Hexameric Assembly of the Orai1 Channel Determined Using Targeted Cryo-Electron Tomography In Situ

The calcium release-activated calcium channel, composed of the pore-forming Orai1 in the plasma membrane and calcium sensor STIM1 in the endoplasmic reticulum membrane, is essential for calcium homeostasis in various organ systems. Despite extensive research on the structure and function of the Orai1 channel over the past decade, its assembly as tetramers or hexamers within the plasma membrane in situ has not been confirmed. To bridge this gap, we developed the technique named targeted cryo-electron tomography in situ, achieved by the integration of cryo-correlative light and electron microscopy and immunogold labeling. Stable cell clones with Orai1 puncta formation in ultrathin lamellipodia were selected for determining the Orai1 stoichiometry in situ. Using targeted cryo-electron tomography, guided precisely to Orai1 puncta by immunogold labeling, we delineated the protein density of the Orai1 and STIM1 complex at plasma membrane-endoplasmic reticulum junctions and verified the hexameric assembly of the native Orai1 channel. This innovative method offers a new approach for in situ structural determination of integral membrane proteins.