Molecular determinants in TRPV5 channel assembly
Molecular determinants in TRPV5 channel assembly
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DOI：
10.1074/jbc.m406222200
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发表时间：
2004-12-24
影响因子：
4.8
通讯作者：
Hoenderop, JGJ
中科院分区：
文献类型：
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作者：
Chang, Q;Gyftogianni, E;Hoenderop, JGJ
上皮Ca2+通道TRPV5和TRPV6介导1,25-二羟基维生素d -3应答上皮内的Ca2+内流，因此在维持机体Ca2+平衡中至关重要。这些Ca2+通道聚集在（异）四聚体通道复合物中，具有不同的功能特征，包括Ca2+依赖性失活，离子选择性和药理阻断。谷胱甘肽s -转移酶下拉和共免疫沉淀表明，细胞内N-和c -尾通过N-N尾、C-C尾和N- c尾之间的物理相互作用，在TRPV5通道组装中发挥了重要作用。人类胚胎肾（HEK293）细胞的片钳分析和共表达TRPV5野生型和截断蛋白的非洲爪蟾卵母细胞Ca-45（2+）摄取实验表明，TRPV5DeltaN（缺失N-tail）和TRPV5DeltaC（缺失C-tail）以显性负向方式降低了野生型TRPV5的通道活性，而TRPV5DeltaNDeltaC（缺失N-tail/C-tail）不影响TRPV5的活性。共表达野生型TRPV5和TRPV5DeltaN或TRPV5DeltaC的卵母细胞在质膜上几乎不表达野生型TRPV5，而共表达野生型TRPV5和TRPV5DeltaNDeltaC的卵母细胞在通道表面表达正常。这表明TRPV5与TRPV5DeltaN或TRPV5DeltaC的组装会干扰TRPV5向质膜的运输，而与TRPV5DeltaNDeltaC的组装则不会。TRPV5通道组装信号分别在N尾的64-77位和c尾的596-601位之间进行了细化。拉下试验和共免疫沉淀表明，缺乏这些关键组装结构域的N-或c -尾突变体无法与TRPV5的尾部相互作用。综上所述，TRPV5的N-尾部（残基64-77）和c -尾部（残基596-601）的两个结构域对于通道亚基组装、随后TRPV5通道复合物向质膜的运输以及通道活性都很重要。
The epithelial Ca2+ channels TRPV5 and TRPV6 mediate the Ca2+ influx in 1,25-dihydroxyvitamin D-3-responsive epithelia and are therefore essential in the maintenance of the body Ca2+ balance. These Ca2+ channels assemble in (hetero) tetrameric channel complexes with different functional characteristics regarding Ca2+-dependent inactivation, ion selectivity, and pharmacological block. Glutathione S-transferase pull-downs and co-immunoprecipitations demonstrated an essential role of the intracellular N- and C-tails in TRPV5 channel assembly by physical interactions between N-N tails, C-C tails, and N-C-tails. Patch clamp analysis in human embryonic kidney (HEK293) cells and Ca-45(2+) uptake experiments in Xenopus laevis oocytes co-expressing TRPV5 wild-type and truncated proteins indicated that TRPV5DeltaN (deleted N-tail) and TRPV5DeltaC (deleted C-tail) decreased channel activity of wild-type TRPV5 in a dominant-negative manner, whereas TRPV5DeltaNDeltaC (deleted N-tail/C-tail) did not affect TRPV5 activity. Oocytes co-expressing wild-type TRPV5 and TRPV5DeltaN or TRPV5DeltaC showed virtually no wild-type TRPV5 expression on the plasma membrane, whereas co-expression of wild-type TRPV5 and TRPV5DeltaNDeltaC displayed normal channel surface expression. This indicates that TRPV5 trafficking toward the plasma membrane was disturbed by assembly with TRPV5DeltaN or TRPV5DeltaC but not with TRPV5DeltaNDeltaC. TRPV5 channel assembly signals were refined between amino acid positions 64-77 and 596-601 in the N- tail and C-tail, respectively. Pull-down assays and co-immunoprecipitations demonstrated that N- or C-tail mutants lacking these critical assembly domains were unable to interact with tails of TRPV5. In conclusion, two domains in the N- tail (residues 64-77) and C-tail (residues 596-601) of TRPV5 are important for channel subunit assembly, subsequent trafficking of the TRPV5 channel complex to the plasma membrane, and channel activity.