Assembly of plant Shaker-like Kout channels requires two distinct sites of the channel α-subunit
Assembly of plant Shaker-like Kout channels requires two distinct sites of the channel α-subunit
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DOI：
10.1529/biophysj.103.037671
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发表时间：
2004-08-01
影响因子：
3.4
通讯作者：
Mueller-Roeber, B
中科院分区：
文献类型：
--
作者：
Dreyer, I;Porée, F;Mueller-Roeber, B
SKOR和SKPK是拟南芥的外向整流钾通道。它们属于电压依赖性K+通道的Shaker超家族。这类通道由四个α亚基组成，亚基组装是通道功能的先决条件。本研究利用酵母双杂交系统和功能检测技术，在非洲爪蟾卵母细胞和酵母中研究了SKOR的组装机制。我们证明，SKOR和PDK的物理相互作用和组装成异聚体的K-出通道。缺失突变体和嵌合蛋白产生的SKOR和K-in通道α-亚基KAT 1显示，细胞质C-末端的SKOR决定通道组装。两个结构域的通道α-亚基KAT 1揭示了细胞质C-末端的SKOR决定通道组装。鉴定了对通道组装至关重要的两个结构域：i）近端相互作用区，其包含推定的环核苷酸结合结构域以及该结构域上游的33个氨基酸，和ii）远端相互作用区，其显示出与KAT 1的K-T结构域的一些相似性。两个区域对通道组装的贡献不同。而近端相互作用区域被发现是活跃的，其本身，远端相互作用区域需要一个完整的近端相互作用区域是活跃的。K-out α-亚基不与K-in α-亚基组装，因为它们的组装位点之间不存在相互作用。
SKOR and GORK are outward-rectifying plant potassium channels from Arabidopsis thaliana. They belong to the Shaker superfamily of voltage-dependent K+ channels. Channels of this class are composed of four alpha-subunits and subunit assembly is a prerequisite for channel function. In this study the assembly mechanism of SKOR was investigated using the yeast two-hybrid system and functional assays in Xenopus oocytes and in yeast. We demonstrate that SKOR and GORK physically interact and assemble into heteromeric K-out channels. Deletion mutants and chimeric proteins generated from SKOR and the K-in channel alpha-subunit KAT1 revealed that the cytoplasmic C-terminus of SKOR determines channel assembly. Two domains thatchannel a-subunit KAT1 revealed that the cytoplasmic C-terminus of SKOR determines channel assembly. Two domains that are crucial for channel assembly were identified: i), a proximal interacting region comprising a putative cyclic nucleotide-binding domain together with 33 amino acids just upstream of this domain, and ii), a distal interacting region showing some resemblance to the K-T domain of KAT1. Both regions contributed differently to channel assembly. Whereas the proximal interacting region was found to be active on its own, the distal interacting region required an intact proximal interacting region to be active. K-out alpha-subunits did not assemble with K-in alpha-subunits because of the absence of interaction between their assembly sites.