Abstract
Low-conductance, highly calcium-selective channels formed by the Orai proteins exist as store-operated CRAC channels and store-independent, arachidonic acid-activated ARC channels. Both are activated by stromal interaction molecule 1 (STIM1), but CRAC channels are activated by STIM1 located in the endoplasmic reticulum membrane, whereas ARC channels are activated by the minor plasma membrane-associated pool of STIM1. Critically, maximally activated CRAC channel and ARC channel currents are completely additive within the same cell, and their selective activation results in their ability to each induce distinct cellular responses. We have previously shown that specific ARC channel activation requires a PKA-mediated phosphorylation of a single threonine residue (Thr389) within the cytoplasmic region of STIM1. Here, examination of the molecular basis of this phosphorylation-dependent activation revealed that phosphorylation of the Thr389 residue induces a significant structural change in the STIM1-Orai-activating region (SOAR) that interacts with the Orai proteins, and it is this change that determines the selective activation of the store-independent ARC channels versus the store-operated CRAC channels. In conclusion, our findings reveal the structural changes underlying the selective activation of STIM1-induced CRAC or ARC channels that determine the specific stimulation of these two functionally distinct Ca2+ entry pathways.
Highlights
Low-conductance, highly calcium-selective channels formed by the Orai proteins exist as store-operated CRAC channels and store-independent, arachidonic acid–activated ARC channels
We have examined the basis for the selective activation by stromal interaction molecule 1 (STIM1) of either the store-operated CRAC channels or the store-independent ARC channels
We focused on the critical CC2-CC3 region of STIM1, which is known to represent the site for the key interactions between STIM1 and the C termini of the Orai proteins that are necessary for the activation of both CRAC [14, 19] and ARC channels [9]
Summary
We began by re-examining the key role of the basic region (BR) lysine residues (Lys382 and Lys384–Lys386) in CRAC channel activation. The same G392A mutation in STIM1 completely eliminated store-operated CRAC channel currents (0.04 Ϯ 0.01 pA/pF at Ϫ80 mV) (Fig. 8B) Together, these data indicate that it is not the phosphorylation status of Thr389 per se that is critical for ARC channel activity, but rather it is the extension of the helical region that is induced by the phosphorylation of residue Thr389. The results obtained showed that arachidonic acid– activated ARC channel currents were indistinguishable from normal wildtype currents (mean Ϯ S.E. ϭ 0.54 Ϯ 0.05 pA/pF at Ϫ80 mV), whereas CRAC channel currents were, once again, essentially negligible (0.02 Ϯ 0.01 pA/pF at Ϫ80 mV) (Fig. 8C) Together, these data indicate that the predicted CC2 extension of the coiled-coil region induced by the G392A mutation fully supports normal ARC channel currents, even in the absence of any phosphorylation of the Thr389 residue, while simultaneously eliminating CRAC channel currents. We conclude that it is the extension of the helical region at the apex of the CC2-CC3 junction of STIM1 that enables the selective activation of the ARC channels versus the CRAC channels, and physiologically this extension is induced by phosphorylation of the Thr389 residue
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