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Acta Pharmacol Sin
2016 Jan 01;371:134-42. doi: 10.1038/aps.2015.134.
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Sensitivity of KATP channels to cellular metabolic disorders and the underlying structural basis.
Li CG
,
Cui WY
,
Wang H
.
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ATP-sensitive potassium (KATP) channels formed by a combination of SUR/Kir6.x subunits play a crucial role in protection against hypoxic or ischemic injuries resulting from cell metabolic disorders. In this study we investigated the effects of Na-azide, a metabolic inhibitor, on KATP channels expressed in Xenopus oocytes, and explored the structure basis for their sensitivity to cell metabolic disorders. Six subtypes of KATP channels (wild SUR1/Kir6.2, SUR2B/Kir6.2, SUR1/Kir6.1, SUR2B/Kir6.1, SUR2A/Kir6.2 and SUR2A/Kir6.1), as well as eleven subtypes of KATP channels with mutant subunits were expressed in Xenopus oocytes. KATP currents were recorded using a two-electrode voltage clamp recording technique. The drugs were applied through bath. Except SUR2A/Kir6.1, five subtypes of KATP channels were activated by Na-azide (3 mmol/L) with an order of the responses: SUR1/Kir6.2>SUR2B/Kir6.2>SUR1/Kir6.1>SUR2B/Kir6.1>SUR2A/Kir6.2, and the opening rate (t1/2) was SUR1/Kir6.x>SUR2B/Kir6.x>SUR2A/Kir6.2. Furthermore, Kir6.2, rather than Kir6.1, had intrinsic sensitivity to Na-azide, and the residues involved in ATP-binding (R50 and K185) or pH-sensing (H175) were associated with the sensitivity of the Kir6.2 subunit to Na-azide. Moreover, the residues (K707 and K1348) within the Walker A (WA) motifs of two nucleotide-binding domains (NBDs) were essential for SUR2B/Kir6.x (especially SUR2B/Kir6.1) channel activation by Na-azide, suggesting a key role for Mg-adenine nucleotide binding and/or hydrolysis in the SUR2B subunit. Among the six subtypes of KATP channels, SUR1/Kir6.2 is the most sensitive, whereas SUR2A/Kir6.1 is insensitive, to cell metabolic disorders. The Kir6.2 subunit, rather than the Kir6.1 subunit, has intrinsic sensitivity to cell metabolic disorders. The residues (K707 and K1348) within the WA motifs of SUR2B are important for the sensitivity of SUR2B/Kir6.x channels to cell metabolic disorders.
Figure 1. Effects of metabolic disorders induced by Na-azide on SUR/Kir6.x channels expressed in Xenopus oocytes. (A) The time courses of metabolic disorders induced by Na-azide at 3 mmol/L on the intracellular concentrations of ATP or (B) the ratio between ADP and ATP in Xenopus oocytes. Each value is the mean of 3–5 samples, each consisting of 5 oocytes (ie, n=15–25). bP<0.05, cP<0.01 vs control (0 min). (C) The opening effects of Na-azide (3 mmol/L) on SUR/Kir6.x channels. The dashed line indicates the control level, n=16–33 in each column. bP<0.05, cP<0.01 vs control. (D) Effects of Na-azide (3 mmol/L) on the opening rates of SUR/Kir6.x channels (all n=15). bP<0.05, cP<0.01 vs SUR2B/Kir6.1. fP<0.01 vs SUR2B/Kir6.2. iP<0.01 vs SUR2A/Kir6.2. No significance (ns) means P>0.05.
Figure 2. Effects of metabolic disorders on Kir6.x channels and the ΔC mutants expressed in Xenopus oocytes. (A) Representative time courses of currents recorded at −80 mV from Xenopus oocytes injected with cRNAs encoding Kir6.1, Kir6.1ΔC45, Kir6.2, and Kir6.2ΔC36. Whole-cell currents were evoked by a series of 300 ms voltage steps (from −120 to +50 mV with 10 mV increments, holding at −10 mV); the raw current traces at the time points (a–d) are shown on right. a: control; b: Na-Azide; c: PNU-37883A; d: BaCl2. (B) The effects of the drugs on currents mediated by Kir6.x channels and the ΔC mutants. Number of oocytes: Kir6.1, n=8; Kir6.1ΔC45, n=15; Kir6.2, n=7; Kir6.2ΔC36, n=17. cP<0.01 vs control. fP<0.01 vs Na-azide (3 mmol/L).
Figure 3. Effects of metabolic disorders on the Kir6.2ΔC36 channel with the mutations R50G, K185E and H175A expressed in Xenopus oocytes. (A) Currents recorded from oocytes expressing Kir6.2ΔC36 and its mutants. Whole-cell currents were evoked by a series of 300 ms voltage steps (from −120 to +50 mV with 10 mV increment, holding at −10 mV). (B) The basal currents of Kir6.2ΔC36 and its mutants recorded at −80 mV. n= 9–14 in each column. cP<0.01 vs Kir6.2ΔC36. fP<0.01 vs H175A. (C) The opening effects of Na-azide on Kir6.2ΔC36 and its mutants, the dashed line indicates the control level, n=9–14 in each column. cP<0.01 vs control. fP<0.01 vs Kir6.2ΔC36.
Figure 4. Effects of metabolic disorders on SUR2B and its mutants co-expressed with Kir6.x in Xenopus oocytes. The currents records and the effects of Na-azide on channel currents recorded from oocytes expressing SUR2B and its mutants with Kir6.1 (A) or Kir6.2 (B). The dashed line indicates the control level. The number of oocytes expressing SUR2B/Kir6.1 channels was: 25 (WT), 19 (K707A), 15 (K1348A) and 15 (K707A/K1348A). In SUR2B/Kir6.2 channels, the number was 18, 25, 12 and 9, respectively. bP<0.05, cP<0.01 vs control. No significance (ns) means P>0.05.
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