Mutation of a single residue promotes gating of vertebrate and invertebrate two-pore domain potassium channels
Mutation of a single residue promotes gating of vertebrate and invertebrate two-pore domain potassium channels
复制标题
DOI：
10.1038/s41467-019-08710-3
复制
发表时间：
2019-02-15
影响因子：
16.6
通讯作者：
Boulin, Thomas
中科院分区：
文献类型：
--
作者：
Ben Soussia, Ismail;El Mouridi, Sonia;Boulin, Thomas
调节离子通道活性的突变是理解控制其门控的生物物理决定因素的必要工具。在这里，我们揭示了位于双孔结构域钾（K2P）通道的第二个跨膜结构域的单个氨基酸位置（TM2.6）所起的保守作用。TM2.6对天冬氨酸或天冬酰胺的突变增加了所有脊椎动物K2P通道的活性。利用双电极电压钳和单通道记录技术，我们发现TM2.6的突变促进了通过选择性滤波门的通道门控，增加了单通道打开的概率。此外，通道门控可以通过使用不同的氨基酸取代来逐步调节。最后，我们利用CRISPR/Cas9基因编辑技术在秀丽隐杆线虫的4个K2P通道中合理设计功能获得突变，证明TM2.6的作用在进化过程中是保守的。因此，这项研究描述了一种简单而有力的策略来系统地操纵整个钾通道家族的活动。
Mutations that modulate the activity of ion channels are essential tools to understand the biophysical determinants that control their gating. Here, we reveal the conserved role played by a single amino acid position (TM2.6) located in the second transmembrane domain of two-pore domain potassium (K2P) channels. Mutations of TM2.6 to aspartate or asparagine increase channel activity for all vertebrate K2P channels. Using two-electrode voltage-clamp and single-channel recording techniques, we find that mutation of TM2.6 promotes channel gating via the selectivity filter gate and increases single channel open probability. Furthermore, channel gating can be progressively tuned by using different amino acid substitutions. Finally, we show that the role of TM2.6 was conserved during evolution by rationally designing gain-of-function mutations in four Caenorhabditis elegans K2P channels using CRISPR/Cas9 gene editing. This study thus describes a simple and powerful strategy to systematically manipulate the activity of an entire family of potassium channels.