Store-Operated Orai1 and IP₃Receptor-Operated TRPC1 Channel: Separation of the Siamese Twins

Abstract

Store-operated channels (SOC) are known to be physiologically activated following agonist-induced IP3 production and depletion of Ca2+ stores. Here we present molecular,biophysical and mechanistic evidence that two ubiquitously expressed plasma membrane channels may be responsible for creating a complex and sometimes controversial SOC image: one being a real SOC encoded by Orai1 and activated exclusively upon depletion of Ca2+ stores (via iPLA2beta -dependent pathway), while the second one is an IP3 receptor-operated channel (IP3ROC) encoded by TRPC1 and activated via its conformational coupling with IP3 receptor. In RBL-2H3 cells endogenously expressing Orai1 and TRPC1, we unmasked and characterized whole-cell current through IP3ROC channels that was hiding behind some familiar fingerprints of ICRAC, a current through the classical Ca2+-selective SOC (CRAC) channels. We discriminated these currents by their molecular identity, selectivity and different requirements for store depletion, IP3, iPLA2beta and conformational coupling to IP3 receptor. New knowledge on the properties and coexistence of Orai1-encoded SOC and TRPC1-encoded IP3ROC, and the use of experimental approaches introduced in this manuscript should help avoid further confusion about these channels, and open new exciting possibilities for their independent studies

The Ca2+release-activated Ca2+current (ICRAC) mediates store-operated Ca2+entry in rat microglia

“…74,75 However, most recent studies conclude that Orai1 forms homomultimers that co-assemble with the stromal interaction molecule 1 (STIM-1) protein, 76 and that TRPC1 and Orai1 produce separate currents. 68 We have demonstrated the conditions necessary for isolating the CRAC current from the numerous other currents that exist in rat microglia, and for comparing it with Ca 2+ -sensitive dye measurements of SOCE. This degree of characterization will be essential for clearly separating its contributions from identified TRP channels and for future studies of regulation of CRAC expression and activity.…”

“…12) but they are less Ca 2+ selective than Orai1/CRAC, with P Ca /P Na ratios of 1.6 for TRPC3, 66 ~5 for TRPC6, 67 and <0.4 for TRPC1. 68 Thus, one crucial test is to compare Ca 2+ -containing and divalent-free solutions.…”

Orai1 and Ca2+-independent phospholipase A2 are required for store-operated Icat-SOC current, Ca2+ entry, and proliferation of primary vascular smooth muscle cells

“…2B). Figure 2A also shows that it can be fully inhibited by diethylstilbestrol (10 M), a known inhibitor of CRAC and cat-SOC channels and SOCE in excitable and nonexcitable cells (29,74,75).…”

“…This requirement is not limited to SMC, as iPLA 2 ␤ was also found to be essential for SOCE in a wide variety of excitable and nonexcitable cells, including Jurkat T lymphocytes (61), rat basophilic leukemia (RBL) cells (19,75), platelets (61), astrocytes (56), keratinocytes (54), skeletal muscle cells (12), fibroblasts (37), prostate cancer cells (66), endothelial cells (11), a neuroblastoma/ glioma cell line (20), and others. Importantly, along with Orai1 and STIM1, iPLA 2 ␤ was identified as a crucial component of SOCE in a comprehensive screen of Drosophila melanogaster genes (68).…”

β Cell Store-Operated Ion Channels

“…TRPC1 also interacts with the type 3 inositol 1,4,5-trisphosphate receptor (IP 3 R3) (Sundivakkam et al 2009), and TRPC3 interacts with IP 3 R1 (Woodard et al 2010). This interaction with IP 3 Rs has been suggested as a conformational coupling gating mechanism for TRPC channels (Zarayskiy et al 2007). Store depletion using either thapsigargin or carbachol also induces translocation of Orai1 into the plasma membrane through a SNAP-25-dependent mechanism that is important for the maintenance, but not the initiation, of store-operated Ca 2+ entry (Woodard et al 2008).…”