Probing allosteric coupling in a constitutively open mutant of the ion channel KcsA using solid-state NMR.
Probing allosteric coupling in a constitutively open mutant of the ion channel KcsA using solid-state NMR.
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使用固态 NMR 探测离子通道 KcsA 的组成型开放突变体中的变构耦合。
DOI：
10.1073/pnas.1908828117
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发表时间：
2020
影响因子：
11.1
通讯作者：
McDermott,AnnE
中科院分区：
文献类型：
--
作者：
Sun,Zhiyu;Xu,Yunyao;Zhang,Dongyu;McDermott,AnnE
跨膜变构偶联是许多关键生物信号事件的特征。在这里，我们测试是否跨膜变构偶联控制的原型钾通道KcsA的C型失活的背景下，钾结合亲和力。KcsA的激活是由质子结合到pH门后，在细胞内pH值下降。许多研究表明，这种质子结合也提示构象转换，导致在选择性过滤器的钾离子的亲和力的损失，因此通道失活。我们使用KcsA突变体（H25 R/E118 A）测试了这种失活机制，该突变体在宽范围的pH值内表现出开放的pH门。我们目前的固态NMR测量这个开放的突变体在中性pH值探测在选择性过滤器的钾的亲和力。该突变体的选择性过滤器中的钾结合亲和力为81 mM，在中性pH下比野生型KcsA弱约四个数量级，并且在低pH（pH ≥ 3.5）下与野生型KcsA的值相当。这一结果有力地支持了我们的断言，即开放的pH门变构影响选择性过滤器的钾结合亲和力。在该突变体中，pH传感器中谷氨酸残基（E120）的质子化状态对钾结合敏感，这表明该突变体在激活门中也具有灵活性，并且受到跨膜变构的影响。
Transmembrane allosteric coupling is a feature of many critical biological signaling events. Here we test whether transmembrane allosteric coupling controls the potassium binding affinity of the prototypical potassium channel KcsA in the context of C-type inactivation. Activation of KcsA is initiated by proton binding to the pH gate upon an intracellular drop in pH. Numerous studies have suggested that this proton binding also prompts a conformational switch, leading to a loss of affinity for potassium ions at the selectivity filter and therefore to channel inactivation. We tested this mechanism for inactivation using a KcsA mutant (H25R/E118A) that exhibits an open pH gate across a broad range of pH values. We present solid-state NMR measurements of this open mutant at neutral pH to probe the affinity for potassium at the selectivity filter. The potassium binding affinity in the selectivity filter of this mutant, 81 mM, is about four orders of magnitude weaker than that of wild-type KcsA at neutral pH and is comparable to the value for wild-type KcsA at low pH (pH ≈ 3.5). This result strongly supports our assertion that the open pH gate allosterically affects the potassium binding affinity of the selectivity filter. In this mutant, the protonation state of a glutamate residue (E120) in the pH sensor is sensitive to potassium binding, suggesting that this mutant also has flexibility in the activation gate and is subject to transmembrane allostery.
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影响因子：
4.3
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通讯作者：
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