Representative single channel recordings of K_ATP currents in native cells. Open channel activity is shown as downward deflection. Membrane potential was held at 0 mV. Closed levels of single channels are marked as 0 pA. A: Single channel recordings of I_KATPn in ventricular myocytes isolated from the SUR2 mutant. B: Single channel recordings of conventional I_KATP in ventricular myocytes isolated from WT. C: Sensitivity of I_KATPn to various concentrations of ATP.

Gating kinetics for K_ATPn. A: Open probability for K_ATPn. B: Single channel conductance for K_ATPn. C: Mean burst duration for K_ATPn. D: Open and closed time histogram for K_ATPn in the SUR2 mutant cells or for K_ATP in WT cells (p<0.05). τ₁ is the time constant for closed intervals within bursts while τ₂ the time constant for closed intervals between bursts.

Design of new SUR2 antibodies and specificity tests. A: The 17-transmembrane helix model for SUR2 topology with positions for SUR2 antibodies shown. B: RT-PCR screening in COS1 cells contained a stably expressed KIR6.2 (top panel) or KIR6.1 (bottom panel). In each panel, Lane 1: PCR products amplified from a mouse cDNA library; Lane 2: Water control; Lanes 3−6: RT-PCR products amplified from selected candidates. Arrows indicate expected sizes of RT-PCR products. C: Western blot analysis to confirm COS1 lines stably expressing either a KIR6.2 (positive from Fig. 5B top panel, Lane 5) or a KIR6.1 (positive from Fig. 5B bottom panel, Lane 6). Arrows indicate detected sizes of Kir6.2 and Kir6.1 proteins. D-F: Specificity tests for T1, BNJ-2, BNJ-39 and BNJ-40 antibodies using isolated proteins from COS1 cells stably expressing a Kir6.2 pore and SUR2A, SUR2B or SUR1 cDNA. In all experiments, ∼25 μg of isolated protein isolated was loaded in each lane of a 4−12% MOPS NuPAGE gel. T1 (1: 2000), BNJ-2 (1:1000), BNJ-39 (1:2000) and BNJ-40 (1:1000) were used as primary antibodies. Secondary antibodies were added at 1:10000−1:12500. Arrows indicate detected protein sizes under our gel system and testing conditions.

Western blot analysis using a membrane fraction isolated from the SUR2 mutant heart tissues. A: A schematic diagram of the targeted disruption in the SUR2 gene. The SUR2 antibodies used in the experiments are labeled by vertical arrows. The position of NBD1 is marked by a dotted line while NBD2 is marked by a solid line. B: Western results by four SUR2 antibodies. In all experiments, ∼25 μg of isolated protein was loaded in each lane of a 4−12% MOPS NuPAGE gel. T1 (Lane 1, 1: 2000), BNJ-2 (Lane 2, 1:1000), BNJ-39 (Lane 3, 1:2000) and BNJ-40 (Lane 4, 1:1000) were used as primary antibodies. Secondary antibodies were added at 1:10000−1:12500. Each sample was tested >3 times. Arrows indicate detected protein sizes under our gel system and testing conditions. Lanes were re-arranged for the purpose of presentation. C: Immunocytochemical studies by a confocal microscope in ventricular myocytes isolated from the SUR2 mutant. I & III: Images taken under transmitted light; II & IV: Green fluorescent images taken under an FITC filter. II: BNJ-39 (1:500) and BNJ-40 (1:250) were used as the primary antibodies. IV: Controls where no primary antibody was added. In all panels, the secondary antibody was the anti-rabbit Alexa-Fluor 488 IgG (1:250). Scale bar represents 5 μm. D: Nested exonic RT-PCR to locate transcripts encoding the SUR2 short forms. Positions of the six forward primers (P1−6) and the reverse primer (P7) are labeled by short horizontal arrows (see Fig. 7A). Top panel: PCR products amplified by all primer pairs using SUR2A cDNA as templates. Middle panel: RT-PCR products amplified by all primer pairs using mRNA isolated from WT mouse hearts. The lower band in Lane 2 was a non-specific product after sequencing. Bottom panel: RT-PCR products amplified by all primer pairs using mRNA isolated from the SUR2 mutant hearts. Arrows indicate sizes for amplified PCR or RT-PCR products.

