A Pathogenic C Terminus-truncated Polycystin-2 Mutant Enhances Receptor-activated Ca2+ Entry via Association with TRPC3 and TRPC7*
A Pathogenic C Terminus-truncated Polycystin-2 Mutant Enhances Receptor-activated Ca2+ Entry via Association with TRPC3 and TRPC7*
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DOI：
10.1074/jbc.m109.015149
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发表时间：
2009-10
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通讯作者：
K. Miyagi;Shigeki Kiyonaka;Kazunori Yamada;T. Miki;E. Mori;Kenta Kato;T. Numata;Yuichi Sawaguchi;T. Numaga;Toru Kimura;Y. Kanai;M. Kawano;M. Wakamori;H. Nomura;I. Koni;M. Yamagishi;Y. Mori
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作者：
K. Miyagi;Shigeki Kiyonaka;Kazunori Yamada;T. Miki;E. Mori;Kenta Kato;T. Numata;Yuichi Sawaguchi;T. Numaga;Toru Kimura;Y. Kanai;M. Kawano;M. Wakamori;H. Nomura;I. Koni;M. Yamagishi;Y. Mori
PKD 2基因突变导致常染色体显性多囊肾病（ADPKD）。PKD 2编码多囊蛋白-2（TRPP 2），其是瞬时受体电位（TRP）阳离子通道蛋白的同源物。在这里，我们确定了一种新的PKD 2突变，产生一个C-末端尾部截短的TRPP 2突变体697 fsX与移码导致一个异常的17个氨基酸添加后，谷氨酸残基697从一个家庭显示轻度ADPKD症状。当在HEK 293细胞中重组表达时，定位于内质网（ER）膜的野生型（WT）TRPP 2在毒蕈碱型乙酰胆碱受体（mAChR）刺激后显著增强ER的Ca 2+释放。与此相反，697 fsX，这表明一个主要的质膜定位特征的TRPP 2突变体与C端缺失，显着增加mAChR激活的Ca 2+内流在细胞中表达TRPC 3或TRPC 7。免疫共沉淀，pulldown测定和交联实验揭示了697 fsX和TRPC 3或TRPC 7之间的物理关联。697 fsX而非WT TRPP 2引起反转电位的去极化偏移和单通道电导的增强，这表明mAChR激活电流的离子渗透孔特性改变。重要的是，在肾上皮LLC-PK 1细胞中，重组679 fsX构建体与天然TRPC 3蛋白共分布在顶膜区域，但WT构建体分布在基底外侧膜和邻近的细胞内区域。我们的研究结果表明，由TRPP 2突变体和TRPC 3或TRPC 7蛋白组成的异聚阳离子通道诱导增强的受体激活的Ca 2+内流，这可能导致ADPKD细胞生长失调。
Mutations in PKD2 gene result in autosomal dominant polycystic kidney disease (ADPKD). PKD2 encodes polycystin-2 (TRPP2), which is a homologue of transient receptor potential (TRP) cation channel proteins. Here we identify a novel PKD2 mutation that generates a C-terminal tail-truncated TRPP2 mutant 697fsX with a frameshift resulting in an aberrant 17-amino acid addition after glutamic acid residue 697 from a family showing mild ADPKD symptoms. When recombinantly expressed in HEK293 cells, wild-type (WT) TRPP2 localized at the endoplasmic reticulum (ER) membrane significantly enhanced Ca2+ release from the ER upon muscarinic acetylcholine receptor (mAChR) stimulation. In contrast, 697fsX, which showed a predominant plasma membrane localization characteristic of TRPP2 mutants with C terminus deletion, prominently increased mAChR-activated Ca2+ influx in cells expressing TRPC3 or TRPC7. Coimmunoprecipitation, pulldown assay, and cross-linking experiments revealed a physical association between 697fsX and TRPC3 or TRPC7. 697fsX but not WT TRPP2 elicited a depolarizing shift of reversal potentials and an enhancement of single-channel conductance indicative of altered ion-permeating pore properties of mAChR-activated currents. Importantly, in kidney epithelial LLC-PK1 cells the recombinant 679fsX construct was codistributed with native TRPC3 proteins at the apical membrane area, but the WT construct was distributed in the basolateral membrane and adjacent intracellular areas. Our results suggest that heteromeric cation channels comprised of the TRPP2 mutant and the TRPC3 or TRPC7 protein induce enhanced receptor-activated Ca2+ influx that may lead to dysregulated cell growth in ADPKD.