Silencing of the Tandem Pore Domain Halothane-inhibited K+ Channel 2 (THIK2) Relies on Combined Intracellular Retention and Low Intrinsic Activity at the Plasma Membrane
Silencing of the Tandem Pore Domain Halothane-inhibited K+ Channel 2 (THIK2) Relies on Combined Intracellular Retention and Low Intrinsic Activity at the Plasma Membrane
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DOI：
10.1074/jbc.m113.503318
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发表时间：
2013-12-06
影响因子：
4.8
通讯作者：
Lesage, Florian
中科院分区：
文献类型：
--
作者：
Chatelain, Franck C.;Bichet, Delphine;Lesage, Florian
背景：THIK2在异源系统中不产生宏观电流。结果：THIK2在其孔区和/或胞浆氨基末端突变时产生K+电流。结论：THIK2的沉默是由于其固有活性低和细胞内滞留所致。意义：THIK2是一个功能正常但沉默的通道。串联孔结构域氟烷抑制的K+通道1(THIK1)产生背景K+电流。尽管THIK1与THIK1有62%的氨基酸同源性，但THIK2在异源表达时不起作用。在这里，我们表明，这种明显的活性缺乏是由于内质网的滞留和质膜上的低内在通道活性的独特组合。THIK2突变体在其第二内螺旋(M2)上含有一个脯氨酸残基(THIK2-A155P)，产生的K+选择性电流具有与THIK1相似的特性，包括被氟烷抑制和对细胞外pH变化不敏感。M2螺旋的另一个突变(I158D)进一步增加了通道的活性，并影响了电流动力学。我们还发现THIK2的细胞质氨基末端区域(NT-THIK2)包含一个富含精氨酸的基序(RRSRRR)，作为保留/恢复信号。THIK2中这个基序的突变导致通道重新定位到质膜，导致可测量的电流，即使在M2螺旋没有突变的情况下也是如此。NT-THIK2-CD161嵌合体的细胞表面递送通过突变保留基序的精氨酸，但也通过将嵌入该基序的丝氨酸转化为天冬氨酸来增加，这表明对THIK2运输的调节依赖于磷酸化。
Background: THIK2 does not generate macroscopic currents in heterologous systems. Results: THIK2 produces K+ currents when mutated in its pore region and/or in its cytoplasmic amino terminus. Conclusion: Silencing of THIK2 is due to low intrinsic activity and intracellular retention. Significance: THIK2 is a functional but silent channel.The tandem pore domain halothane-inhibited K+ channel 1 (THIK1) produces background K+ currents. Despite 62% amino acid identity with THIK1, THIK2 is not active upon heterologous expression. Here, we show that this apparent lack of activity is due to a unique combination of retention in the endoplasmic reticulum and low intrinsic channel activity at the plasma membrane. A THIK2 mutant containing a proline residue (THIK2-A155P) in its second inner helix (M2) produces K+-selective currents with properties similar to THIK1, including inhibition by halothane and insensitivity to extracellular pH variations. Another mutation in the M2 helix (I158D) further increases channel activity and affects current kinetics. We also show that the cytoplasmic amino-terminal region of THIK2 (Nt-THIK2) contains an arginine-rich motif (RRSRRR) that acts as a retention/retrieval signal. Mutation of this motif in THIK2 induces a relocation of the channel to the plasma membrane, resulting in measurable currents, even in the absence of mutations in the M2 helix. Cell surface delivery of a Nt-THIK2-CD161 chimera is increased by mutating the arginines of the retention motif but also by converting the serine embedded in this motif to aspartate, suggesting a phosphorylation-dependent regulation of THIK2 trafficking.