Electrostatic state of the cytoplasmic domain influences inactivation at the selectivity filter of the KcsA potassium channel.
Electrostatic state of the cytoplasmic domain influences inactivation at the selectivity filter of the KcsA potassium channel.
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DOI：
10.1016/j.bbamem.2018.07.011
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发表时间：
2019-01
期刊：
影响因子：
--
通讯作者：
M. Hirano;T. Ide
中科院分区：
文献类型：
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作者：
M. Hirano;T. Ide
KcsA是一种质子活化的K+通道，受两个门的调控：位于孔内入口的激活门和位于选择性过滤器的失活门。在此之前，我们发现KcsA的胞质结构域（CPD）感知质子，CPD的静电变化影响激活门的开启和关闭。然而，我们之前的研究并没有揭示CPD对失活门的影响，因为我们使用了非失活突变体（E71A）。在本研究中，我们使用了不含E71A突变的突变体，并表明CPD的静电状态会影响失活门。三个新的CPD突变体，其中一些带负电荷的氨基酸被中性氨基酸取代。这些CPD突变体进行K+，但表现出不同的失活特性。与野生型KcsA相似，携带D149N突变的突变体开放概率高，失活速度慢，未携带D149N突变的突变体开放概率低，失活速度快。此外，含有D149N的突变体对K+的选择性较差，允许Na+流动。这些结果表明，D149N突变引起的CPD的静电变化引发了快速失活的丧失和选择性过滤器构象的改变。此外，D149N诱导的快速失活的丧失被R153A突变逆转，这表明不仅D149的静电状态影响失活，R153的静电状态也影响失活。
KcsA is a proton-activated K+channel that is regulated at two gates: an activation gate located in the inner entrance of the pore and an inactivation gate at the selectivity filter. Previously, we revealed that the cytoplasmic domain (CPD) of KcsA senses proton and that electrostatic changes of the CPD influences the opening and closing of the activation gate. However, our previous studies did not reveal the effect of CPD on the inactivation gate because we used a non-inactivating mutant (E71A). In the present study, we used mutants that did not harbor the E71A mutation, and showed that the electrostatic state of the CPD influences the inactivation gate. Three novel CPD mutants were generated in which some negatively charged amino acids were replaced with neutral amino acids. These CPD mutants conducted K+, but showed various inactivation properties. Mutants carrying the D149N mutation showed high open probability and slow inactivation, whereas those without the D149N mutation showed low open probability and fast inactivation, similar to wild-type KcsA. In addition, mutants with D149N showed poor K+selectivity, and permitted Na+to flow. These results indicated that electrostatic changes in the CPD by D149N mutation triggered the loss of fast inactivation and changes in the conformation of selectivity filter. Additionally, the loss of fast inactivation induced by D149N was reversed by R153A mutation, suggesting that not only the electrostatic state of D149, but also that of R153 affects inactivation.