Supra-molecular assemblies of ORAI1 at rest precede local accumulation into punctae after activation

The Ca2+ selective channel ORAI1 and endoplasmic reticulum (ER)-resident STIM proteins form the core of the channel complex mediating store operated Ca2+ entry (SOCE). Using liquid phase electron microscopy (LPEM) the distribution of ORAI1 proteins was examined at rest and after SOCE-activation at nanoscale resolution. The analysis of over seven hundred thousand of ORAI1 positions showed that already at rest, a majority of the ORAI1 channels formed STIM-independent distinct supra-molecular clusters. Upon SOCE activation and in the presence of STIM proteins, ORAI1 assembled in micron-sized two-dimensional (2D) structures, such as the known punctae at the ER plasma membrane contact zones, but also in divergent structures such as strands, and ring-like shapes. Our results thus question the hypothesis that stochastically migrating single ORAI1 channels are trapped at regions containing activated STIM, and we propose instead that supra-molecular ORAI1 clusters fulfill an amplifying function for creating dense ORAI1 accumulations upon SOCE-activation. STATEMENT OF SIGNIFICANCEORAI1 proteins form channels mediating store operated Ca2+ entry, an important trigger for many cellular functions, especially in the immune system. ORAI1 channels at rest are assumed to be randomly distributed in the plasma membrane, while they accumulate into so-called "punctae" upon activation, where binding by STIM proteins activate the Ca2+ channels. Using liquid phase electron microscopy, we discovered that ORAI1 forms small, elongated clusters indicating the existence of supramolecular assemblies. The role of such supramolecular organization of ORAI1 is possibly an amplifying function for the effective creation of ORAI1 accumulations in punctae, since the binding of only one ORAI1 protein would trap a multiple of channels.