ABSTRACT

Arabidopsis thaliana contains five tandem-pore domain potassium channels, TPK1-TPK5 and the related one-pore domain potassium channel, KCO3. Although KCO3 is unlikely to be an active channel, it still has a physiological role in plant cells. TPK2 is most similar to KCO3 and both are localized to the tonoplast. However, their function remains poorly understood. Here, taking advantage of the similarities between TPK2 and KCO3, we evaluated Ca²⁺ binding to the EF hands in TPK2, and the elements of KCO3 required for K⁺ channel activity. Presence of both EF-hand motifs in TPK2 resulted in Ca²⁺ binding, but EF1 or EF2 alone failed to interact with Ca²⁺. The EF hands were not required for K⁺ transport activity. EF1 contains two cysteines separated by two amino acids. Replacement of both cysteines with serines in TPK2 increased Ca²⁺ binding. We generated a two-pore domain chimeric K⁺ channel by replacing the missing pore region in KCO3 with a pore domain of TPK2. Alternatively, we generated two versions of simple one-pore domain K⁺ channels by removal of an extra region from KCO3. The chimera and one of the simple one-pore variants were functional channels. This strongly suggests that KCO3 is not a pseudogene and KCO3 retains components required for the formation of a functional K⁺ channel and oligomerization. Our results contribute to our understanding of the structural properties required for K⁺ channel activity.

摘要

拟南芥包含五个串联孔结构域钾通道TPK1-TPK5和相关的单孔结构域钾通道KCO3。尽管KCO3不太可能成为活性通道，但它在植物细胞中仍具有生理作用。TPK2与KCO3最相似，两者都位于液泡膜中。但是，它们的功能仍然知之甚少。在这里，利用TPK2和KCO3之间的相似性，我们评估了Ca ²⁺与TPK2中EF手的结合以及K ⁺通道活性所需的KCO3元素。TPK2中同时存在两个EF-手基序会导致Ca ²⁺结合，但单独的EF1或EF2无法与Ca ²⁺相互作用。K ⁺不需要EF手运输活动。EF1包含被两个氨基酸分隔的两个半胱氨酸。在TPK2中用丝氨酸取代两个半胱氨酸可增加Ca ^2+的结合。通过用TPK2的孔结构域替换KCO3中缺失的孔结构，我们生成了一个两孔结构域的嵌合K ⁺通道。另外，我们通过从KCO3去除额外的区域，生成了两个版本的简单的单孔域K ⁺通道。嵌合体和简单的单孔变体之一是功能通道。这强烈表明KCO3不是假基因，并且KCO3保留了形成功能性K ⁺所需的组分通道和低聚。我们的结果有助于我们理解K ⁺通道活性所需的结构性质。