Function and regulation of TRPP2 ion channel revealed by a gain-of-function mutant
Function and regulation of TRPP2 ion channel revealed by a gain-of-function mutant
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DOI：
10.1073/pnas.1517066113
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发表时间：
2016-04-26
影响因子：
11.1
通讯作者：
Yu, Yong
中科院分区：
文献类型：
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作者：
Pavel, Mahmud Arif;Lv, Caixia;Yu, Yong
多囊蛋白-1和瞬时受体电位多囊蛋白2（TRPP 2）的突变解释了几乎所有临床鉴定的常染色体显性多囊肾病（ADPKD）病例，ADPKD是最常见的人类遗传疾病之一。TRPP 2在其同源复合物和TRPP 2/多囊蛋白-1受体/离子通道复合物中起阳离子通道的作用。TRPP 2的激活机制尚不清楚，这极大地限制了对其功能和调控的研究。在这里，我们通过对S4-S5接头和S5跨膜结构域进行诱变扫描，产生了TRPP 2的组成型活性功能获得性（GOF）突变体，并研究了GOF TRPP 2通道的功能特性。我们发现细胞外二价离子，包括Ca 2+，通过直接阻断TRPP 2通道孔来抑制单价离子的渗透。我们还发现，D 643，一个带负电荷的氨基酸在孔，是至关重要的通道渗透性。通过将单点ADPKD致病突变引入GOF TRPP 2，我们发现不同的突变对通道活性可能具有完全不同的影响。在斑马鱼胚胎中研究了GOF TRPP 2的体内功能。结果表明，与野生型（WT）相比，GOF TRPP 2更有效地挽救形态异常，包括卷曲尾和囊肿形成的原肾，引起内源性TRPP 2表达下调。因此，我们建立了一个GOF TRPP 2通道，可以作为一个强大的工具，研究TRPP 2的功能和调节。GOF通道也可能具有开发ADPKD新治疗策略的潜在应用。
Mutations in polycystin-1 and transient receptor potential polycystin 2 (TRPP2) account for almost all clinically identified cases of autosomal dominant polycystic kidney disease (ADPKD), one of the most common human genetic diseases. TRPP2 functions as a cation channel in its homomeric complex and in the TRPP2/polycystin-1 receptor/ion channel complex. The activation mechanism of TRPP2 is unknown, which significantly limits the study of its function and regulation. Here, we generated a constitutively active gain-of-function (GOF) mutant of TRPP2 by applying a mutagenesis scan on the S4-S5 linker and the S5 transmembrane domain, and studied functional properties of the GOF TRPP2 channel. We found that extracellular divalent ions, including Ca2+, inhibit the permeation of monovalent ions by directly blocking the TRPP2 channel pore. We also found that D643, a negatively charged amino acid in the pore, is crucial for channel permeability. By introducing single-point ADPKD pathogenic mutations into the GOF TRPP2, we showed that different mutations could have completely different effects on channel activity. The in vivo function of the GOF TRPP2 was investigated in zebrafish embryos. The results indicate that, compared with wild type (WT), GOF TRPP2 more efficiently rescued morphological abnormalities, including curly tail and cyst formation in the pronephric kidney, caused by down-regulation of endogenous TRPP2 expression. Thus, we established a GOF TRPP2 channel that can serve as a powerful tool for studying the function and regulation of TRPP2. The GOF channel may also have potential application for developing new therapeutic strategies for ADPKD.