Dynamic coupling of the putative coiled-coil domain of ORAI1 with STIM1 mediates ORAI1 channel activation.
STIM1 and ORAI1 (also termed CRACM1) are essential components of the classical calcium release-activated calcium current; however, the mechanism of the transmission of information of STIM1 to the calcium release-activated calcium/ORAI1 channel is as yet unknown. Here we demonstrate by Förster resonance energy transfer microscopy a dynamic coupling of STIM1 and ORAI1 that culminates in the activation of Ca(2+) entry. Förster resonance energy transfer imaging of living cells provided insight into the time dependence of crucial events of this signaling pathway comprising Ca(2+) store depletion, STIM1 multimerization, and STIM1-ORAI1 interaction. Accelerated store depletion allowed resolving a significant time lag between STIM1-STIM1 and STIM1-ORAI1 interactions. Store refilling reversed both STIM1 multimerization and STIM1-ORAI1 interaction. The cytosolic STIM1 C terminus itself was able, in vitro as well as in vivo, to associate with ORAI1 and to stimulate channel function, yet without ORAI1-STIM1 cluster formation. The dynamic interaction occurred via the C terminus of ORAI1 that includes a putative coiled-coil domain structure. An ORAI1 C terminus deletion mutant as well as a mutant (L273S) with impeded coiled-coil domain formation lacked both interaction as well as functional communication with STIM1 and failed to generate Ca(2+) inward currents. An N-terminal deletion mutant of ORAI1 as well as the ORAI1 R91W mutant linked to severe combined immune deficiency syndrome was similarly impaired in terms of current activation despite being able to interact with STIM1. Hence, the C-terminal coiled-coil motif of ORAI1 represents a key domain for dynamic coupling to STIM1.
Muik
Martin
M
Institute of Biophysics, University of Linz, A-4040 Linz, Austria.
Frischauf
Irene
I
Derler
Isabella
I
Fahrner
Marc
M
Bergsmann
Judith
J
Eder
Petra
P
Schindl
Rainer
R
Hesch
Clemens
C
Polzinger
Bernhard
B
Fritsch
Reinhard
R
Kahr
Heike
H
Madl
Josef
J
Gruber
Hermann
H
Groschner
Klaus
K
Romanin
Christoph
C
D020816:Amino Acid Motifs / Q000502:physiology; D019943:Amino Acid Substitution; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D003593:Cytoplasm / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D020125:Mutation, Missense; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism*; D000071740:ORAI1 Protein; D011485:Protein Binding / Q000502:physiology; D017434:Protein Structure, Tertiary / Q000502:physiology; D000071737:Stromal Interaction Molecule 1
D015220:Calcium Channels; D008565:Membrane Proteins; D009363:Neoplasm Proteins; D000071740:ORAI1 Protein; C510746:ORAI1 protein, human; C103530:STIM1 protein, human; D000071737:Stromal Interaction Molecule 1; D002118:Calcium