Modulation of MthK potassium channel activity at the intracellular entrance to the pore
Modulation of MthK potassium channel activity at the intracellular entrance to the pore
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DOI：
10.1074/jbc.m603109200
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发表时间：
2006-07-28
影响因子：
4.8
通讯作者：
Rothberg, Brad S.
中科院分区：
文献类型：
--
作者：
Parfenova, Lyubov V.;Crane, Brittany M.;Rothberg, Brad S.
我们使用大肠杆菌 LB2003 K+ 摄取缺陷菌株进行细菌互补筛选，分析对热自养甲烷杆菌钾通道 (MthK) 功能至关重要的残基。通过 SDS-PAGE 和蛋白质印迹分析突变体的通道表达和相对结构完整性，并使用单通道记录分析突变体通道功能改变的机制。我们观察到野生型 MthK 表达弥补了 K 吸收缺陷。尽管 MthK 功能之前被认为需要毫摩尔范围内的 Ca2+，但我们证明，在升高的温度下，对 Ca2+ 的需求会变得低得多。 MthK 孔细胞质口的突变会削弱互补作用，表明这些突变通道无法支持 K+ 的吸收。相反，与野生型 MthK 相比，MthK RCK 结构域中 Ca2+ 结合位点的取代不会降低互补性。我们重点关注残基 Glu-92 和 Glu-96 的突变，它们可能在通道关闭状态下形成孔的最窄部分。这些残基的突变可能会导致单通道电导发生轻微变化，但不一定与对细菌互补的影响相关。然而，Glu-92 的突变也可能改变通道开放概率，并且这些变化与互补效应相关。这些突变中最引人注目的是 E92A，它通过降低 MthK 的开放概率几乎消除了细菌互补。我们的结果表明，K+通道束交叉点处的小疏水性丙氨酸侧链可能会产生本质上稳定的结构，从而将关闭到打开状态的平衡转变为关闭状态。
We used a bacterial complementation screen with the LB2003 K+ uptake-deficient strain of Escherichia coli to analyze residues that are critical to Methanobacterium thermoautotrophicum potassium channel (MthK) function. Channel expression and relative structural integrity of mutants were analyzed by SDS-PAGE and Western blot, and mechanisms underlying altered mutant channel function were analyzed using single-channel recording. We observed that wild-type MthK expression complements K uptake deficiency. Although MthK function was previously thought to require Ca2+ in the millimolar range, we demonstrate that at elevated temperatures the requirement for Ca2+ becomes much lower. Mutations at the cytoplasmic mouth of the MthK pore can blunt complementation, indicating that those mutant channels cannot support K+ uptake. In contrast, substitutions at the Ca2+-binding site in the MthK RCK domain did not decrease complementation compared with wild-type MthK. We focused on mutations to residues Glu-92 and Glu-96, which may form the narrowest part of the pore in the channel's closed state. Mutations at these residues can yield slight changes in single-channel conductance that do not necessarily correlate with effects on bacterial complementation. However, mutations at Glu-92 could also change channel open probability, and these changes correlated with complementation effects. The most striking of these mutations was E92A, which nearly eliminated bacterial complementation by decreasing the open probability of MthK. Our results suggest that the small, hydrophobic alanine side chain at the K+ channel bundle crossing may generate an intrinsically stable structure, which in turn shifts the closed-to-open-state equilibrium toward the closed state.