Differential Redox Regulation of ORAI Ion Channels: A Mechanism to Tune Cellular Calcium Signaling

Abstract

The following resources related to this article are available online at http://stke.sciencemag.org. Reactive oxygen species (ROS) are involved in many physiological and pathophysiological cellular processes. We used lymphocytes, which are exposed to highly oxidizing environments during inflammation, to study the influence of ROS on cellular function. Calcium ion (Ca 2+ ) influx through Ca 2+ release-activated Ca 2+ (CRAC) channels composed of proteins of the ORAI family is essential for the activation, proliferation, and differentiation of T lymphocytes, but whether and how ROS affect ORAI channel function have been unclear. Here, we combined Ca 2+ imaging, patch-clamp recordings and measurements of cell proliferation and cytokine secretion to determine the effects of hydrogen peroxide (H 2 O 2 ) on ORAI channel activity and human T helper lymphocyte (T H cell) function. ORAI1, but not ORAI3, channels were inhibited by oxidation by H 2 O 2 . The differential redox sensitivity of ORAI1 and ORAI3 channels depended mainly on an extracellularly located reactive cysteine, which is absent in ORAI3. T H cells became progressively less redox-sensitive after differentiation into effector cells, a shift that would allow them to proliferate, differentiate, and secrete cytokines in oxidizing environments. The decreased redox sensitivity of effector T H cells correlated with increased expression of Orai3 and increased abundance of several cytosolic antioxidants. Knockdown of ORAI3 with small-interfering RNA rendered effector T H cells more redox-sensitive. The differential expression of Orai isoforms between naïve and effector T H cells may tune cellular responses under oxidative stress. Article Toolshttp INTRODUCTION Intracellular Ca2+ is a second messenger involved in the regulation of a diverse range of functions (1). One of the major Ca 2+ entry pathways into nonexcitable cells, such as lymphocytes and epithelial cells, is through ubiquitously expressed Ca 2+ release-activated Ca 2+ (CRAC) channels that are localized in the plasma membrane. Ca 2+ influx through CRAC channels is activated when inositol 1,4,5-trisphosphate (IP 3 ) triggers Ca 2+ release from intracellular stores in the lumen of the endoplasmic reticulum (ER) (2-4).The concomitant decrease in ER luminal Ca 2+ triggers accumulation of the ER Ca 2+ sensor protein stromal interaction molecule (STIM1) into puncta close to the plasma membrane (5, 6). These clustered STIM1 proteins directly activate Ca 2+ influx through CRAC channels, which are encoded by the Orai gene family (7-11). ORAI proteins contain four transmembrane domains with both N-and C-terminal intracellular domains (12, 13) that contain putative N-terminal calmodulinbinding domains and C-terminal coiled-coil motifs. Orai family members are highly homologous, and Orai1 and 3 are widely expressed at the messenger RNA (mRNA) level, with Orai2 showing a somewhat more restricted expression pattern (14, 15). ORAI1 proteins contain the longest intracellular N termini with two proline-rich regions an...

Redox regulation of calcium ion channels: Chemical and physiological aspects

“…Mutation of all three cysteines in Orai1 render the channels functional but completely insensitive to preincubation with H 2 O 2 , providing evidence that in a heterologous expression system, longer extracellular preincubation with H 2 O 2 mediates its effects on CRAC solely through oxidation of Orai1. Increased expression of the insensitive Orai3 enables primary effector T cells to reduce their SOCE sensitivity towards extracellular H 2 O 2 [187]. Acute application of H 2 O 2 to T cells also induced a rise in internal Ca 2+ , which is likely independent of Orai1, as siRNA knock-down of Orai1 did not reduce this acute increase in intracellular Ca 2+ (unpublished data).…”

“…One of the major reasons for this, as mentioned above, was the unknown molecular identity of the CRAC channels. Recently, using electrophysiology, Ca 2+ -imaging and site-directed mutagenesis we found that Orai1, but not Orai3 activation is inhibited by preincubation with H 2 O 2 [187]. Cysteine 195 which is present in Orai1 but absent in Orai3 was identified as a major redox sensor of Orai1, although C126 and C143 also contributed to redox sensitivity.…”

The Redox Biochemistry of Protein Sulfenylation and Sulfinylation

“…For example, human T-helper lymphocyte ORAI1 channels, a member of the CRAC (Ca 2ϩ release-activated Ca 2ϩ ) channel family, are inhibited by oxidation of the extracellular Cys-195 residue (49), although the nature of this Cys oxidation remains unknown.…”

Store-Operated Calcium Channels: New Perspectives on Mechanism and Function

“…In this way STIM1 may act as a cellular sensor for oxidant stress, although an indirect effect of oxidants on SOCE through depletion of Ca 2þ stores has not been excluded. On the other hand, oxidation also inhibits SOCE by reducing Orai1 activity (Bogeski et al 2010). This effect has been traced primarily to C195, and occurs with Orai1 but not Orai3.…”