Resting state Orai1 diffuses as homotetramer in the plasma membrane of live mammalian cells.
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.
Madl
Josef
J
Biophysics Institute, Johannes Kepler University Linz, Altenbergerstrasse 69, A-4040 Linz, Austria.
Weghuber
Julian
J
Fritsch
Reinhard
R
Derler
Isabella
I
Fahrner
Marc
M
Frischauf
Irene
I
Lackner
Barbara
B
Romanin
Christoph
C
Schütz
Gerhard J
GJ
D000818:Animals; D016466:CHO Cells; D015220:Calcium Channels / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000235:genetics / Q000378:metabolism*; D006224:Cricetinae; D003412:Cricetulus; D057809:HEK293 Cells; D006801:Humans; D000071740:ORAI1 Protein; D055503:Protein Multimerization / Q000502:physiology*; D020836:Protein Structure, Quaternary
D015220:Calcium Channels; D000071740:ORAI1 Protein; C510746:ORAI1 protein, human