TRPC3 and TRPC4 associate to form a redox-sensitive cation channel - Evidence for expression of native TRPC3-TRPC4 heteromeric channels in endothelial cells
TRPC3 and TRPC4 associate to form a redox-sensitive cation channel - Evidence for expression of native TRPC3-TRPC4 heteromeric channels in endothelial cells
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DOI：
10.1074/jbc.m512205200
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发表时间：
2006-05-12
影响因子：
4.8
通讯作者：
Groschner, K
中科院分区：
文献类型：
--
作者：
Poteser, M;Graziani, A;Groschner, K
经典瞬时受体电位蛋白（TRPC）被认为可在多种组织（包括血管内皮）中形成同聚体或异聚体阳离子通道。TRPC多聚体的组装尚未完全了解。特别是，远缘相关的TRPC异构体的异聚体组装仍然是一个有争议的问题。因为我们之前曾提出TRPC蛋白是猪主动脉内皮细胞（PAECs）氧化还原激活阳离子电导的基础，所以我们着手分析内源性内皮TRPC通道的TRPC亚基组成，并在此报告一种对氧化还原敏感的TRPC3 - TRPC4通道复合物。TRPC3和TRPC4蛋白能够结合并形成阳离子传导孔复合物，这得到了以下四条证据的支持：1）在PAECs和HEK293细胞中进行的免疫共沉淀实验表明TRPC3和TRPC4在同一复合物中相互关联。2）荧光共振能量转移分析表明TRPC3 - TRPC4存在关联，涉及TRPC4的N末端和TRPC3亚基的C末端之间的紧密接近。3）在HEK293细胞中共表达TRPC3和TRPC4产生了一个具有独特生物物理和调节特性的通道。4）显性负性TRPC4蛋白的表达抑制了HEK293表达系统和天然内皮细胞中与TRPC3相关的通道活性。具体而言，一种细胞外带有血凝素（HA）标签的TRPC4突变体（对抗 - HA抗体的阻断敏感）被发现可将抗 - HA敏感性转移给HEK293表达系统中与TRPC3相关的电流以及PAECs的氧化还原敏感阳离子电导。我们提出TRPC3和TRPC4是受细胞氧化还原状态调控的天然内皮阳离子通道的亚基。
Canonical transient receptor potential proteins (TRPC) have been proposed to form homo- or heteromeric cation channels in a variety of tissues, including the vascular endothelium. Assembly of TRPC multimers is incompletely understood. In particular, heteromeric assembly of distantly related TRPC isoforms is still a controversial issue. Because we have previously suggested TRPC proteins as the basis of the redox-activated cation conductance of porcine aortic endothelial cells (PAECs), we set out to analyze the TRPC subunit composition of endogenous endothelial TRPC channels and report here on a redox- sensitive TRPC3-TRPC4 channel complex. The ability of TRPC3 and TRPC4 proteins to associate and to form a cation-conducting pore complex was supported by four lines of evidence as follows: 1) Co-immunoprecipitation experiments in PAECs and in HEK293 cells demonstrated the association of TRPC3andTRPC4in the same complex. 2) Fluorescence resonance energy transfer analysis demonstrated TRPC3-TRPC4 association, involving close proximity between the N terminus of TRPC4 and the C terminus of TRPC3 subunits. 3) Co expression of TRPC3 and TRPC4 in HEK293 cells generated a channel that displayed distinct biophysical and regulatory properties. 4) Expression of dominantnegative TRPC4 proteins suppressed TRPC3-related channel activity in the HEK293 expression system and in native endothelial cells. Specifically, an extracellularly hemagglutinin (HA)-tagged TRPC4 mutant, which is sensitive to blockage by anti-HA-antibody, was found to transfer anti- HA sensitivity to both TRPC3-related currents in the HEK293 expression system and the redox-sensitive cation conductance of PAECs. We propose TRPC3 and TRPC4 as subunits of native endothelial cation channels that are governed by the cellular redox state.