Intracellular traffic of the K+ channels TASK‐1 and TASK‐3: role of N‐ and C‐terminal sorting signals and interaction with 14‐3‐3 proteins
Intracellular traffic of the K+ channels TASK‐1 and TASK‐3: role of N‐ and C‐terminal sorting signals and interaction with 14‐3‐3 proteins
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K+ 通道 TASK-1 和 TASK-3 的细胞内交通：N 端和 C 端分选信号的作用以及与 14-3-3 蛋白的相互作用
DOI：
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发表时间：
2009
期刊：
影响因子：
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通讯作者：
R. Preisig
中科院分区：
文献类型：
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作者：
Marylou Zuzarte;K. Heusser;Vijay Renigunta;G. Schlichthörl;S. Rinné;E. Wischmeyer;J. Daut;B. Schwappach;R. Preisig
两孔结构域钾通道TASK-1(KCNK3)和TASK-3(KCNK9)调节神经元和许多其他类型细胞的电活动。我们在非洲爪哇卵母细胞、哺乳动物细胞系和不同的酵母菌株中表达了TASK-1、TASK-3和相关的报告结构，以研究它们向表膜转运的机制和14-3-3蛋白的作用。我们用电压钳技术测量钾电流，并将通道的N-端和C-端片段与不同的报告蛋白融合，以研究亚细胞定位和表面表达的变化。突变分析表明，14-3-3蛋白与TASK-1和TASK-3的末端结合掩盖了一个三碱基基序KRR，该基序在几个重要方面不同于典型的基于精氨酸(RxR)或基于赖氨酸(KKxx)的保留信号。GST融合蛋白的下拉实验表明，TASK-3通道C-末端的KRR基序能够与COPI辅基结合。禁用14-3-3的结合，从而暴露KRR基序，导致GFP标记的通道蛋白主要定位于高尔基复合体。TASK-1和TASK-3还具有一个二碱性N-末端保留信号KR，其功能不依赖于14-3-3的结合。用显性-负通道突变体抑制通道表面表达表明，与14-3-3的相互作用对通道的二聚体组装没有显著影响。我们的结果全面描述了14-3-3蛋白与N-端和C-端分选信号一起控制TASK-1和TASK-3细胞内流量的机制。
The two‐pore‐domain potassium channels TASK‐1 (KCNK3) and TASK‐3 (KCNK9) modulate the electrical activity of neurons and many other cell types. We expressed TASK‐1, TASK‐3 and related reporter constructs in Xenopus oocytes, mammalian cell lines and various yeast strains to study the mechanisms controlling their transport to the surface membrane and the role of 14‐3‐3 proteins. We measured potassium currents with the voltage‐clamp technique and fused N‐ and C‐terminal fragments of the channels to various reporter proteins to study changes in subcellular localisation and surface expression. Mutational analysis showed that binding of 14‐3‐3 proteins to the extreme C‐terminus of TASK‐1 and TASK‐3 masks a tri‐basic motif, KRR, which differs in several important aspects from canonical arginine‐based (RxR) or lysine‐based (KKxx) retention signals. Pulldown experiments with GST fusion proteins showed that the KRR motif in the C‐terminus of TASK‐3 channels was able to bind to COPI coatomer. Disabling the binding of 14‐3‐3, which exposes the KRR motif, caused localisation of the GFP‐tagged channel protein mainly to the Golgi complex. TASK‐1 and TASK‐3 also possess a di‐basic N‐terminal retention signal, KR, whose function was found to be independent of the binding of 14‐3‐3. Suppression of channel surface expression with dominant‐negative channel mutants revealed that interaction with 14‐3‐3 has no significant effect on the dimeric assembly of the channels. Our results give a comprehensive description of the mechanisms by which 14‐3‐3 proteins, together with N‐ and C‐terminal sorting signals, control the intracellular traffic of TASK‐1 and TASK‐3.
影响因子：
11.4
作者：
JACKSON, MR;NILSSON, T;PETERSON, PA
通讯作者：
PETERSON, PA
影响因子：
13.8
作者：
Bayliss DA;Barrett PQ
通讯作者：
Barrett PQ
影响因子：
33.6
作者：
Vacher H;Mohapatra DP;Trimmer JS
通讯作者：
Trimmer JS