Thiol dependent intramolecular locking of Orai1 channels

Abstract

Store-operated Ca2+ 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 Ca2+ 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.

A Thioacetal Photocage Designed for Dual Release: Application in the Quantitation of Therapeutic Release by Synchronous Reporter Decaging

“…These applications range from the control of redox biology, [43] enzymes, [15, 44] and ion channel permeability. [45] As discussed above, the values of Φ uncaging which were determined for TNB-caged small thiols including 1 , 2 (Table S1) are comparable to ONB caged molecules [6b] and are supportive of the compatibility of such application. Future efforts will focus on extending and validating the scope of this TNB strategy in photocaged small molecules for chemical biology, and in nanoscale systems designed for receptor targeted anticancer therapeutic agents.…”

Crosstalk between calcium and reactive oxygen species signaling in cancer

“…The lesser-studied ORAI2 isoform had a similar inhibition profile to ORAI1 [6]. In subsequent work the authors proposed that pretreatment of cells with H 2 O 2 leads to intramolecular interaction of C195 with S239, locking the channel in a closed conformation [226]. It should be noted that the high concentrations of H 2 O 2 (1mM) used in this work may contribute to the irreversible state of this redox modification [226].…”

“…In subsequent work the authors proposed that pretreatment of cells with H 2 O 2 leads to intramolecular interaction of C195 with S239, locking the channel in a closed conformation [226]. It should be noted that the high concentrations of H 2 O 2 (1mM) used in this work may contribute to the irreversible state of this redox modification [226]. C143 and C126 have also been implicated with electrophilic interactions and inhibition of CRAC currents by curcumin and caffeic acid phenethyl ester [227].…”

ORAI1 channel gating and selectivity is differentially altered by natural mutations in the first or third transmembrane domain

“…; Alansary et al . ). Natural substitutions of TM1 residues facing the channel pore alter channel conductance and selectivity, but mutations in TM2, TM3 or TM4 also alter channel conductance (Nesin et al .…”