Subunit Stoichiometry of Human Orai1 and Orai3 in Closed and Open States

Abstract

We applied single-molecule photobleaching to investigate the stoichiometry of human Orai1 and Orai3 channels tagged with eGFP and expressed in HEK cells. At low expression, GFP-tagged subunits were detected in TIRF microscopy as single fluorescent spots that decayed in a step-wise manner as individual GFP molecules bleached. By counting the number of photobleaching steps, the number of subunits per channel complex could be deduced. In fixed cells, Orai1 was detected primarily as dimers when expressed alone and as tetramers when co-expressed with the cytosolic STIM1 fragment, C-STIM1, to activate Ca2+ influx, as previously found for Drosophila Orai with and without activation by C-Stim (Penna et al., 2008, Nature 456:116-120). When co-expressed with full-length STIM1, Orai1 was also found to be predominantly dimeric in living cells under resting conditions with Ca2+ stores filled. We also investigated Orai3 alone and when activated by either C-STIM1 to form a Ca2+-selective channel in its store-operated mode, or by addition of 2-APB to form a Ca2+-permeable but relatively nonselective cation channel in its STIM1-independent mode. Similar to our observations with Orai1, eGFP-Orai3 alone was detected mostly as dimers under basal conditions, but predominantly as tetramers when co-expressed with C-STIM1. On the other hand, cells expressing only eGFP-Orai3 that were exposed to 2APB before fixation showed a distribution of bleaching steps closely similar to that observed without 2APB, with a predominance of dimers. These results indicate a predominantly dimeric state for Orai3 at rest or when activated by 2-APB, and a tetrameric channel when activated by C-STIM1. We conclude that the human Orai1 and Orai3 channels undergo a dimer-to-tetramer transition to form a Ca2+-selective pore during store-operated activation, and that Orai3 forms a dimeric non-selective cation pore upon activation by 2-APB.