Abstract
The essential role of ORAI1 channels in receptor-evoked Ca2+ signaling is well understood, yet little is known about the physiological activation of the ORAI channel trio natively expressed in all cells. The roles of ORAI2 and ORAI3 have remained obscure. We show that ORAI2 and ORAI3 channels play a critical role in mediating the regenerative Ca2+ oscillations induced by physiological receptor activation, yet ORAI1 is dispensable in generation of oscillations. We reveal that ORAI2 and ORAI3 channels multimerize with ORAI1 to expand the range of sensitivity of receptor-activated Ca2+ signals, reflecting their enhanced basal STIM1-binding and heightened Ca2+-dependent inactivation. This broadened bandwidth of Ca2+ influx is translated by cells into differential activation of NFAT1 and NFAT4 isoforms. Our results uncover a long-sought role for ORAI2 and ORAI3, revealing an intricate control mechanism whereby heteromerization of ORAI channels mediates graded Ca2+ signals that extend the agonist-sensitivity to fine-tune transcriptional control.
Highlights
The essential role of ORAI1 channels in receptor-evoked Ca2+ signaling is well understood, yet little is known about the physiological activation of the ORAI channel trio natively expressed in all cells
Nuclear translocation of each nuclear factor of activated T-cells (NFAT)-green fluorescent protein (GFP) fusion protein was recorded over time by monitoring the ratio of nuclear to wholecell GFP fluorescence (Fig. 7). We show that both NFAT1 (Fig. 7a–g) and NFAT4 (Fig. 7h–n) translocation to the nucleus was significantly faster and higher in ORAI2-SKO, ORAI3-SKO, and ORAI2,3-SKO cells when compared with WT HEK293 cells, essentially causing significant NFAT1 activation at lower agonist concentrations that normally cause selective activation of the faster isoform NFAT4
Data from our rescue experiments of single tk-driven ORAIs in ORAI-TKO cells and data from single and double KO cells suggest that each ORAI isoform can support a specific range of Ca2+ signaling events: ORAI3 and ORAI2 are critical for low- and mid-range oscillatory responses, whereas ORAI1 mediates high-range plateaus
Summary
The essential role of ORAI1 channels in receptor-evoked Ca2+ signaling is well understood, yet little is known about the physiological activation of the ORAI channel trio natively expressed in all cells. To reliably measure SOCE function with fluorescent dyes, most studies have relied on protocols that cause maximal SOCE activation This is achieved by the use of either pharmacological tools such as thapsigargin, which is a sarcoplasmic/ER Ca2+ ATPase (SERCA) blocker or high agonist concentrations (AgHigh; 30–100 μM carbachol (Cch) in HEK293 cells), both of which maximally deplete ER Ca2+ stores[13]. To determine the signaling functions of native ORAI3, ORAI2, and ORAI1, we generated multiple single, double, and triple ORAI isoform knockouts in HEK293 cells using CRISPR/Cas[9] technology Using this system, we show that ORAI1 is required for SOCE activated by maximal store depletion, surprisingly it is dispensable for Ca2+ oscillations.
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