Function of Orai/Stim Proteins Studied in Transgenic Animal Models

Abstract

Calcium signaling is an indispensable signaling cascade for an organism. Since the concentration of free intracellular calcium ions ([Ca2+]i) is maintained at a very low level under resting conditions (100–200 nM), the activation of calcium signaling requires the elevation of [Ca2+]i, which is regulated by selective or nonselective Ca2+-permeable channels and transporters upon stimulation. Studies have shown that the dysregulated function of Ca2+ channels or their modulators causes channelopathies, such as immunodeficiency and Timothy syndrome [1].A ligand binding to cell surface receptors, for example, immunoreceptors such as the T cell receptors (TCR) or G-protein-coupled receptors, induces the depletion of Ca2+ stores in the endoplasmic reticulum (ER), which in turn induces a large and sustained Ca2+ entry from extracellular space. This Ca2+ entry is called “store-operated Ca2+” (SOC) entry, formerly coined “capacitative” Ca2+ entry [2] (see Chapter 16). SOC entry has been observed in a variety of non-excitable and excitable cells and is now known to contribute to the function of many cell types including lymphocytes [3], pancreatic acinar cells [4], and skeletal muscle cells [5]. The prototypical SOC channel is the Ca2+-release-activated Ca2+ (CRAC) channel originally characterized in T lymphocytes and mast cells [6,7]. Although the molecular mechanism and identity remained a mystery for over 20 years, several groups identified two key proteins of the SOC entry, stromal interaction molecule 1 (STIM1) as the ER Ca2+ sensor [8,9] and ORAI1 (also known as CRACM1 or TMEM142A) as a pore-forming subunit of the CRAC channels [10–12] a decade ago (see Chapters 2 and 3).The aim of this chapter is to summarize how an Orai/Stim gene-deficient mouse is established and to describe a general protocol to make mouse embryonic fibroblasts (MEFs). Functions that have been elucidated using knockout mice from selected examples are also discussed. Readers interested in the details of gene targeting using ES cells are referred to an excellent textbook [13].