title: Genetically targeted single-channel optical recording reveals multiple Orai1 gating states and oscillations in calcium influx.
abstract: 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.
authors.affiliation: Department of Physiology and Biophysics, University of California, Irvine, CA 92697.
authors.forename: Joseph L
authors.initials: JL
authors.lastname: Dynes
authors.affiliation: Department of Physiology and Biophysics, University of California, Irvine, CA 92697.
authors.forename: Anna
authors.initials: A
authors.lastname: Amcheslavsky
authors.affiliation: Department of Physiology and Biophysics, University of California, Irvine, CA 92697 Institute for Immunology, University of California, Irvine, CA 92697 mcahalan@uci.edu.
authors.forename: Michael D
authors.initials: MD
authors.lastname: Cahalan
mesh_terms: D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000737:chemistry / Q000378:metabolism*; D020013:Calcium Signaling; D002462:Cell Membrane / Q000378:metabolism; D005453:Fluorescence; D049452:Green Fluorescent Proteins / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D015640:Ion Channel Gating; D000071740:ORAI1 Protein; D062308:Optogenetics / Q000379:methods*; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013997:Time Factors; D014162:Transfection
chemical_list: D015220:Calcium Channels; D000071740:ORAI1 Protein; C510746:ORAI1 protein, human; D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D002118:Calcium
keywords: CRAC channel; Orai; Stim; local Ca2+; store-operated Ca2+ entry