Cross-linking of Orai1 channels by STIM proteins

Abstract

The transmembrane docking of endoplasmic reticulum (ER) Ca-sensing STIM proteins with plasma membrane (PM) Orai Ca channels is a critical but poorly understood step in Ca signal generation. STIM1 protein dimers unfold to expose a discrete STIM-Orai activating region (SOAR1) that tethers and activates Orai1 channels within discrete ER-PM junctions. We reveal that each monomer within the SOAR dimer interacts independently with single Orai1 subunits to mediate cross-linking between Orai1 channels. Superresolution imaging and mobility measured by fluorescence recovery after photobleaching reveal that SOAR dimer cross-linking leads to substantial Orai1 channel clustering, resulting in increased efficacy and cooperativity of Orai1 channel function. A concatenated SOAR1 heterodimer containing one monomer point mutated at its critical Orai1 binding residue (F394H), although fully activating Orai channels, is completely defective in cross-linking Orai1 channels. Importantly, the naturally occurring STIM2 variant, STIM2.1, has an eight-amino acid insert in its SOAR unit that renders it functionally identical to the F394H mutant in SOAR1. Contrary to earlier predictions, the SOAR1-SOAR2.1 heterodimer fully activates Orai1 channels but prevents cross-linking and clustering of channels. Interestingly, combined expression of full-length STIM1 with STIM2.1 in a 5:1 ratio causes suppression of sustained agonist-induced Ca oscillations and protects cells from Ca overload, resulting from high agonist-induced Ca release. Thus, STIM2.1 exerts a powerful regulatory effect on signal generation likely through preventing Orai1 channel cross-linking. Overall, STIM-mediated cross-linking of Orai1 channels is a hitherto unrecognized functional paradigm that likely provides an organizational microenvironment within ER-PM junctions with important functional impact on Ca signal generation.

Pore properties of Orai1 calcium channel dimers and their activation by the STIM1 ER calcium sensor

“…Hence, our understanding of the molecular coupling and function of the two proteins has major translational significance. Although we have recently determined much on the function and organization of STIM and Orai proteins (11)(12)(13)(14), there is still considerable uncertainty about how the proteins associate within junctions and how these interactions mediate opening of Orai channels and the generation of Ca 2ϩ signals (1-4, 15, 16).…”

Reaction-diffusion model for STIM-ORAI interaction: The role of ROS and mutations

“…Other experiments indicate that CRAC channels can occur as a a mixture of hexameric, tetrameric and dimeric channel complexes or as hexamers with different conducting states [17,23]. A very recent report provides evidence for the existence of different conductance states of hexameric channels due to the ability of STIM1 dimers to crosslink neighbouring ORAI1 hexamers to provide a more efficient activation for a given number of STIM1 molecules [36]. Until now, there has been no model taking these new findings into account.…”

Understanding the regulation of STIM1-ORAI1 interaction using a reaction-diffusion model

“…Other experiments indicate that CRAC channels can occur as a a mixture of hexameric, tetrameric and dimeric channel complexes or as hexamers with different conducting states [16,34]. A very recent report provides evidence for the existence of different conductance states of hexameric channels due to the ability of STIM1 dimers to crosslink neighbouring ORAI1 hexamers to provide a more efficient activation for a given number of STIM1 molecules [35]. Until now, there has been no model taking these new findings into account.…”