A novel EF-hand protein, CRACR2A, is a cytosolic Ca2+ sensor that stabilizes CRAC channels in T cells

Abstract

Orai1 and STIM1 are critical components of Ca2+ release-activated Ca2+ (CRAC) channels that mediate store-operated Ca2+ entry (SOCE) in immune cells. While Orai1 and STIM1 co-cluster and physically interact to mediate SOCE, the cytoplasmic machinery modulating these functions remains poorly understood. We sought to find modulators of Orai1 and STIM1 using affinity protein purification and identified a novel EF-hand protein, CRACR2A (CRAC regulator 2A, EFCAB4B, FLJ33805). We show that CRACR2A directly interacts with Orai1 and STIM1, forming a ternary complex that dissociates at elevated Ca2+ concentrations. Studies using siRNA-mediated knockdown and mutagenesis show that CRACR2A is important for clustering of Orai1 and STIM1 upon store depletion. Expression of an EF-hand mutant of CRACR2A enhanced STIM1 clustering, elevated cytoplasmic Ca2+ and induced cell death, suggesting its active interaction with CRAC channels. These observations implicate CRACR2A, a novel Ca2+ binding protein, highly expressed in T cells and conserved in vertebrates, as a key regulator of CRAC channel-mediated SOCE.

Blockade of NOX2 and STIM1 signaling limits lipopolysaccharide-induced vascular inflammation

“…While Stim1 ΔEC mice displayed normal baseline characteristics up to 8 weeks, future studies are necessary to understand the role of STIM1 in aging, cardiovascular, and chronic lung diseases. Although Orai channels are the major STIM targets, new information reveals the additional STIM/Orai complex regulators including POST, junctate, SARAF, and CRACR2A (19,(63)(64)(65)(66) could also be considered as potential therapeutic targets. Importantly, therapeutic strategies targeting the Ca 2+ signaling machinery have great advantage over antioxidant-based strategies, since they do not interfere with the microbicidal role of ROS.…”

Alternative splicing converts STIM2 from an activator to an inhibitor of store-operated calcium channels

“…Alternative splicing converts STIM2 from an activator to an inhibitor of store-operated calcium channels (Srikanth et al, 2010;Palty et al, 2012;Miao et al, 2013). Significantly, all of these mechanisms modulate the activity of STIM proteins without altering their role as activators of SOCE.…”

Pore waters regulate ion permeation in a calcium release-activated calcium channel

“…Later, the Orai protein was identified as the pore subunit of the channel (7-9). CRAC regulator 2A was then discovered to reinforce the binding of the two aforementioned key components at elevated Ca 2+ levels to promote the SOCE, and to inhibit the influx at low Ca 2+ levels by dissociating from the complex (10). The newly published crystal structure of the CRAC channel pore subunit, Orai, from Drosophila melanogaster at 3.35 Å resolution, provides groundbreaking insights into a molecular architecture that is distinct from that of all of the other ion channels (11).…”