obtained with the following values of KO,WT and mWT: KO,WT = 5.0 mM−1; mWT = 2.7 (1WT:3M); KO,WT = 4.2 mM−1; mWT = 2.7 (2WT:2M cis); KO,WT = 5.3 mM−1; mWT = 2.7 (2WT:2M trans); KO,WT = 2.9 mM−1; mWT = 2.7 (3WT:1M); KO,WT = 3.1 mM−1; mWT = 2.8 (4WT). Log scale. The lines were generated with Eq. 14 using KO,WT = 3.1 mM−1; mWT = 2.8 for wild-type subunits and KO,M = 3.3mM−1; mM = 1.13 for K185E subunits (see text for details).
	Figure 6. Macroscopic properties of connected tetramers. (A) Macroscopic dose–response curves for ATP inhibition of connected KATP channels containing different numbers of WT and K185E (M) subunits (◃, 4WT; ▪, 3WT:1M; •, 2WT:2M cis; ▴, 2WT:2M trans; ▾, 1WT:3M; ♦, 4M). n = 5 for all data. The lines are best fit of the data with Eq. 5 with IC50 = 2.6 mM, h = 1.18, P = 0.48 (4M); IC50 = 690 μM, h = 0.84, P = 0.08 (1WT:3M); IC50 = 14 μM, h = 1.36, P = 0 (4WT); IC50 = 31 μM, h = 1.27, P = 0 (3WT:1M); IC50 = 97 μM, h = 0.94, P = 0 (2WT:2M cis); IC50 = 122 μM, h = 0.98, P = 0 (2WT:2M trans). (B) Pedestal amplitude as a function of the number of wild-type subunits in the connected tetramer. Open circles, 2WT:2M trans; filled circles, all other species. The pedestal was estimated from data in A as (a) the fraction of unblocked current at 30mM ATP or (b) from fitting Eq. 5 to the data (4M, 1WT:3M). Solid line, spline function fit through the data; dotted line, prediction of the MWC model (Eq. 15) with PO = 0.75, F = 0.21, E0 = 0.12 (Table I) and values KC,WT/KO,WT = 51 and KC,M/KO,M = 1.9 obtained from the fit of homomeric wild-type and mutant tetramers.

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