Abstract
Store-operated calcium entry is essential for many signaling processes in nonexcitable cells. The best studied store-operated calcium current is the calcium release-activated calcium (CRAC) current in T-cells and mast cells, with Orai1 representing the essential pore forming subunit. Although it is known that functional CRAC channels in store-depleted cells are composed of four Orai1 subunits, the stoichiometric composition in quiescent cells is still discussed controversially: both a tetrameric and a dimeric stoichiometry of resting state Orai1 have been reported. We obtained here robust and similar FRET values on labeled tandem repeat constructs of Orai1 before and after store depletion, suggesting an unchanged tetrameric stoichiometry. Moreover, we directly visualized the stoichiometry of mobile Orai1 channels in live cells using a new single molecule recording modality that combines single molecule tracking and brightness analysis. By alternating imaging and photobleaching pulses, we recorded trajectories of single, fluorescently labeled Orai1 channels, with each trajectory consisting of bright and dim segments, corresponding to higher and lower numbers of colocalized active GFP label. The according brightness values were used for global fitting and statistical analysis, yielding a tetrameric subunit composition of mobile Orai1 channels in resting cells.
Highlights
Store-operated calcium entry is a mechanism where the decrease of the calcium concentration in the endoplasmic reticulum (ER)3 triggers the influx of extracellular calcium into the cytoplasm [1]
Our rationale was that mobile subfractions provide the cleanest pool of data, devoid of contributions, e.g. from potentially preassociated STIM1-Orai1 complexes or vesicles attached to the plasma membrane; resting state Orai1 is predominantly mobile [8], and mobility appears critical for the lateral redistribution upon store depletion
Store Depletion Does Not Affect Association State of Covalently Linked Orai1 Homodimers—In an attempt to approach the dimer-to-tetramer transition of Orai1, which might occur in response to store depletion, we initially generated a covalently linked Orai1 homodimer and followed a potential alteration of FRET between CFP/YFP-labeled dimers upon stimulation of their coupling to STIM1 by thapsigargin
Summary
Store-operated calcium entry is a mechanism where the decrease of the calcium concentration in the endoplasmic reticulum (ER)3 triggers the influx of extracellular calcium into the cytoplasm [1]. The store-dependent function of Orai1-mGFP was confirmed in HEK-293 cells coexpressing STIM1 (supplemental Fig. 2).
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
