Molecular components of the store-operated CRAC channel

描述（由申请人提供）：1992年发现了典型的储存操作钙内流途径ICRAC（即“Ca2+释放激活Ca2+电流”）。从那时起，关于ICRAC的生理和临床重要性的大量信息已经获得，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蛋白质在储存操作的Ca2+内流中是必不可少的，即基质相互作用分子（STIM1）和CRAC调节剂1 （（CRACM1）或Orai1）。STIM1和CRACM1的联合过表达极大地放大了储存操作电流，这些电流具有ICRAC最明显的特征。随后，证明了CRACM1是CRAC通道的一个成孔亚基。我们的初步数据进一步表明，CRACM1同源物CRACM2和CRACM3也形成具有不同性质的存储操作通道，并且STIM2可以以存储依赖性和存储非依赖性的方式瞬时激活CRACM1蛋白。因此，我们假设CRACM和STIM同源物代表了一组介导具有不同功能特性的存储操作Ca2+进入的蛋白质。Specific Aim 1验证了STIM1和CRACM1的特定结构特征决定它们的功能和相互作用的假设。我们将通过修饰分子位点来评估三个crac同源物的性质，这些位点已被确定为ICRAC特定的已知功能特征的可能结构决定因素。CRACM突变体将在HEK293细胞中与STIM1共表达，并使用生物物理方法（包括全细胞膜片钳和钙成像）进行功能分析。下一步，我们将研究导致CRAC通道激活的STIM1/2和CRACM1/2/3的分子偶联元件。提出的CRACM突变体将在HEK293细胞中与wt STIM1/2共表达，而STIM1/2突变体将与wt CRACM1/2/3共表达。特异性Aim 2验证了STIM1和STIM2，以及CRACM1、CRACM2和CRACM3是CRAC通道的交替分子组分的假设，赋予了天然细胞系统中不同的通道特性和Ca2+信号。为此，人类细胞系统Jurkat T、HEK293和HeLa细胞将用于将储存操作的Ca2+通道补体与激动剂介导的Ca2+信号相关联。此外，细胞（DT40）和动物敲除模型（CRACM和STIM KO小鼠）将用于评估CRACM和STIM的补体和功能。这项工作将进一步加深我们对CRAC钙通道的分子定义的理解，极大地改善了在过敏、炎症和自身免疫性疾病中开发涉及储存操作的Ca2+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.