Intramolecular shielding maintains STIM1 in an inactive conformation

Abstract

SummaryStore-operated calcium entry (SOCE) represents a major calcium influx pathway in non-excitable cells and is central to many physiological processes such as T cell activation and mast cell degranulation. SOCE is activated through intricate coordination between the Ca 2+ sensor on the ER membrane (stromal interaction molecule 1, STIM1) and the plasma membrane channel Orai1. When Ca 2+ stores are depleted, STIM1 oligomerizes and physically interacts with Orai1 through its SOAR/CAD domain, resulting in Orai1 gating and Ca 2+ influx. Here, we describe novel inter-and intramolecular FRET sensors in the context of the full-length membrane-anchored STIM1, and show that STIM1 undergoes a conformational change in response to store depletion to adopt a stretched 'open' conformation that exposes SOAR/CAD and allows it to interact with Orai1. Mutational analyses reveal that electrostatic interactions between the predicted first and third coiled-coil domains of STIM1 are not involved in maintaining the 'closed' inactive conformation. In addition, the results argue that an amphipathic a-helix between residues 317 and 336 in the so-called inhibitory domain is important to maintain STIM1 in a closed conformation at rest. Indeed, mutations that alter the amphipathic properties of this helix result in a STIM1 variant that is unable to respond to store depletion in terms of forming puncta, translocation to the cortical ER or activating Orai1.

Application of fluorescence resonance energy transfer in protein studies

“…Thoughtfully designed FRET measurements have been applied in studies on voltage-gated K v 2.1 channels [126], voltage-gated Ca v 1.2 channels [127], CLC chloride channels [46, 51], ryanodine receptors [128], L-type Ca 2+ channels [129], P 2 X channels [130], gramicidin channels [131, 132], Large-conductance voltage- and calcium-dependent potassium channels (BK channels) [133], and store-operated calcium channels [134]. …”

Critical role for Orai1 C-terminal domain and TM4 in CRAC channel gating

“…The process begins when depletion of calcium from the ER leads to calcium disassociation from the EF hand of STIM1, facilitating dimerization of SAM domains [21]. Dimerization of STIM1 ER luminal domains triggers extensive conformational changes in the cytoplasmic domains that lead to the exposure of CAD [20,[22][23][24][25][26]. CAD binds with low affinity to a site in the Orai1 N terminal (NBD, residues 73-87; Figure 1A) and with higher affinity to a site in the Orai1 C terminal (CBD, residues 267-292; Figure 1A) [14,15,28,29].…”

Cooperative Binding of Stromal Interaction Molecule 1 (STIM1) to the N and C Termini of Calcium Release-activated Calcium Modulator 1 (Orai1)

“…Each Orai1 subunit has four transmembrane segments, with the N and C termini of the protein facing the cytosol, and Orai1 channels are assembled from hexamers of Orai1 subunits. The activation of CRAC channels starts when depletion of calcium from the ER causes rearrangement of STIM1 and unmasking of CAD (3)(4)(5)(6)(7)(8). When exposed, CAD binds to a site in the Orai1 N terminus (N-terminal binding domain (NBD), residues 74 -87) and to a second site in the Orai1 C terminus (C-terminal binding domain (CBD), residues 267-292) (1,9).…”