Molecular identification of the CRAC channel by altered ion selectivity in a mutant of Orai
Molecular identification of the CRAC channel by altered ion selectivity in a mutant of Orai
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DOI：
10.1038/nature05108
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发表时间：
2006-09-14
期刊：
影响因子：
64.8
通讯作者：
Cahalan, Michael D.
中科院分区：
文献类型：
--
作者：
Yeromin, Andriy V.;Zhang, Shenyuan L.;Cahalan, Michael D.
最近的RNA干扰筛选已经鉴定了几种对果蝇和哺乳动物中钙库操纵的Ca 2+内流和Ca 2+释放激活的Ca 2+（CRAC）通道活性至关重要的蛋白质，包括跨膜蛋白Stim（基质相互作用分子）（1，2）和奥赖（3-5）。刺激可能作为管腔Ca 2+含量的传感器发挥作用，并在Ca 2+储存耗尽后触发表面膜中CRAC通道的激活（1，6）。在奥赖（也称为olf 186-F）的三种人类同源物中，发现12号染色体上的Orai 1在严重联合免疫缺陷病患者中发生突变，野生型Orai 1的表达恢复了患者T细胞中的Ca 2+内流和CRAC通道活性（3）。Stim和奥赖的过表达一起显著增加CRAC电流（5，7 -9）。然而，尚不清楚Stim或奥赖是否实际上形成CRAC通道，或者它们的表达是否仅仅限制由不同的通道形成亚基介导的CRAC通道活性。在这里，我们表明，野生型Stim和奥赖之间的相互作用，通过免疫共沉淀法评估，大大增强后处理毒胡萝卜素诱导钙库耗尽。通过定点突变，我们发现，从谷氨酸到天冬氨酸在位置180的保守的S1 - S2环的奥赖的点突变转换的离子选择性特性的CRAC电流从钙离子选择性与内向整流是选择性的一价阳离子和外向整流。同一位置的电荷中和突变（谷氨酸至丙氨酸）充当显性负性不导电亚基。在同一个循环中的其他电荷中和突变体表达大的内向整流CRAC电流，其中两个表现出对通道阻断剂Gd 3+的敏感性降低。这些结果表明，奥赖本身形成CRAC通道的Ca 2+选择性过滤器。
Recent RNA interference screens have identified several proteins that are essential for store-operated Ca2+ influx and Ca2+ release-activated Ca2+ ( CRAC) channel activity in Drosophila and in mammals, including the transmembrane proteins Stim ( stromal interaction molecule)(1,2) and Orai(3-5). Stim probably functions as a sensor of luminal Ca2+ content and triggers activation of CRAC channels in the surface membrane after Ca2+ store depletion(1,6). Among three human homologues of Orai ( also known as olf186-F), ORAI1 on chromosome 12 was found to be mutated in patients with severe combined immunodeficiency disease, and expression of wild-type Orai1 restored Ca2+ influx and CRAC channel activity in patient T cells(3). The overexpression of Stim and Orai together markedly increases CRAC current(5,7-9). However, it is not yet clear whether Stim or Orai actually forms the CRAC channel, or whether their expression simply limits CRAC channel activity mediated by a different channel-forming subunit. Here we show that interaction between wild-type Stim and Orai, assessed by coimmunoprecipitation, is greatly enhanced after treatment with thapsigargin to induce Ca2+ store depletion. By site-directed mutagenesis, we show that a point mutation from glutamate to aspartate at position 180 in the conserved S1 - S2 loop of Orai transforms the ion selectivity properties of CRAC current from being Ca2+-selective with inward rectification to being selective for monovalent cations and outwardly rectifying. A charge-neutralizing mutation at the same position ( glutamate to alanine) acts as a dominant-negative non-conducting subunit. Other charge-neutralizing mutants in the same loop express large inwardly rectifying CRAC current, and two of these exhibit reduced sensitivity to the channel blocker Gd3+. These results indicate that Orai itself forms the Ca2+-selectivity filter of the CRAC channel.