Genetically targeted single-channel optical recording reveals multiple Orai1 gating states and oscillations in calcium influx

Proc Natl Acad Sci U S A. 2016 Jan 12;113(2):440-5. doi: 10.1073/pnas.1523410113. Epub 2015 Dec 28.


Orai1 comprises the pore-forming subunit of the Ca(2+) release-activated Ca(2+) (CRAC) channel. When bound and activated by stromal interacting molecule 1 (STIM1), an endoplasmic reticulum (ER)-resident calcium sensor, Orai1 channels possess high selectivity for calcium but extremely small conductance that has precluded direct recording of single-channel currents. We have developed an approach to visualize Orai1 activity by fusing Orai1 to fluorescent, genetically encoded calcium indicators (GECIs). The GECI-Orai1 probes reveal local Ca(2+) influx at STIM1-Orai1 puncta. By whole cell recording, these fusions are fully functional as CRAC channels. When GECI-Orai1 and the CRAC-activating domain (CAD) of STIM1 were coexpressed at low levels and imaged using a total internal reflectance fluorescence microscope, cells exhibited sporadic fluorescence transients the size of diffraction-limited spots and the brightness of a few activated GECI proteins. Transients typically rose rapidly and fell into two classes according to duration: briefer "flickers" lasting only a few hundred milliseconds, and longer "pulses" lasting one to several seconds. The size, intensity, trace shape, frequency, distribution, physiological characteristics, and association with CAD binding together demonstrate that GECI-Orai1 fluorescence transients correspond to single-channel Orai1 responses. Single Orai1 channels gated by CAD, and small Orai1 puncta gated by STIM1, exhibit repetitive fluctuations in single-channel output. CAD binding supports a role in open state maintenance and reveals a second phase of CAD/STIM1 binding after channel opening. These first recordings of single-channel Orai1 currents reveal unexpected dynamics, and when paired with CAD association, support multiple single-channel states.

Keywords: CRAC channel; Orai; Stim; local Ca2+; store-operated Ca2+ entry.

Publication types

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

MeSH terms

  • Calcium / metabolism*
  • Calcium Channels / chemistry
  • Calcium Channels / metabolism*
  • Calcium Signaling*
  • Cell Membrane / metabolism
  • Fluorescence
  • Green Fluorescent Proteins / metabolism
  • HEK293 Cells
  • Humans
  • Ion Channel Gating*
  • ORAI1 Protein
  • Optogenetics / methods*
  • Protein Structure, Tertiary
  • Recombinant Fusion Proteins / metabolism
  • Time Factors
  • Transfection


  • Calcium Channels
  • ORAI1 Protein
  • ORAI1 protein, human
  • Recombinant Fusion Proteins
  • enhanced green fluorescent protein
  • Green Fluorescent Proteins
  • Calcium