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J Physiol
2018 Dec 01;59624:6205-6217. doi: 10.1113/JP276708.
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Role of the C-terminus of SUR in the differential regulation of β-cell and cardiac KATP channels by MgADP and metabolism.
Vedovato N
,
Rorsman O
,
Hennis K
,
Ashcroft FM
,
Proks P
.
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KEY POINTS: β-Cell KATP channels are partially open in the absence of metabolic substrates, whereas cardiac KATP channels are closed. Using cloned channels heterologously expressed in Xenopus oocytes we measured the effect of MgADP on the MgATP concentration-inhibition curve immediately after patch excision. MgADP caused a far more striking reduction in ATP inhibition of Kir6.2/SUR1 channels than Kir6.2/SUR2A channels; this effect declined rapidly after patch excision. Exchanging the final 42 amino acids of SUR was sufficient to switch the Mg-nucleotide regulation of Kir6.2/SUR1 and Kir6.2/SUR2A channels, and partially switch their sensitivity to metabolic inhibition. Deletion of the C-terminal 42 residues of SUR abolished MgADP activation of both Kir6.2/SUR1 and Kir6.2/SUR2A channels. We conclude that the different metabolic sensitivity of Kir6.2/SUR1 and Kir6.2/SUR2A channels is at least partially due to their different regulation by Mg-nucleotides, which is determined by the final 42 amino acids.
ABSTRACT: ATP-sensitive potassium (KATP ) channels couple the metabolic state of a cell to its electrical activity and play important physiological roles in many tissues. In contrast to β-cell (Kir6.2/SUR1) channels, which open when extracellular glucose levels fall, cardiac (Kir6.2/SUR2A) channels remain closed. This is due to differences in the SUR subunit rather than cell metabolism. As ATP inhibition and MgADP activation are similar for both types of channels, we investigated channel inhibition by MgATP in the presence of 100 μm MgADP immediately after patch excision [when the channel open probability (PO ) is near maximal]. The results were strikingly different: 100 μm MgADP substantially reduced MgATP inhibition of Kir6.2/SUR1, but had no effect on MgATP inhibition of Kir6.2/SUR2A. Exchanging the final 42 residues of SUR2A with that of SUR1 switched the channel phenotype (and vice versa), and deleting this region abolished Mg-nucleotide activation. This suggests the C-terminal 42 residues are important for the ability of MgADP to influence ATP inhibition at Kir6.2. This region was also necessary, but not sufficient, for activation of the KATP channel in intact cells by metabolic inhibition (azide). We conclude that the ability of MgADP to impair ATP inhibition at Kir6.2 accounts, in part, for the differential metabolic sensitivities of β-cell and cardiac KATP channels.
Figure 1. MgATP sensitivity of Kir6.2/SUR1 and Kir6.2/SUR2A channelsRepresentative traces showing time points at which rundown (A) and instantaneous (B) ATP sensitivities were measured for Kir6.2/SUR1. The arrow indicates patch excision. In the case of the instantaneous concentration–response relationship only a single ATP concentration was tested per patch, whereas a full concentration–response relationship was performed in each patch following rundown. C and D, rundown (C) and instantaneous (D) concentration–response relationships for MgATP inhibition of Kir6.2/SUR1 (circles) or Kir6.2/SUR2A channels (squares). Current is measured in excised patches and expressed relative to that in the absence of nucleotide. The continuous lines are the best fit of eqn (1) to the mean data. C: ●, IC
50 = 13.9 μm, h = 1.1 (n = 8); ■, IC
50 = 28.7 μm, h = 1.3 (n = 8). D: ●, IC
50 = 23.6 μm, h = 0.82 (n = 5); ■, IC
50 = 66.8 μm, h = 1.2 (n = 5).
Figure 2. Effects of MgADP on MgATP inhibition of Kir6.2/SUR1 and Kir6.2/SUR2A channels
A and B, representative traces of Kir6.2/SUR1 currents recorded in the continuous presence of 100μM MgADP and (as indicated) 1mM MgATP following rundown (A) or immediately after patch excision (B). Rundown (C) and instantaneous (D) concentration–response relationships for MgATP inhibition of Kir6.2/SUR1 channels in the absence (open symbols) and presence (filled symbols) of 100 μm MgADP. C: ○, IC
50 = 14 μm, h = 1.1 (n = 8); ●, IC
50 = 120 μm, h = 1.5 (n = 6). D: ○, IC
50 = 24 μm, h = 0.83 (n = 6); ●, IC
50 = 504 μm, h = 1.4 (n = 6). E and F, representative traces of Kir6.2/SUR2A currents recorded in the continuous presence of 100 μM MgADP and (as indicated) 1 mM MgATP following rundown (E) or immediately after patch excision (F). Rundown (G) and instantaneous (H) concentration–response relationships for MgATP inhibition of Kir6.2/SUR2A channels in the absence (open symbols) and presence (filled symbols) of 100 μm MgADP. G: □, IC
50 = 29€μm, h = 1.3 (n = 6); ■, IC
50 = 59 μm, h = 1.1 (n = 6). H: □, IC
50 = 67 μm, h = 1.2 (n = 6); ■, IC
50 = 64 μm, h = 1.2 (n = 6). Currents are expressed relative to that in the absence of MgATP. The continuous lines are the best fit of eqn (1) to the mean data.
