Targeting of calcineurin to an NFAT-like docking site is required for the calcium-dependent activation of the background K+channel, TRESK
Targeting of calcineurin to an NFAT-like docking site is required for the calcium-dependent activation of the background K+channel, TRESK
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DOI：
10.1074/jbc.m602495200
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发表时间：
2006-05-26
影响因子：
4.8
通讯作者：
Enyedi, Peter
中科院分区：
文献类型：
--
作者：
Czirjak, Gabor;Enyedi, Peter
双孔结构域 K+ 通道 TRESK（TWIK 相关脊髓 K+ 通道）响应钙/钙调蛋白依赖性蛋白磷酸酶（钙调神经磷酸酶）的钙信号而被激活。在本研究中，我们报告除了其酶促作用外，钙调神经磷酸酶还通过类似 NFAT 的对接位点与 TRESK 相互作用。在其细胞内环中，小鼠 TRESK 具有氨基酸序列 PQIVID，该序列与钙调神经磷酸酶结合共有基序 PXIXIT（其中 X 表示任何氨基酸）相似，是 NFAT（激活 T 细胞的核因子）激活和核转位所必需的。当在非洲爪蟾卵母细胞中的钙信号之后进行测量时，TRESK 的 PQIVID 序列突变为 PQIVIA、PQIVAD 或 PQAVAD，按所列顺序逐渐恶化钙依赖性激活，并相应降低苯佐卡因敏感性（区分激活通道和静止通道的特性）。显微注射 VIVIT 肽，旨在特异性抑制 NFAT-钙调神经磷酸酶相互作用，也消除了 TRESK 激活。 TRESK 的细胞内环以 GST 融合蛋白的形式表达，在体外与具有活性的钙调神经磷酸酶结合。 PQAVAD 突变以及向反应中添加 VIVIT 肽会消除这种钙调神经磷酸酶结合。在钙和钙调蛋白存在的情况下，野生型钙调神经磷酸酶被招募至 GST-TRESK 环。这些结果表明 PQIVID 序列是钙调神经磷酸酶的对接位点，并且其占据是 TRESK 的钙依赖性调节所必需的。为靶向钙调神经磷酸酶的 NFAT 结合位点而开发的免疫抑制化合物，除了对 NFAT 产生预期效果外，预计还会干扰 TRESK 调节。
The two-pore domain K+ channel, TRESK (TWIK-related spinal cord K+ channel) is activated in response to the calcium signal by the calcium/calmodulin-dependent protein phosphatase, calcineurin. In the present study we report that calcineurin also interacts with TRESK via an NFAT-like docking site, in addition to its enzymatic action. In its intracellular loop, mouse TRESK possesses the amino acid sequence, PQIVID, which is similar to the calcineurin binding consensus motif, PXIXIT (where X denotes any amino acids), necessary for NFAT (nuclear factor of activated T cells) activation and nuclear translocation. Mutations of the PQIVID sequence of TRESK to PQIVIA, PQIVAD, or PQAVAD increasingly deteriorated the calcium-dependent activation in the listed order and correspondingly reduced the benzocaine sensitivity (a property discriminating activated channels from resting ones), when it was measured after the calcium signal in Xenopus oocytes. Microinjection of VIVIT peptide, designed to inhibit the NFAT-calcineurin interaction specifically, also eliminated TRESK activation. The intracellular loop of TRESK, expressed as a GST fusion protein, bound constitutively active calcineurin in vitro. PQAVAD mutation as well as addition of VIVIT peptide to the reaction abrogated this calcineurin binding. Wild type calcineurin was recruited to GST-TRESK-loop in the presence of calcium and calmodulin. These results indicate that the PQIVID sequence is a docking site for calcineurin, and its occupancy is required for the calcium-dependent regulation of TRESK. Immunosuppressive compounds, developed to target the NFAT binding site of calcineurin, are also expected to interfere with TRESK regulation, in addition to their desired effect on NFAT.