Molecular components of the store-operated CRAC channel

描述（由申请人提供）：1992年确定了原型钙池操作的钙内流途径ICRAC（“Ca 2+释放激活的Ca 2+电流”）。从那时起，大量的信息已经获得了ICRAC的生理和临床的重要性，然而，其分子组成仍然难以捉摸。直到最近才有突破性的发现，确定了两种在钙池操纵的钙内流中必不可少的蛋白质，即基质相互作用分子（STIM 1）和CRAC调节剂1（（CRACM 1）或Orai 1）。STIM 1和CRACM 1的组合过表达极大地放大了存储操作电流，并且这些具有ICRAC的最确定特征。随后，证明了CRACM 1是CRAC通道的成孔亚基。我们的初步数据进一步表明，CRACM 1同源物CRACM 2和CRACM 3也形成具有不同特性的存储操作通道，并且STIM 2可以以存储依赖和存储不依赖的方式瞬时激活CRACM蛋白。因此，我们假设，CRACM和STIM同源物代表一组蛋白质，介导存储操作的Ca 2+进入具有不同的功能特性。具体目标1测试了STIM 1和CRACM 1的特定结构特征决定其功能和相互作用的假设。我们将通过修改已被确定为可能的结构决定因素的具体的，已知的功能特性的ICRAC的分子位点，评估三个CRACM同系物的属性。CRACM突变体构建体将与STIM 1在HEK 293细胞中共表达，并使用生物物理方法进行功能分析，包括全细胞膜片钳和钙成像。在下一步中，将研究导致CRAC通道激活的STIM 1/2和CRACM 1/2/3的分子偶联元件。所提出的CRACM突变体构建体将与wt STIM 1/2在HEK 293细胞中共表达，并且STIM 1/2突变体将与wt CRACM 1/2/3共表达。特定目的2检验了以下假设：STIM 1和STIM 2以及CRACM 1、CRACM 2和CRACM 3是CRAC通道的替代分子组分，在天然细胞系统中赋予不同的通道特征和Ca 2+信号。为此，将使用人细胞系统Jurkat T、HEK 293和HeLa细胞将钙池操纵的Ca 2+通道补体与激动剂介导的Ca 2+信号相关联。此外，细胞（DT 40）和动物敲除模型（CRACM和STIM KO小鼠）将用于评估CRACM和STIM补体和功能。这项工作将进一步加深我们对CRAC钙通道分子定义的理解，大大改善开发涉及过敏，炎症和自身免疫性疾病中钙库操作的Ca 2+内流的治疗策略的前景。

DESCRIPTION (provided by applicant): The prototypical store-operated calcium-influx pathway ICRAC (for "Ca2+-release activated Ca2+ current") was identified in 1992. Since then, substantial information has been acquired about ICRAC's physiological and clinical importance, however, its molecular composition has remained elusive. Only recently did break-through findings identify two proteins that are essential in store-operated Ca2+ influx, namely stromal interaction molecule (STIM1) and CRAC Modulator 1 ((CRACM1) or Orai1). The combined overexpression of STIM1 and CRACM1 greatly amplifies store-operated currents and these possess the most defining characteristics of ICRAC. Subsequently, it was demonstrated that CRACM1 is a pore-forming subunit of the CRAC channel. Our preliminary data further suggest that the CRACM1 homologs CRACM2 and CRACM3 also form store-operated channels with distinct properties and that STIM2 can transiently activate CRACM proteins in store-dependent and store-independent manners. We therefore hypothesize that CRACM and STIM homologs represent a group of proteins that mediate store-operated Ca2+ entry with distinct functional properties. Specific Aim 1 tests the hypothesis that specific structural features of STIM1 and CRACM1 determine their function and interaction. We will assess the properties of the three CRACM homologs by modifying molecular sites that have been identified as possible structural determinants of specific, known functional characteristics of ICRAC. The CRACM mutant constructs will be co-expressed with STIM1 in HEK293 cells and functionally analyzed using biophysical approaches, including whole-cell patch-clamp and calcium imaging. In a next step, the molecular coupling elements of STIM1/2 and CRACM1/2/3 that result in CRAC channel activation will be investigated. The proposed CRACM mutant constructs will be co-expressed with wt STIM1/2 in HEK293 cells and STIM1/2 mutants will be co-expressed with wt CRACM1/2/3. Specific Aim 2 tests the hypothesis that STIM1 and STIM2, as well as CRACM1, CRACM2 and CRACM3 are alternate molecular components of the CRAC channel conferring different channel characteristics and Ca2+ signals in native cell systems. To this end, the human cell systems Jurkat T, HEK293, and HeLa cells will be used to correlate store-operated Ca2+ channel complements with agonist-mediated Ca2+ signals. Furthermore, cellular (DT40) and animal knock-out models (CRACM and STIM KO mice) will be used to assess CRACM and STIM complement and function. The proposed work will further our understanding of the molecular definition of CRAC calcium channels, greatly improving the prospects for developing therapeutic strategies involving store-operated Ca2+ influx in allergy, inflammation and autoimmune diseases.