Biogenesis and topology of the transient receptor potential Ca2+ channel TRPC1
Biogenesis and topology of the transient receptor potential Ca2+ channel TRPC1
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DOI：
10.1074/jbc.m312456200
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发表时间：
2004-03-26
影响因子：
4.8
通讯作者：
Turner, RJ
中科院分区：
文献类型：
--
作者：
Dohke, Y;Oh, YS;Turner, RJ
TRPC离子通道是响应细胞内Ca 2+库耗竭而激活的钙库操纵的Ca 2+进入途径的候选者。亲水性分析表明，这些蛋白质含有8个疏水区（HR），可能形成α-螺旋跨膜段。基于与其他离子通道有限的序列相似性，已经提出八个HR中只有六个实际上跨越膜，并且最后两个跨膜段（HR 6和8）与离子传导孔（HR 7形成其一部分）相邻。在这里，我们研究了人TRPC 1的生物发生和跨膜拓扑结构来测试这个模型。我们采用了截断突变体的方法结合插入糖基化位点到全长TRPC 1。在我们的截短突变体中，含有一个或多个HR的TRPC 1序列的部分融合在增强的绿色荧光蛋白和C-末端糖基化标签之间。这些嵌合体在人胚胎细胞系HEK-293 T中瞬时表达。标签的糖基化用于监测其相对于内质网腔的位置，从而监测HR取向。我们的数据表明，HR 1，4和6从胞质溶胶到ER腔穿过膜，HR 2，5和8具有相反的方向，HR 3在胞质溶胶侧离开膜。我们的研究结果还表明，HR 8的下游序列在将其C-末端锚定在膜的胞质侧上起着重要作用。这种效应似乎阻止HR 7跨越双层，并导致其形成先前设想的TRPC通道类型的孔样结构。我们推测，类似的机制可能是负责其他离子通道孔的形成。
The TRPC ion channels are candidates for the store-operated Ca2+ entry pathway activated in response to depletion of intracellular Ca2+ stores. Hydropathy analyses indicate that these proteins contain eight hydrophobic regions (HRs) that could potentially form alpha-helical membrane-spanning segments. Based on limited sequence similarities to other ion channels, it has been proposed that only six of the eight HRs actually span the membrane and that the last two membrane-spanning segments (HRs 6 and 8) border the ion-conducting pore of which HR 7 forms a part. Here we study the biogenesis and transmembrane topology of human TRPC1 to test this model. We have employed a truncation mutant approach combined with insertions of glycosylation sites into full-length TRPC1. In our truncation mutants, portions of the TRPC1 sequence containing one or more HRs were fused between the enhanced green fluorescent protein and a C-terminal glycosylation tag. These chimeras were transiently expressed in the human embryonic cell line HEK-293T. Glycosylation of the tag was used to monitor its location relative to the lumen of the endoplasmic reticulum and thereby HR orientation. Our data indicate that HRs 1, 4, and 6 cross the membrane from cytosol to the ER lumen, that HRs 2, 5, and 8 have the opposite orientation, and that HR 3 is left out of the membrane on the cytosolic side. Our results also show that the sequence downstream of HR 8 plays an important role in anchoring its C-terminal end on the cytosolic side of the membrane. This effect appears to prevent HR 7 from spanning the bilayer and to result in its forming a pore-like structure of the type previously envisioned for the TRPC channels. We speculate that a similar mechanism may be responsible for the formation of other ion channel pores.