Store-operated Ca2+ entry (SOCE) through Ca2+ release-activated Ca2+ (CRAC) channels contributes to calcium signaling in immune cells. CRAC channels consist of the Ca2+-binding protein STIM1 and the Ca2+ channel Orai. When Ca2+ stores in the endoplasmic reticulum (ER) have been depleted, STIM1 oligomerize and translocate to ER-plasma membrane junctions to cluster and activate Orai to elicit Ca2+ influx. By affinity purifying cross-linked Orai1-STIM1 complexes from stably transfected HeLa cells, Srikanth et al. identified CRAC regulator 2A (CRACR2A) as a binding partner of Orai. CRACR2A was predicted to contain two Ca2+-binding EF hand domains. Immunoprecipitations indicated that the interaction of CRACR2A with Orai1 (like that of STIM1) was enhanced after depletion of ER Ca2+ stores by thapsigargin treatment. In vitro binding assays showed that the association between CRACR2A and Orai1 was stronger in the absence of Ca2+ than in its presence, an effect that was not observed with a form of CRACR2A with mutations that abrogated Ca2+ binding to the EF hand domains (CRACR2AEF2MUT). Knockdown of CRACR2A in Jurkat cells by small interfering RNA (siRNA) reduced the clustering of transfected STIM1 and Orai1 in response to depletion of ER Ca2+ stores by thapsigargin treatment, whereas transfection of CRACR2AEF2MUT caused clustering of STIM1 in the absence of thapsigargin treatment. Expression of CRACR2A increased SOCE and apoptosis in Jurkat cells, and both effects were enhanced when CRACR2AEF2MUT was expressed instead of the wild-type form. In the associated commentary, Carrasco and Meyer note that these findings help to explain why CRAC channels are inhibited by high cytosolic Ca2+ concentrations.
S. Srikanth, H.-J. Jung, K.-D. Kim, P. Souda, J. Whitelegge, Y. Gwack, A novel EF-hand protein, CRACR2A, is a cytosolic Ca2+ sensor that stabilizes CRAC channels in T cells. Nat. Cell Biol. 12, 436–446 (2010). [PubMed]
S. Carrasco, T. Meyer, Cracking CRAC. Nat. Cell Biol. 12, 416–418 (2010). [PubMed]