Rajan Sindhu, Preisig-Müller Regina, Wischmeyer Erhard, Nehring Ralf, Hanley Peter J, Renigunta Vijay, Musset Boris, Schlichthörl Günter, Derst Christian, Karschin Andreas, Daut Jürgen
Institute of Physiology, Marburg University, Deutschhausstrasse 2, 35037 Marburg, Germany.
J Physiol. 2002 Nov 15;545(1):13-26. doi: 10.1113/jphysiol.2002.027052.
The two-pore-domain potassium channels TASK-1, TASK-3 and TASK-5 possess a conserved C-terminal motif of five amino acids. Truncation of the C-terminus of TASK-1 strongly reduced the currents measured after heterologous expression in Xenopus oocytes or HEK293 cells and decreased surface membrane expression of GFP-tagged channel proteins. Two-hybrid analysis showed that the C-terminal domain of TASK-1, TASK-3 and TASK-5, but not TASK-4, interacts with isoforms of the adapter protein 14-3-3. A pentapeptide motif at the extreme C-terminus of TASK-1, RRx(S/T)x, was found to be sufficient for weak but significant interaction with 14-3-3, whereas the last 40 amino acids of TASK-1 were required for strong binding. Deletion of a single amino acid at the C-terminal end of TASK-1 or TASK-3 abolished binding of 14-3-3 and strongly reduced the macroscopic currents observed in Xenopus oocytes. TASK-1 mutants that failed to interact with 14-3-3 isoforms (V411*, S410A, S410D) also produced only very weak macroscopic currents. In contrast, the mutant TASK-1 S409A, which interacts with 14-3-3-like wild-type channels, displayed normal macroscopic currents. Co-injection of 14-3-3zeta cRNA increased TASK-1 current in Xenopus oocytes by about 70 %. After co-transfection in HEK293 cells, TASK-1 and 14-3-3zeta (but not TASK-1DeltaC5 and 14-3-3zeta) could be co-immunoprecipitated. Furthermore, TASK-1 and 14-3-3 could be co-immunoprecipitated in synaptic membrane extracts and postsynaptic density membranes. Our findings suggest that interaction of 14-3-3 with TASK-1 or TASK-3 may promote the trafficking of the channels to the surface membrane.
双孔结构域钾通道TASK-1、TASK-3和TASK-5具有一个由五个氨基酸组成的保守C末端基序。TASK-1的C末端截短后，在非洲爪蟾卵母细胞或HEK293细胞中进行异源表达后所测得的电流大幅降低，并且绿色荧光蛋白标记的通道蛋白的表面膜表达也减少。酵母双杂交分析表明，TASK-1、TASK-3和TASK-5的C末端结构域（而非TASK-4）与衔接蛋白14-3-3的亚型相互作用。在TASK-1的最末端C末端发现一个五肽基序RRx(S/T)x足以与14-3-3进行微弱但显著的相互作用，而TASK-1的最后40个氨基酸是强结合所必需的。在TASK-1或TASK-3的C末端缺失单个氨基酸会消除14-3-3的结合，并大幅降低在非洲爪蟾卵母细胞中观察到的宏观电流。无法与14-3-3亚型相互作用的TASK-1突变体（V411* [此处可能有误，推测应为V411A之类更合理的突变形式，因为原文后面提到了S410A、S410D等类似突变形式]、S410A、S410D）也仅产生非常微弱的宏观电流。相反，与14-3-3样野生型通道相互作用的突变体TASK-1 S409A表现出正常的宏观电流。共注射14-3-3ζ的cRNA可使非洲爪蟾卵母细胞中的TASK-1电流增加约70%。在HEK293细胞中共转染后，TASK-1和14-3-3ζ（但不是TASK-1DeltaC5和14-3-3ζ）可以进行共免疫沉淀。此外，在突触膜提取物和突触后致密膜中，TASK-1和14-3-3也可以进行共免疫沉淀。我们的研究结果表明，14-3-3与TASK-1或TASK-3的相互作用可能促进通道向表面膜的转运。