Current density and IC₅₀ for K_ATPn. A: Number of patches that contain K_ATP currents in ventricular myocytes isolated from the SUR2 mutant or WT. B: Mean amplitude of K_ATP currents per patch in the mutant or WT cells (p<0.015). C: Determination of the half-maximal ATP inhibition value (IC₅₀) for K_ATPn in the SUR2 mutant cells or IC₅₀ for K_ATP in WT cells (n=8 per concentration, p<0.05).

Resting potential and response to glibenclamide for K_ATPn. A: Resting potential for K_ATPn. B: I_KATP recorded from WT cells in the absence or presence of 10 μM glibenclamide. Note that one channel was glibenclamide-insensitive. C: I_KATPn recorded from the SUR2 mutant cells showing sensitivity to ATP but resistance to 10 μM glibenclamide. Solution changes were applied as marked by solid lines.

Western blot analysis using a membrane fraction isolated from WT mouse heart tissues. In all experiments, ∼25 μg of isolated protein was loaded in each lane of a 4−12% MOPS NuPAGE gel. Arrows indicate detected protein sizes under our gel system and testing conditions. Lanes were re-arranged for the purpose of presentation due to the numbers of different antibodies and samples tested. A: Western results by markers and controls. Anti-Na/K ATPase (Lane 1, 1:1000), anti-VDAC1 (Lane 2, 1:250) and anti-COXIV (Lane 3, 1:5000) were used as primary antibodies to cross-react with the cardiac membrane fraction. In separate blots, anti-Na_v1.5 (Lane 4, 1: 200) and anti-HCN4 (Lane 5, 1:200) were used as the primary antibodies to cross-react with the same fraction. B: Western results by various SUR2-specific antibodies. T1 (Lane 1, 1: 2000), BNJ-2 (Lane 2, 1:1000), BNJ-39 (Lane 5, 1:2000) and BNJ-40 (Lane 6, 1:1000) were used as primary antibodies to cross-react with the cardiac membrane fraction. BNJ-39 and BNJ-40 were also used to cross-react with a whole heart cell lysate (Lanes 3 & 4). In Lanes 1−4: data shown represents a molecular weight range >100-kDa. Each sample was tested >3 times. C: Co-IP experiments using the WT cardiac membrane fraction. In the forward IP experiment, 5 μg T1 (Lane 1) or control IgG (Lane 2) was used to IP ∼100 μg purified membrane proteins followed by a Western using BNJ-39 (1:2000). In the reverse IP experiment, 5 μg BNJ-39 (Lane 3) or control IgG (Lane 4) was used to IP ∼100 μg purified membrane proteins followed by a Western using T1 (1:2000). Arrows indicate detected protein sizes under our gel system and testing conditions. D: Immunocytochemical studies by a confocal microscope in ventricular myocytes isolated from WT hearts. I & III: Images taken under transmitted light; II & IV: Green fluorescent images taken under an FITC filter. II: BNJ-39 (1:500) and BNJ-40 (1:250) were used as primary antibodies. IV: Controls where no primary antibody was added. In all panels, the secondary antibody was the anti-rabbit Alexa-Fluor 488 IgG (1:250). Scale bar represents 5 μm.

Co-IP results of the SUR2 short forms with Kir6.1 or Kir6.2. 5 μg of a specific antibody or control IgG was used to IP ∼100 μg purified membrane proteins isolated from the SUR2 mutant hearts. A: Control IgG, BNJ-39 and BNJ-40 were used in the forward IP experiments followed by a Western using anti-Kir6.2 (1:200). Anti-Kir6.2 was used in the reverse IP experiments followed by Westerns using either BNJ-39 (1:2000) or BNJ-40 (1:1000). B: Control IgG, BNJ-39 and BNJ-40 were used in the forward IP experiments followed by a Western using anti-Kir6.1 (1:200). Anti-Kir6.1 was used in the reverse IP experiment followed by a Western using BNJ-39 (1:2000). In all panels, secondary antibodies were added at 1:10000−1:12500. Arrows refer to detected protein sizes under our gel system and testing conditions. C: A schematic diagram of each deduced K_ATP complex based on results from this work. (I): 150/Kir6.1 or Kir6.2, (II) 150/28A/Kir6.1 or Kir6.2; (III): 68A/Kir6.1 or Kir6.2; (IV): 28B/Kir6.2.