The Ca²⁺ sensor protein calmodulin interacts in a Ca²⁺-dependent manner with a large number of proteins that among them encompass a diverse assortment of functions and subcellular localizations. A method for monitoring calmodulin-protein interactions as they occur throughout a living cell would thus uniquely enable investigations of the intracellular landscape of [Ca²⁺] and its relationship to cell function. We have developed such a method based on capture of calmodulin-protein interactions by rapid photoactivated cross-linking (t_1/2 ∼7s) in cells stably expressing a tandem affinity tagged calmodulin that have been metabolically labeled with a photoreactive methionine analog. Tagged adducts are stringently enriched, and captured calmodulin interactors are then identified and quantified based on tandem mass spectrometry data for their tryptic peptides. In this paper we show that the capture behaviors of interactors in cells are consistent with the presence of basal microdomains of elevated [Ca²⁺]. Ca²⁺ sensitivities for capture were determined, and these suggest that [Ca²⁺] levels are above ∼1 μM in these regions. Although the microdomains appear to affect capture of most proteins, capture of some is at an apparent Ca²⁺-dependent maximum, suggesting they are targeted to the domains. Removal of extracellular Ca²⁺ has both immediate (5 min) and delayed (30 min) effects on capture, implying that the microdomains are supported by a combination of Ca²⁺ influx across the cell membrane and Ca²⁺ derived from internal stores. The known properties of the presumptive microdomain targeted proteins suggestroles in a variety of Ca²⁺-dependent basal metabolism and in formation and maintenance of the domains.

Ca ²⁺传感器蛋白钙调蛋白以Ca ²⁺依赖性方式与大量蛋白质相互作用，其中包括多种功能和亚细胞定位。一种监测钙调蛋白-蛋白质相互作用的方法，因为它们发生在整个活细胞中，因此可以独特地研究 [Ca ²⁺ ] 的细胞内景观及其与细胞功能的关系。我们开发了一种基于通过快速光活化交联（t _1/2∼7s）在稳定表达串联亲和标记的钙调蛋白的细胞中，该钙调蛋白已被光反应性蛋氨酸类似物代谢标记。标记的加合物经过严格富集，然后根据胰蛋白酶肽的串联质谱数据识别和量化捕获的钙调蛋白相互作用物。在本文中，我们表明细胞中相互作用物的捕获行为与升高的 [Ca ²⁺ ] 的基础微区的存在是一致的。测定了对捕获的Ca ²⁺敏感性，这些表明在这些区域中 [Ca ²⁺ ] 水平高于～1 μM。尽管微结构域似乎会影响大多数蛋白质的捕获，但对一些蛋白质的捕获是明显的 Ca ²⁺-依赖最大值，表明它们是针对域的。细胞外 Ca ²⁺的去除对捕获具有立即（5 分钟）和延迟（30 分钟）影响，这意味着微区受到跨细胞膜的 Ca ²⁺流入和来自内部储存的 Ca ^{2+的组合的支持。}假定的微结构域靶向蛋白的已知特性表明在多种Ca ²⁺依赖性基础代谢以及结构域的形成和维持中起作用。