Optogenetic Approaches to Control Calcium Entry in Non-Excitable Cells.

The calcium ion serves as a versatile and universal second messenger to control a myriad of biological processes, including muscle contraction, neurotransmission, hormone secretion, immune cell activation, cell motility, and apoptosis [1&#x2013;3]. The calcium release-activated calcium (CRAC) channel mediated by ORAI and stromal interaction molecule (STIM) constitutes one of the primary Ca²⁺ entry routes in non-excitable cells [4&#x2013;6] (see Chapter 2). The CRAC channel is regarded as a prototypical example of store-operated Ca²⁺ entry (SOCE), in which the depletion of Ca²⁺ store in the endoplasmic reticulum (ER) induces calcium influx from the extracellular space. Over the past decade, tremendous progress has been made with respect to the critical steps required to activate SOCE (Figure 8.1) [5&#x2013;7]. Under resting conditions, the STIM1 luminal EF-SAM domain is loaded with Ca²⁺ and exists largely as a monomer [8]. The STIM1 cytoplasmic domain (STIM1ct), consisting of a coiled-coil region (CC1), a minimal ORAI1-activating region (SOAR or CAD), and a C-terminal polybasic tail (PB), adopts a folded-back configuration that keeps itself inactive through autoinhibition [9,10]. Upon ER Ca²⁺ store depletion, dissociation of Ca²⁺ from the EF-SAM domain initiates a destabilization-coupled oligomerization process in the ER lumen [8]. Conformational changes in the canonical EF-hand Ca²⁺-binding motif disrupt the intramolecular interaction between the EF-hands and SAM domains, thereby causing aggregation of the luminal EF-SAM domains. The luminal domain oligomerization further triggers conformational changes that propagate throughout STIM1ct. STIM1ct redeploys itself and adopts a more extended conformation by exposing the SOAR/CAD domain, as well as the polybasic C-tail. Next, the activated STIM1 multimerizes and moves toward the ER-plasma membrane (PM) junctional sites, where it recruits and gates ORAI1 channels through direct physical association with ORAI1. This process is also facilitated by the interaction between its polybasic C-tail and the negatively charged phosphoinositides in the inner leaflet of the plasma membrane (see Chapters 2 and 3). Sustained Ca²⁺ influx through ORAI1 channels activates downstream effectors such as calcineurin, a Ca²⁺-dependent phosphatase that dephosphorylates the nuclear factor of activated T cells (NFAT) and triggers the nuclear translocation of NFAT to regulate gene expression during lymphocyte activation [4] (see Chapter 5). Two critical steps during SOCE activation, oligomerization of STIM1 luminal domain and conformational switch within STIM1ct, can be mimicked by photosensitive domains that undergo light-inducible oligomerization or allosteric regulation to devise photoactivatable CRAC channels (termed Opto-CRAC), thereby enabling remote and optical control of calcium signaling [11&#x2013;13]. Compared to the existing microbial opsin-based optogenetic tools, Opto-CRAC has several distinctive features such as the following: Unlike the widely used channelrhodopsin (ChR)-based tools [14] that exhibit less stringent ion selectivity and tend to perturb intracellular pH due to their high proton permeability, Opto-CRAC is engineered from a <i>bona fide</i> Ca²⁺ channel that is regarded as one of the most Ca²⁺-selective ion channels (see Chapter 1). The Opto-CRAC tool is among the smallest optogenetic tools (&lt;1 kb, compared to &gt;2.2 kb of ChR) and is thus compatible with viral vectors used for <i>in vivo</i> gene delivery. Its tunable and relatively slow kinetics make Opto-CRAC most suitable for generating customized Ca²⁺ oscillation patterns in non-excitable cell types, such as cells of the immune and hematopoietic systems. In this chapter, we present a brief overview of the design principles of Opto-CRAC constructs based on two different photoresponsive domains (CRY2 and LOV2) and illustrate how to use them to remotely control Ca²⁺ influx with at high spatiotemporal precision and subsequent nuclear translocation of a master transcriptional factor, the nuclear factor of activated T cells (NFAT), to fine-tune NFAT-dependent gene expression and control the function of immune cells.