Subtype-selective regulation of IP(3) receptors by thimerosal via cysteine residues within the IP(3)-binding core and suppressor domain.
Subtype-selective regulation of IP(3) receptors by thimerosal via cysteine residues within the IP(3)-binding core and suppressor domain.
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DOI：
10.1042/bj20121600
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发表时间：
2013-04-15
期刊：
影响因子：
--
通讯作者：
Taylor CW
中科院分区：
文献类型：
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作者：
Khan SA;Rossi AM;Riley AM;Potter BV;Taylor CW
IP 3R（IP 3 [肌醇1，4，5-三磷酸]受体）和兰尼碱受体是最广泛表达的细胞内Ca 2+通道，两者都受巯基试剂的调节。在稳定表达哺乳动物IP 3R单一亚型的DT 40细胞中，低浓度的硫柳汞（也称为硫柳汞）可氧化硫醇形成硫柳汞乙基复合物，通过IP 3R 1和IP 3R 2增加IP 3诱发的Ca 2+释放的敏感性，但抑制IP 3R 3。IP 3 R的激活由IP 3与IBC结合（IP 3结合核心;残基224-604）启动，并通过IBC和SD之间界面的重排（抑制结构域;残基1-223）进行。硫柳汞（100 μM）刺激IP 3与IP 3R 1和IP 3R 2的分离NT（N-末端;残基1-604）结合，但不与IP 3R 3结合。竞争性拮抗剂（肝素）或部分激动剂（二聚体-IP 3）与NT 1的结合不受硫柳汞的影响，这表明硫柳汞的作用与IP 3R活化特别相关。IP 3与其中所有半胱氨酸残基被丙氨酸取代的NT 1结合对硫柳汞不敏感，其中半胱氨酸残基在SD或IBC中被取代的NT 1也是如此。这表明硫代巴比妥在SD和IBC中都与半胱氨酸直接相互作用。嵌合蛋白，其中SD的IP 3R被替换的结构相关的A结构域的ryanodine受体的功能，但硫柳汞抑制IP 3结合嵌合NT和IP 3-诱发的Ca 2+释放的嵌合IP 3R。这是第一次系统分析硫醇试剂对每种IP 3R亚型的影响。我们得出结论，硫柳汞选择性敏化IP 3R 1和IP 3R 2 IP 3通过修改内的SD和IBC的半胱氨酸残基，从而稳定的受体的活性构象。
IP3R (IP3 [inositol 1,4,5-trisphosphate] receptors) and ryanodine receptors are the most widely expressed intracellular Ca2+ channels and both are regulated by thiol reagents. In DT40 cells stably expressing single subtypes of mammalian IP3R, low concentrations of thimerosal (also known as thiomersal), which oxidizes thiols to form a thiomercurylethyl complex, increased the sensitivity of IP3-evoked Ca2+ release via IP3R1 and IP3R2, but inhibited IP3R3. Activation of IP3R is initiated by IP3 binding to the IBC (IP3-binding core; residues 224–604) and proceeds via re-arrangement of an interface between the IBC and SD (suppressor domain; residues 1–223). Thimerosal (100 μM) stimulated IP3 binding to the isolated NT (N-terminal; residues 1–604) of IP3R1 and IP3R2, but not to that of IP3R3. Binding of a competitive antagonist (heparin) or partial agonist (dimeric-IP3) to NT1 was unaffected by thiomersal, suggesting that the effect of thimerosal is specifically related to IP3R activation. IP3 binding to NT1 in which all cysteine residues were replaced by alanine was insensitive to thimerosal, so too were NT1 in which cysteine residues were replaced in either the SD or IBC. This demonstrates that thimerosal interacts directly with cysteine in both the SD and IBC. Chimaeric proteins in which the SD of the IP3R was replaced by the structurally related A domain of a ryanodine receptor were functional, but thimerosal inhibited both IP3 binding to the chimaeric NT and IP3-evoked Ca2+ release from the chimaeric IP3R. This is the first systematic analysis of the effects of a thiol reagent on each IP3R subtype. We conclude that thimerosal selectively sensitizes IP3R1 and IP3R2 to IP3 by modifying cysteine residues within both the SD and IBC and thereby stabilizing an active conformation of the receptor.