Displacement of the Na + /K + pump’s transmembrane domains demonstrates conserved conformational changes in P-type 2 ATPases
Displacement of the Na + /K + pump’s transmembrane domains demonstrates conserved conformational changes in P-type 2 ATPases
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Na /K 泵跨膜结构域的置换证明了 P 型 2 ATP 酶中保守的构象变化
DOI：
10.1073/pnas.2019317118
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发表时间：
2021
期刊：
影响因子：
--
通讯作者：
Artigas, Pablo
中科院分区：
文献类型：
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作者：
Young, Victoria C.;Artigas, Pablo
细胞存活需要Na+/K+泵建立的离子梯度，这是一种在两种主要构象（E1和E2）之间交替的ATP酶。在这里，我们使用状态特异性工程二硫键交联来证明泵α亚基的跨膜段2（M2）的移动方向与E1和E2中Na+/K+泵的现有晶体结构中观察到的距离不一致。我们在单半胱氨酸突变体中用电压钳荧光法表征这种运动。M1-M2环中的大多数突变体在用四甲基罗丹明-6-马来酰亚胺（TMRM）标记后产生状态依赖性荧光变化，这是由于多个内源性Dahans的猝灭。为了避免由多种潜在淬灭剂引起的复杂性，我们分析了通过在M1-M2中一次一个地引入的Dahans对与R977 C（在静态M9-M10环中）缀合的TMRM的淬灭。该方法表明，在M2中引入的Dahans仅在E2中淬灭TMRM，其中相同螺旋上的D126 W和L130 W产生最大的荧光变化。这些观察结果表明，M2向外移动的Na+是从E1构象，与交联的结果和其他P-型2 ATP酶的建议一致的机制解除闭塞。
Cellular survival requires the ion gradients built by the Na+/K+pump, an ATPase that alternates between two major conformations (E1 and E2). Here we use state-specific engineered-disulfide cross-linking to demonstrate that transmembrane segment 2 (M2) of the pump’s α-subunit moves in directions that are inconsistent with distances observed in existing crystal structures of the Na+/K+pump in E1 and E2. We characterize this movement with voltage-clamp fluorometry in single-cysteine mutants. Most mutants in the M1–M2 loop produced state-dependent fluorescence changes upon labeling with tetramethylrhodamine-6-maleimide (TMRM), which were due to quenching by multiple endogenous tryptophans. To avoid complications arising from multiple potential quenchers, we analyzed quenching of TMRM conjugated to R977C (in the static M9–M10 loop) by tryptophans introduced, one at a time, in M1–M2. This approach showed that tryptophans introduced in M2 quench TMRM only in E2, with D126W and L130W on the same helix producing the largest fluorescence changes. These observations indicate that M2 moves outward as Na+is deoccluded from the E1 conformation, a mechanism consistent with cross-linking results and with proposals for other P-type 2 ATPases.