Figure 3. Comparison of nucleotide sensitivity in inside‐out and cell‐attached patches
A and B, MgATP inhibition of Kir6.2/SUR1 (●) and Kir6.2/SUR2A (■) channels in the presence of 100 μm MgADP measured after rundown (A) or immediately after patch excision (B). Data as in Fig. 2. The grey box indicates the physiological range of MgATP concentrations (1–10 mm). C–F, representative KATP currents recorded at −60 mV in a cell‐attached (above) and subsequently inside‐out (below) patch from Xenopus oocytes expressing Kir6.2/SUR1 (C,E) or Kir6.2/SUR2A (D,F). Oocytes were preincubated in 3 mm azide for 30 min prior to the recording. The number of channels in E and F was estimated by noise analysis (see Methods). The dotted line represents the zero current level.
Figure 4. The C‐terminus modulates nucleotide interactions with the KATP channels (data from instantaneous measurements in inside‐out patches)
A, sequence alignment of the C‐termini of rat SUR1, rat SUR2A and rat SUR2B. The grey box indicates the 7 amino acids previously identified by Matsushita et al. (2002). B, concentration–response relationships for MgATP inhibition of Kir6.2/SUR1 (●), Kir6.2/SUR2A (■), Kir6.2/SUR1‐T2A (○) and Kir6.2/SUR2A‐T1 (□) channels measured in the presence of 100 μm MgADP immediately after patch excision. ●, IC
50 = 504 μm, h = 1.4 (n = 6); ■, IC
50 = 58.8 μm, h = 1.1 (n = 6); ○, IC
50 = 430 μm, h = 1.5 (n = 6); □, IC
50 = 74.4 μm, h = 1.3 (n = 6). C, concentration–response relationships for MgATP inhibition of Kir6.2/SUR1 (●), Kir6.2/SUR2A (■), Kir6.2/SUR1‐7aa (○) and Kir6.2/SUR2A‐7aa (□) channels measured in the presence of 100 μm MgADP immediately after patch excision. ●, IC
50 = 504 μm, h = 1.4 (n = 6); ■, IC
50 = 58.8 μm, h = 1.1 (n = 6); ○, IC
50 = 430 μm, h = 1.3 (n = 6); □, IC
50 = 63 μm, h = 1.2 (n = 6).
Figure 5. Metabolic inhibition of wild‐type and chimeric KATP channels
A–D, representative whole‐cell currents recorded from Xenopus oocytes expressing Kir6.2/SUR1 (A), Kir6.2/SUR2A (B), Kir6.2/SUR1‐T2A (C) or Kir6.2/SUR2A‐T1 (D) channels. The horizontal bars indicate the presence of sodium azide (3 mm), K‐channel openers (340 μm diazoxide or 100 μm pinacidil) and sulphonylureas (0.5 mm tolbutamide or 50 μm glibenclamide). Different activators and inhibitors were used because pinacidil is specific for SUR2A and tolbutamide for SUR1. The dotted line indicates the zero current level. E, mean whole‐cell currents recorded before (dark grey bar) and after (grey bar) azide application, then after addition of a K‐channel opener (white bar) and finally in the presence of azide and a sulphonylurea (black bar). F, surface expression of Kir6.2‐HA/SUR1 and Kir6.2‐HA/SUR2A KATP channels (white bars) plotted as relative luminescence units (RLU) measured in the same batch of oocytes as in E. Black bars, control oocyte expressing Kir6.2/SUR1 and Kir6.2/SUR2A. G, mean whole‐cell currents normalised to the average RLU. H, mean whole‐cell currents recorded from Xenopus oocytes before (dark grey bar) and after (grey bar) azide application, and in the presence of azide + sulphonylurea (black bar), expressed as a percentage of that in the presence of azide + K‐channel opener. Numbers in parentheses denote the number of experiments. **
P < 0.01; ***
P < 0.001.
Figure 6. The last 42 residues of SUR are required for MgADP activation
A, representative Kir6.2/SUR1Δ42 (left) and Kir6.2/SUR2AΔ42 (right) currents recorded at −60 mv in inside‐out patches from Xenopus oocytes; 100 μm MgADP was added as indicated. The dotted line indicates the zero current level. B, surface expression of HA‐tagged (white) and untagged (black) Kir6.2/SUR1, Kir6.2/SUR1Δ42, Kir6.2/SUR2A and Kir6.2/SUR2AΔ42 KATP channels, plotted as relative luminescence units (RLU) (n = 6–13). **
P < 0.01; ***
P < 0.001. C, fractional currents remaining in the presence of 100 μm ADP recorded at −60 mV in inside‐out patches from Xenopus oocytes expressing wild‐type (WT) or truncated (Δ42) KATP channels, in the presence or absence of Mg2+. White bars: SUR1‐containing channels; black bars: SUR2A‐containing channels. Current is expressed as a fraction of that in nucleotide‐free solution (n = 6–13).
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