Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, England, UK.
J Gen Physiol. 2010 Oct;136(4):389-405. doi: 10.1085/jgp.201010475.
The mechanism of adenosine triphosphate (ATP)-sensitive potassium (K(ATP)) channel activation by Mg-nucleotides was studied using a mutation (G334D) in the Kir6.2 subunit of the channel that renders K(ATP) channels insensitive to nucleotide inhibition and has no apparent effect on their gating. K(ATP) channels carrying this mutation (Kir6.2-G334D/SUR1 channels) were activated by MgATP and MgADP with an EC(50) of 112 and 8 µM, respectively. This activation was largely suppressed by mutation of the Walker A lysines in the nucleotide-binding domains of SUR1: the remaining small (∼10%), slowly developing component of MgATP activation was fully inhibited by the lipid kinase inhibitor LY294002. The EC(50) for activation of Kir6.2-G334D/SUR1 currents by MgADP was lower than that for MgATP, and the time course of activation was faster. The poorly hydrolyzable analogue MgATPγS also activated Kir6.2-G334D/SUR1. AMPPCP both failed to activate Kir6.2-G334D/SUR1 and to prevent its activation by MgATP. Maximal stimulatory concentrations of MgATP (10 mM) and MgADP (1 mM) exerted identical effects on the single-channel kinetics: they dramatically elevated the open probability (P(O) > 0.8), increased the mean open time and the mean burst duration, reduced the frequency and number of interburst closed states, and eliminated the short burst states. By comparing our results with those obtained for wild-type K(ATP) channels, we conclude that the MgADP sensitivity of the wild-type K(ATP) channel can be described quantitatively by a combination of inhibition at Kir6.2 (measured for wild-type channels in the absence of Mg(2+)) and activation via SUR1 (determined for Kir6.2-G334D/SUR1 channels). However, this is not the case for the effects of MgATP.
研究了 Mg-核苷酸对三磷酸腺苷（ATP）敏感钾（K(ATP)）通道激活的机制，使用通道 Kir6.2 亚基中的突变（G334D），该突变使 K(ATP)通道对核苷酸抑制不敏感，并且对其门控没有明显影响。携带这种突变（Kir6.2-G334D/SUR1 通道）的 K(ATP)通道被 MgATP 和 MgADP 激活，EC(50)分别为 112 和 8 μM。这种激活在很大程度上受到 SUR1 核苷酸结合结构域 Walker A 赖氨酸突变的抑制：脂质激酶抑制剂 LY294002 完全抑制剩余的小（约 10%）、缓慢发展的 MgATP 激活的组成部分。MgADP 激活 Kir6.2-G334D/SUR1 电流的 EC(50)低于 MgATP，并且激活的时间过程更快。水解不良的类似物 MgATPγS 也激活了 Kir6.2-G334D/SUR1。AMPPCP 既不能激活 Kir6.2-G334D/SUR1，也不能阻止其被 MgATP 激活。最大刺激浓度的 MgATP（10 mM）和 MgADP（1 mM）对单通道动力学产生相同的影响：它们极大地提高了开放概率（P(O) > 0.8），增加了平均开放时间和平均爆发持续时间，降低了爆发之间的频率和关闭状态的数量，并消除了短暂爆发状态。通过将我们的结果与野生型 K(ATP)通道的结果进行比较，我们得出结论，野生型 K(ATP)通道的 MgADP 敏感性可以通过 Kir6.2 的抑制（在没有 Mg(2+)的情况下测量野生型通道）和 SUR1 的激活（确定 Kir6.2-G334D/SUR1 通道）的组合来定量描述。然而，对于 MgATP 的影响情况并非如此。
