Critical regions for activation gating of the inositol 1,4,5-trisphosphate receptor
Critical regions for activation gating of the inositol 1,4,5-trisphosphate receptor
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DOI：
10.1074/jbc.m300646200
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发表时间：
2003-05-09
影响因子：
4.8
通讯作者：
Mikoshiba, K
中科院分区：
文献类型：
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作者：
Uchida, K;Miyauchi, H;Mikoshiba, K
为了了解配体诱导的三磷酸肌醇（IP 3）受体（IP 3 R）/Ca 2+释放通道门控的分子机制，我们分析了保留IP（3）R1的IP 3结合和通道形成结构域的缺失突变体的通道特性。使用固有IP 3R缺陷的细胞作为受体表达的宿主细胞，我们确定了六个突变体，即缺少残基1-223、651-1130、1267-2110、1845-2042、1845-2216和2610-2748的突变体，没有表现出任何可测量的Ca 2+释放活性，而缺少残基1131-1379和2736-2749的突变体保留了活性。有限的胰蛋白酶消化表明，不仅缺乏残基1131-1379和2736-2749的IP 3门控Ca 2+渗透突变体，而且缺乏残基1-223和651-1130的两个非功能性突变体，至少保留了C-末端通道形成结构域的正常折叠结构。这些结果表明IP（3）R1的两个区域，即残基1-223和651-1130，对于IP 3诱导的门控是关键的。我们还确定了一个高度保守的半胱氨酸残基在位置2613，这是位于C-末端的尾巴，是必不可少的通道开放。基于这些结果，我们提出了一种新的五域结构模型，其中N端和内部耦合结构域都将配体结合信号传递到C端尾部，C端尾部充当看门人，触发IP（3）R1在IP 3结合后激活门的打开。
To understand the molecular mechanism of ligand-induced gating of the inositol 1,4,5-trisphosphate (IP3) receptor (IP3R)/Ca2+ release channel, we analyzed the channel properties of deletion mutants retaining both the IP3-binding and channel-forming domains of IP(3)R1. Using intrinsically IP3R-deficient cells as the host cells for receptor expression, we determined that six of the mutants, those lacking residues 1-223, 651-1130, 1267-2110, 1845-2042, 1845-2216, and 2610-2748, did not exhibit any measurable Ca2+ release activity, whereas the mutants lacking residues 1131-1379 and 2736-2749 retained the activity. Limited trypsin digestion showed that not only the IP3-gated Ca2+-permeable mutants lacking residues 1131-1379 and 2736-2749, but also two nonfunctional mutants lacking residues 1-223 and 651-1130, retained the normal folding structure of at least the C-terminal channel-forming domain. These results indicate that two regions of IP(3)R1, viz. residues 1-223 and 651-1130, are critical for IP3-induced gating. We also identified a highly conserved cysteine residue at position 2613, which is located within the C-terminal tail, as being essential for channel opening. Based on these results, we propose a novel five-domain structure model in which both N-terminal and internal coupling domains transduce ligand-binding signals to the C-terminal tail, which acts as a gatekeeper that triggers opening of the activation gate of IP(3)R1 following IP3 binding.