Thiol dependent intramolecular locking of Orai1 channels.
Store-operated Ca(2+) entry mediated by STIM1-gated Orai1 channels is essential to activate immune cells and its inhibition or gain-of-function can lead to immune dysfunction and other pathologies. Reactive oxygen species interacting with cysteine residues can alter protein function. Pretreatment of the Ca(2+) selective Orai1 with the oxidant H2O2 reduces ICRAC with C195, distant to the pore, being its major redox sensor. However, the mechanism of inhibition remained elusive. Here we combine experimental and theoretical approaches and show that oxidation of Orai1 leads to reduced subunit interaction, slows diffusion and that either oxidized C195 or its oxidomimetic mutation C195D located at the exit of transmembrane helix 3 virtually eliminates channel activation by intramolecular interaction with S239 of transmembrane helix 4, thereby locking the channel in a closed conformation. Our results demonstrate a novel mechanistic model for ROS-mediated inhibition of Orai1 and identify a candidate residue for pharmaceutical intervention.
Molecular Biophysics, Saarland University, 66421 Homburg, Germany.
Dalia
D
Alansary
Molecular Biophysics, Saarland University, 66421 Homburg, Germany.|Department of Biophysics, Saarland University, 66421 Homburg, Germany.|Department of Theoretical Physics, Saarland University, 66041 Saarbrücken, Germany.
Barbara
B
Schmidt
Molecular Biophysics, Saarland University, 66421 Homburg, Germany.
Kathrin
K
Dörr
Department of Biophysics, Saarland University, 66421 Homburg, Germany.
Ivan
I
Bogeski
Department of Theoretical Physics, Saarland University, 66041 Saarbrücken, Germany.
Heiko
H
Rieger
Gruenenthal Innovation, Drug Discovery Technologies, Gruenenthal GmbH, 52078 Aachen, Germany.
Achim
A
Kless
Molecular Biophysics, Saarland University, 66421 Homburg, Germany.
Barbara A
BA
Niemeyer
D057809:HEK293 Cells; D006801:Humans; D008698:Mesylates / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D009363:Neoplasm Proteins / Q000378:metabolism; D000071740:ORAI1 Protein / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010084:Oxidation-Reduction; D011485:Protein Binding; D021122:Protein Subunits / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism; D012694:Serine / Q000378:metabolism; D000071737:Stromal Interaction Molecule 1 / Q000378:metabolism; D013438:Sulfhydryl Compounds / Q000378:metabolism*
D008698:Mesylates; D009363:Neoplasm Proteins; D000071740:ORAI1 Protein; D021122:Protein Subunits; D017382:Reactive Oxygen Species; C103530:STIM1 protein, human; D000071737:Stromal Interaction Molecule 1; D013438:Sulfhydryl Compounds; C109153:(2-sulfonatoethyl)methanethiosulfonate; D012694:Serine