Screening of SUR1 residues identifies the E203K mutation that when combined with Q52E in Kir6.2 gives rise to channels with extremely high ATP sensitivity. (A) Charged residues located near the SUR1 sliding helix (see Fig. S1, A and B) were mutated to the opposite charge and tested for their effects on ATP sensitivity with WT (top)-, Q52E (middle)-, and Q52R-Kir6.2 (bottom) using inside-out patch voltage-clamp recording. Current in 1, 0.1, and 0.01 mM ATP relative to nucleotide-free Kint/EDTA is shown with error bars representing SEM. Only 1 mM ATP was tested in the cases of E203K-SUR1//Q52R-Kir6.2 and K205E-SUR1//Q52R-Kir6.2 because of extremely low ATP sensitivity. The number of patches tested is given below each condition. (B) Representative traces from WT or Q52E-Kir6.2//E203K-SUR1 K_ATP channels exposed to various concentrations of ATP or nucleotide-free Kint/EDTA control solution; note difference in ATP concentrations used. Dashed lines represent zero current. (C) Dose–response data illustrating the mean currents in several ATP concentrations relative to maximum in nucleotide-free solution for WT and Q52E-Kir6.2//E203K-SUR1 K_ATP channels. Best fit curves were generated using the Hill equation (IC₅₀: 11 ± 1 µM for WT and 140 ± 5 nM for Q52E-Kir6.2//E203K-SUR1). n = 3–9 for each data point. For comparison, the best fit curve for Q52E-Kir6.2 (see Fig. 1) and mean currents for the three ATP concentrations tested on E203K-SUR1 channels (see A) were also included. Error bars represent SEM, and some are smaller than the symbols.