Homo- and heterotetrameric architecture of the epithelial Ca2+ channels TRPV5 and TRPV6
Homo- and heterotetrameric architecture of the epithelial Ca2+ channels TRPV5 and TRPV6
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DOI：
10.1093/emboj/cdg080
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发表时间：
2003-02-17
期刊：
影响因子：
11.4
通讯作者：
Bindels, RJM
中科院分区：
文献类型：
--
作者：
Hoenderop, JGJ;Voets, T;Bindels, RJM
研究了上皮细胞钙通道(TRPV5和TRPV6)的分子组装，以确定亚单位的化学计量和组成。对表达TRPV5和TRPV6的非洲爪哇卵母细胞进行免疫印迹分析，发现两条大小分别为75 kDa和85-100 kDa的特异条带，分别对应于核心蛋白和糖基化蛋白。随后，将这些卵母细胞的膜沉淀在蔗糖梯度上。免疫印迹显示，TRPV5和TRPV6复合体以摩尔迁移率迁移。WT为400 kDa，符合四聚体结构。四聚体化学计量比在HEK293细胞的电生理分析中得到证实，该细胞共表达串联通道和一个降低了CD2+敏感性和电压依赖门控的TRPV5孔突变体。用共表达TRPV5和TRPV6的卵母细胞的膜部分进行免疫沉淀表明，这两种通道都可以形成异构体复合体。所有可能的异四聚体TRPV5/6复合体在HEK293细胞中的表达导致了钙通道的变化，包括钙依赖的失活、Ba2+的选择性和药物阻断。因此，共表达TRPV5和TRPV6的钙离子转运上皮细胞可以产生一组具有不同钙离子转运动力学的功能异构体通道。
The molecular assembly of the epithelial Ca2+ channels (TRPV5 and TRPV6) was investigated to determine the subunit stoichiometry and composition. Immunoblot analysis of Xenopus laevis oocytes expressing TRPV5 and TRPV6 revealed two specific bands of 75 and 85-100 kDa, corresponding to the core and glycosylated proteins, respectively, for each channel. Subsequently, membranes of these oocytes were sedimented on sucrose gradients. Immunoblotting revealed that TRPV5 and TRPV6 complexes migrate with a mol. wt of 400 kDa, in line with a tetrameric structure. The tetrameric stoichiometry was confirmed in an electrophysiological analysis of HEK293 cells co-expressing concatemeric channels together with a TRPV5 pore mutant that reduced Cd2+ sensitivity and voltage-dependent gating. Immunoprecipitations using membrane fractions from oocytes co-expressing TRPV5 and TRPV6 demonstrated that both channels can form heteromeric complexes. Expression of all possible heterotetrameric TRPV5/6 complexes in HEK293 cells resulted in Ca2+ channels that varied with respect to Ca2+-dependent inactivation, Ba2+ selectivity and pharmacological block. Thus, Ca2+-transporting epithelia co-expressing TRPV5 and TRPV6 can generate a pleiotropic set of functional heterotetrameric channels with different Ca2+ transport kinetics.