The inactivation domain of STIM1 is functionally coupled with the Orai1 pore to enable Ca2+-dependent inactivation
Journal of Cell Biology
Ca 2+ release-activated Ca 2+ (CRAC) channels are prototypic store operated channels that are encoded by the Orai family of plasma membrane proteins and activated by depletion of Ca 2+ from the ER (Prakriya and Lewis, 2015) . The ER protein STIM1 is best recognized for its function in sensing ER Ca 2+ depletion and, in response, activating CRAC channels in the plasma membrane. However, an early clue that STIM1 is more than an activating ligand for Orai1 was the observation that channels
... at channels activated by an isolated fragment of STIM1 (the CRAC activation domain or CAD; STIM1 aa 342-448), failed to show characteristic fast Ca 2+ -dependent inactivation (CDI) in response to Ca 2+ entry . A series of C-terminal truncations identified a region of STIM1 (aa 470-491) that is required for CDI, termed the inactivation domain of STIM, or ID STIM . Additional evidence for a role of STIM1 in CDI came from observations that in heterologous systems the STIM1/Orai1 expression ratio influences the extent of CDI, with a ratio of 2-4:1 being Correspondence to Richard S. Lewis: r s l e w i s @ s t a n f o r d . e d u ; or Franklin M. Mullins: f m u l l i n s @ s t a n f o r d . e d u Abbreviations used in this paper: CDI, Ca 2+ -dependent inactivation; CRAC, Ca 2+ release-activated Ca 2+ ; DVF, divalent-free; ID STIM , inactivation domain of STIM. required for CDI to approach the level seen for native CRAC channels (Scrimgeour et al., 2009; Hoover and Lewis, 2011) . A highly negatively charged region within ID STIM ( 475 DDVDDMDEE 483 ) attracted attention from several groups as a potential Ca 2+ binding site for CDI, based on analogy to the highly acidic Ca 2+ bowl binding site of the BK channel (Cox, 2011) . Consistent with such a role, alanine or glycine substitutions for various combinations of acidic residues within this region reduced CDI, and full neutralization of all acidic residues eliminated CDI (Derler et al., 2009b; Lee et al., 2009; Mullins et al., 2009) . However, an attempt to test whether the acidic region is the Ca 2+ sensor for CDI yielded equivocal results. 45 Ca 2+ overlay experiments showed that Ca 2+ bound weakly to a peptide containing this region, and mutations that reduced or eliminated CDI reduced 45 Ca 2+ binding. However, other mutations (STIM1 482/ 483 EE>AA) paradoxically increased the Ca 2+ sensitivity of CDI while reducing 45 Ca 2+ binding, raising doubts The inactivation domain of STIM1 (ID STIM : amino acids 470-491) has been described as necessary for Ca 2+ -dependent inactivation (CDI) of Ca 2+ release-activated Ca 2+ (CRAC) channels, but its mechanism of action is unknown. Here we identify acidic residues within ID STIM that control the extent of CDI and examine functional interactions of ID STIM with Orai1 pore residues W76 and Y80. Alanine scanning revealed three ID STIM residues (D476/D478/ D479) that are critical for generating full CDI. Disabling ID STIM by a triple alanine substitution for these three residues ("STIM1 3A") or by truncation of the entire domain (STIM1 1-469 ) reduced CDI to the same residual level observed for the Orai1 pore mutant W76A (approximately one third of the extent seen with full-length STIM1). Results of noise analysis showed that STIM1 1-469 and Orai1 W76A mutants do not reduce channel open probability or unitary Ca 2+ conductance, factors that determine local Ca 2+ accumulation, suggesting that they diminish CDI instead by inhibiting the CDI gating mechanism. We tested for functional coupling between ID STIM and the Orai1 pore by double-mutant cycle analysis. The effects on CDI of mutations disabling ID STIM or W76 were not additive, demonstrating that ID STIM and W76 are strongly coupled and act in concert to generate full-strength CDI. Interestingly, disabling ID STIM and W76 separately gave opposite results in Orai1 Y80A channels: channels with W76 but lacking ID STIM generated approximately two thirds of the WT extent of CDI but those with ID STIM but lacking W76 completely failed to inactivate. Together, our results suggest that Y80 alone is sufficient to generate residual CDI, but acts as a barrier to full CDI. Although ID STIM is not required as a Ca 2+ sensor for CDI, it acts in concert with W76 to progress beyond the residual inactivated state and enable CRAC channels to reach the full extent of inactivation.