STIM1-Induced Clustering of Orai1 Channels

Abstract

Store operated Ca2+ entry is an evolutionarily conserved mechanism in all eukaryotic cells. Decreased ER Ca2+ promotes a transmembrane conformational shift and unfolding of the cytoplasmic STIM1 domain to expose the STIM-Orai Activating Region (SOAR) of STIM1. SOAR1 couples to and activates PM Orai1 channels, mediating Ca2+ entry signals. The F394H mutation in SOAR prevents it coupling with and activating Orai1. SOAR concatemer-dimers containing a single F394H mutated SOAR unit, fully couple to and activate Orai1, suggesting only one functional monomer of the STIM1 dimer is required for coupling to and activating Orai1 channels, consistent with a stoichiometry of one STIM1 dimer binding to each Orai1 monomer in the hexameric Orai1 channel. Using high-resolution fluorescence imaging approaches, we reveal the existence of dense clusters of YFP-tagged concatenated SOAR-dimers dependent on Orai1-His stably expressed in HEK cells. These clusters are absent in cells expressing heterodimers of concatenated-SOAR dimers containing one single F394H-SOAR residue. Concatenated-SOAR dimers with both monomers mutated with F394H, give no visible clustering. Using HEK cells stably expressing Orai1 C-terminally labeled with CFP, wildtype SOAR dimers did not undergo clustering. Thus, the CFP-label appears to sterically block cluster formation mediated by SOAR dimers. 2-APB is able overcome the inhibitory effect of the F394H mutation in SOAR. Interestingly, 2-APB induced recovery of Orai1-dependent clustering of SOAR concatemer dimers heteromeric for the F394H mutation. The results suggest that the second Orai1 binding site within a SOAR dimer can interact with adjacent Orai1 channels to form clusters of Orai1 channels in the PM. This theory is corroborated by recent electron microscopy data showing Orai1 channels spaced approximately the distance of a SOAR1 dimer. Physiologically, Orai1 cluster formation in ER-PM junctions may be important enhancing activation and deactivation kinetics of Orai1 channels and/or amplifying local Ca2+ signals.