Nanoscale patterning of STIM1 and Orai1 during store-operated Ca2+ entry

Proc Natl Acad Sci U S A. 2015 Oct 6;112(40):E5533-42. doi: 10.1073/pnas.1515606112. Epub 2015 Sep 8.


Stromal interacting molecule (STIM) and Orai proteins constitute the core machinery of store-operated calcium entry. We used transmission and freeze-fracture electron microscopy to visualize STIM1 and Orai1 at endoplasmic reticulum (ER)-plasma membrane (PM) junctions in HEK 293 cells. Compared with control cells, thin sections of STIM1-transfected cells possessed far more ER elements, which took the form of complex stackable cisternae and labyrinthine structures adjoining the PM at junctional couplings (JCs). JC formation required STIM1 expression but not store depletion, induced here by thapsigargin (TG). Extended molecules, indicative of STIM1, decorated the cytoplasmic surface of ER, bridged a 12-nm ER-PM gap, and showed clear rearrangement into small clusters following TG treatment. Freeze-fracture replicas of the PM of Orai1-transfected cells showed extensive domains packed with characteristic "particles"; TG treatment led to aggregation of these particles into sharply delimited "puncta" positioned upon raised membrane subdomains. The size and spacing of Orai1 channels were consistent with the Orai crystal structure, and stoichiometry was unchanged by store depletion, coexpression with STIM1, or an Orai1 mutation (L273D) affecting STIM1 association. Although the arrangement of Orai1 channels in puncta was substantially unstructured, a portion of channels were spaced at ∼15 nm. Monte Carlo analysis supported a nonrandom distribution for a portion of channels spaced at ∼15 nm. These images offer dramatic, direct views of STIM1 aggregation and Orai1 clustering in store-depleted cells and provide evidence for the interaction of a single Orai1 channel with small clusters of STIM1 molecules.

Keywords: Orai1; SOCE; STIM1; electron microscopy; nanoscale patterning.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Calcium / metabolism*
  • Calcium Channels / genetics
  • Calcium Channels / metabolism*
  • Calcium-Transporting ATPases / antagonists & inhibitors
  • Calcium-Transporting ATPases / metabolism
  • Cell Membrane / metabolism
  • Cell Membrane / ultrastructure
  • Endoplasmic Reticulum / metabolism
  • Endoplasmic Reticulum / ultrastructure
  • Enzyme Inhibitors / pharmacology
  • Freeze Fracturing
  • HEK293 Cells
  • Humans
  • Luminescent Proteins / genetics
  • Luminescent Proteins / metabolism
  • Membrane Proteins / genetics
  • Membrane Proteins / metabolism*
  • Microscopy, Electron
  • Microscopy, Fluorescence / methods
  • Monte Carlo Method
  • Mutation
  • Neoplasm Proteins / genetics
  • Neoplasm Proteins / metabolism*
  • ORAI1 Protein
  • Protein Binding
  • Protein Transport / drug effects
  • Stromal Interaction Molecule 1
  • Thapsigargin / pharmacology
  • Videotape Recording


  • Calcium Channels
  • Enzyme Inhibitors
  • Luminescent Proteins
  • Membrane Proteins
  • Neoplasm Proteins
  • ORAI1 Protein
  • ORAI1 protein, human
  • STIM1 protein, human
  • Stromal Interaction Molecule 1
  • Thapsigargin
  • Calcium-Transporting ATPases
  • Calcium