Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, United Kingdom.
Elife. 2019 Feb 21;8:e41103. doi: 10.7554/eLife.41103.
The response of ATP-sensitive K channels (K) to cellular metabolism is coordinated by three classes of nucleotide binding site (NBS). We used a novel approach involving labeling of intact channels in a native, membrane environment with a non-canonical fluorescent amino acid and measurement (using FRET with fluorescent nucleotides) of steady-state and time-resolved nucleotide binding to dissect the role of NBS2 of the accessory SUR1 subunit of K in channel gating. Binding to NBS2 was Mg-independent, but Mg was required to trigger a conformational change in SUR1. Mutation of a lysine (K1384A) in NBS2 that coordinates bound nucleotides increased the for trinitrophenyl-ADP binding to NBS2, but only in the presence of Mg, indicating that this mutation disrupts the ligand-induced conformational change. Comparison of nucleotide-binding with ionic currents suggests a model in which each nucleotide binding event to NBS2 of SUR1 is independent and promotes K activation by the same amount.
ATP 敏感性钾通道 (K) 对细胞代谢的反应由三类核苷酸结合位点 (NBS) 协调。我们使用了一种新方法，涉及用非典型荧光氨基酸标记完整通道在天然、膜环境中，并测量（使用荧光核苷酸的 FRET）稳态和时间分辨的核苷酸结合，以剖析辅助 SUR1 亚基的 K 通道门控中 NBS2 的作用。NBS2 的结合对 Mg 不依赖，但 Mg 是触发 SUR1 构象变化所必需的。在与结合核苷酸配位的 NBS2 中的赖氨酸 (K1384A) 的突变增加了三硝基苯-ADP 与 NBS2 的结合，但仅在存在 Mg 的情况下，表明该突变破坏了配体诱导的构象变化。核苷酸结合与离子电流的比较表明了一个模型，其中 SUR1 的 NBS2 上的每个核苷酸结合事件都是独立的，并以相同的量促进 K 的激活。
