Calcium regulation of inositol 1,4,5-trisphosphate receptors
Calcium regulation of inositol 1,4,5-trisphosphate receptors
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DOI：
10.1016/j.ceca.2005.07.007
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发表时间：
2005-12-01
期刊：
影响因子：
4
通讯作者：
Khan, MT
中科院分区：
文献类型：
--
作者：
Joseph, SK;Brownell, S;Khan, MT
Ca ~（2+）对I型IP_3R通道活性具有刺激和抑制作用。然而，IP（3）Rs中Ca 2+感应的结构决定因素尚未完全了解。其他人先前的研究已经鉴定了I型IP 3R的八个结构域，当表达为GST融合蛋白时，其结合Ca-45（2+）。我们突变了6个高度保守的酸性残基内的第二个这些域（aa 378 -450）在全长IP 3R和测量的Ca 2+调节的IP 3介导的Ca 2+释放COS-7细胞。用最大[IP 3]（1 μ M）测量的Ca-45（2+）通量测定表明，其中一个突变体保留了与对照（E411 Q）没有显著差异的Ca 2+敏感性，其中三个突变体显示出增强的Ca 2+抑制（D426 N、E428 Q和E439 Q），其中两个突变体对Ca 2+抑制相对不敏感（D442 N和D444 N）。IP 3剂量-反应关系表明，对Ca 2+抑制的敏感性和对IP 3的亲和力与三种构建体相关。具有增强的IP 3敏感性的其它突变体（例如R441 Q和II/I型IP 3 IP 3R嵌合体）对Ca 2+抑制也较不敏感。我们的结论是酸性残基内的aa 378 -450段是不可能代表一个单一的功能性钙离子结合域，并没有贡献的钙离子激活的受体。突变的不同影响可能与它们在配体结合结构域的晶体结构中鉴定的两簇酸性残基内的位置有关[I. Bosanac，J.R. Alattia，T.K. Mal等人，与其配体复合的肌醇1，4，5-三磷酸受体结合核心的结构，Nature 420（2002）696-700]。这些数据支持以下观点：所有IP 3R亚型可能显示出一系列Ca 2+敏感性，这些敏感性由蛋白质内的多个位点决定，并受到IP 3受体亲和力的显著影响（c）2005 Elsevier Ltd.保留所有权利。
Ca2+ exerts both a stimulatory and inhibitory effect on type-I IP3R channel activity. However, the structural determinants of Ca2+ sensing in IP(3)Rs are not fully understood. Previous studies by others have identified eight domains of the type-I IP3R that bind Ca-45(2+) when expressed as GST-fusion proteins. We have mutated six highly conserved acidic residues within the second of these domains (aa378-450) in the full-length IP3R and measured the Ca2+ regulation of IP3-mediated Ca2+ release in COS-7 cells. Ca-45(2+) flux assays measured with a maximal [IP3] (1 mu M) indicate that one of the mutants retained a Ca2+ sensitivity that was not significantly different from control (E411Q), three of the mutants show an enhanced Ca2+ inhibition (D426N, E428Q and E439Q) and two of the mutants were relatively insensitive to Ca2+ inhibition (D442N and D444N). IP3 dose-response relationships indicated that the sensitivity to Ca2+ inhibition and affinity for IP3 were correlated for three of the constructs. Other mutants with enhanced IP3 Sensitivity (e.g. R441Q and a type-II/I IP3 IP3R chimera) were also less sensitive to Ca2+ inhibition. We conclude that the acidic residues within the aa378-450 segment are unlikely to represent a single functional Ca2+ binding domain and do not contribute to Ca2+ activation of the receptor. The different effects of the mutations may be related to their location within two clusters of acidic residues identified in the crystal structure of the ligand-binding domain [I. Bosanac, J.R. Alattia, T.K. Mal, et al., Structure of the inositol 1,4,5-trisphosphate receptor binding core in complex with its ligand, Nature 420 (2002) 696-700]. The data support the view that all IP3R isoforms may display a range of Ca2+ sensitivities that are determined by multiple sites within the protein and markedly influenced by the affinity of the receptor for IP3 (c) 2005 Elsevier Ltd. All rights reserved.