Detecting single ORAI1 proteins within the plasma membrane reveals higher-order channel complexes.

ORAI1 proteins form highly selective Ca²⁺ channels in the plasma membrane. Crystallographic data point towards a hexameric stoichiometry of ORAI1 channels, whereas optical methods postulated ORAI1 channels to reside as dimers at rest, and other data suggests that they have a tetrameric configuration. Here, liquid-phase scanning transmission electron microscopy (STEM) and quantum dot (QD) labeling was utilized to study the conformation of ORAI1 proteins at rest. To address the question of whether ORAI1 was present as a dimer, experiments were designed using single ORAI1 monomers and covalently linked ORAI1 dimers with either one or two label-binding positions. The microscopic data was statistically analyzed via the pair correlation function. Label pairs were found in all cases, even for concatenated dimers with one label-binding position, which is only possible if a significant fraction of ORAI1 was assembled in larger order oligomers than dimers, binding at least two QDs. This interpretation of the data was consistent with Blue Native PAGE analysis showing that ORAI1 is mainly present as a complex of an apparent molecular mass larger than that calculated for a dimer.

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