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

Abstract

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.