Abstract
The endoplasmic reticulum (ER) Ca2+ sensor, STIM1, becomes activated when ER-stored Ca2+ is depleted and translocates into ER–plasma membrane junctions where it tethers and activates Orai1 Ca2+ entry channels. The dimeric STIM1 protein contains a small STIM-Orai-activating region (SOAR)—the minimal sequence sufficient to activate Orai1 channels. Since SOAR itself is a dimer, we constructed SOAR concatemer–dimers and introduced mutations at F394, which is critical for Orai1 coupling and activation. The F394H mutation in both SOAR monomers completely blocks dimer function, but F394H introduced in only one of the dimeric SOAR monomers has no effect on Orai1 binding or activation. This reveals an unexpected unimolecular coupling between STIM1 and Orai1 and argues against recent evidence suggesting dimeric interaction between STIM1 and two adjacent Orai1 channel subunits. The model predicts that STIM1 dimers may be involved in crosslinking between Orai1 channels with implications for the kinetics and localization of Orai1 channel opening.
Highlights
The endoplasmic reticulum (ER) Ca2 þ sensor, STIM1, becomes activated when ER-stored Ca2 þ is depleted and translocates into ER–plasma membrane junctions where it tethers and activates Orai[1] Ca2 þ entry channels
The mutation in whole STIM1 was shown to curtail Orai[1] coupling, and to unfold the entire STIM1 C terminus allowing its K-rich region to be exposed and for the mutated STIM1 molecule to constitutively move into ER–plasma membrane (PM) junctions[19]
The R429C mutation clearly altered the secondary structure of STIM-Orai-activating region (SOAR) as determined by circular dichroism spectral analysis and thermal stability
Summary
The endoplasmic reticulum (ER) Ca2 þ sensor, STIM1, becomes activated when ER-stored Ca2 þ is depleted and translocates into ER–plasma membrane junctions where it tethers and activates Orai[1] Ca2 þ entry channels. The F394H mutation in both SOAR monomers completely blocks dimer function, but F394H introduced in only one of the dimeric SOAR monomers has no effect on Orai[1] binding or activation This reveals an unexpected unimolecular coupling between STIM1 and Orai[1] and argues against recent evidence suggesting dimeric interaction between STIM1 and two adjacent Orai[1] channel subunits. We took a simple approach to understand the STIM1–Orai[1] interaction, based on recent identification of a powerful point mutation (F394H) in the Orai1-binding site of STIM1, that completely prevents STIM1 binding to and activation of Orai channels[20] This mutation does not cause any change in the resting state of STIM1 or its ability to undergo activation by store depletion, or to move into and be retained within ER–PM junctions[20]. STIM1 C-terminus undergo dimeric interactions with Orai[1] dimers[17,18], our findings indicate a rather different view of the STIM1–Orai[1] interface from that recently put forward
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