M. Bozem

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

Redox sensitivity of T cells decreases through ORAI Ca2+ channel subunit switching during T cell differentiation. Adapting to Oxidizing Environments Reactive oxygen species (ROS) were thought for many years to be only detrimental, causing damage to DNA and proteins. However, it has become clear that ROS, particularly H2O2, can act as intracellular signaling molecules that link cellular redox state to such processes as proliferation and differentiation. Bogeski et al. have uncovered a role for ROS in regulating calcium channel activity—and intracellular Ca2+ signals crucial to the immune response—in T lymphocytes. They found that activity of ORAI1 calcium channels was blocked by H2O2, whereas that of the related ORAI3 channels was not. Redox sensitivity decreased as naïve human T helper lymphocytes differentiated into effector T helper lymphocytes, which was associated with an increase in the abundance of mRNA encoding the insensitive ORAI3 protein. The authors suggest that changes in the specific complement of ORAI channels, and thereby sensitivity to ROS, could enable T lymphocytes to fine tune cellular responses in oxidizing environments such as those found during inflammation. 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 (Ca2+) influx through Ca2+ release–activated Ca2+ (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 Ca2+ imaging, patch-clamp recordings, and measurements of cell proliferation and cytokine secretion to determine the effects of hydrogen peroxide (H2O2) on ORAI channel activity and human T helper lymphocyte (TH cell) function. ORAI1, but not ORAI3, channels were inhibited by oxidation by H2O2. The differential redox sensitivity of ORAI1 and ORAI3 channels depended mainly on an extracellularly located reactive cysteine, which is absent in ORAI3. TH 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 TH cells correlated with increased expression of Orai3 and increased abundance of several cytosolic antioxidants. Knockdown of ORAI3 with small-interfering RNA rendered effector TH cells more redox-sensitive. The differential expression of Orai isoforms between naïve and effector TH cells may tune cellular responses under oxidative stress.

Glucose modulation of spike activity independently from changes in slow waves of membrane potential in mouse B-cells.

In mouse B-cells glucose induces a typical electrical activity consisting of slow waves of the membrane potential with spikes superimposed on the plateau. As the concentration of glucose is raised the number of spikes per minute increases. However, this increase could simply be due to the concomitant lengthening of the slow waves. We thus investigated whether glucose can influence spike activity when no slow waves occur. Persistent depolarization to the plateau potential was achieved at 3 mM glucose by tolbutamide or at 10 mM glucose by low Ca2+, by arginine or by ouabain. Under all these conditions, raising the concentration of glucose increased the spike frequency without changing the plateau potential. Similar effects were produced by tolbutamide which does not affect B-cell metabolism but directly blocks K+-ATP channels. The spike frequency could also be increased by arginine, which, however, consistently depolarized the membrane. In conclusion, spike activity in B-cells can be influenced by glucose independently from changes in slow wave duration. This indicates that some K+-ATP channels, a target for both glucose and tolbutamide, are still open when the membrane is depolarized at the plateau, or that these two agents share another yet unidentified target involved in spike generation.

Inosine partially mimics the effects of glucose on ionic fluxes, electrical activity, and insulin release in mouse pancreatic B-cells

The purine ribonucleoside inosine is known to be metabolized in islet cells (its ribose moiety feeds into the pentose-phosphate cycle) and stimulate insulin release, but the mechanisms of this stimulation have not been established. These were investigated with mouse islets. In the absence of glucose, 5 mM inosine decreased86Rb+ efflux from islet cells, depolarized the B-cell membrane, induced electrical activity (slow waves of membrane potential with bursts of spikes on the plateau), accelarated45Ca2+ efflux and stimulated insulin release with the same efficiency as 10 mM glucose. Raising the concentration of inosine to 20 mM only had a slight further effect and, in particular, failed to cause persistent depolarization of the B-cell membrane. The electrical activity triggered by inosine was blocked by cobalt, and the stimulation of45Ca2+ efflux and insulin release was abolished in a Ca2+-free medium. The effects of 10 mM glucose on electrical activity,45Ca2+ efflux and insulin release were augmented by as little as 0.5 mM inosine. All effects of inosine were abolished by an inhibitor of nucleoside transport (nitrobenzylthioguanosine) and markedly impaired by inhibitors of nucleoside phosphorylase (formycin B) or of glycolysis (iodoacetate). In conclusion, inosine metabolism in B-cells induces insulin release by triggering the same sequence of events as glucose metabolism: a decrease of K+ permeability of the B-cell membrane, leading to depolarization and activation of voltage-dependent Ca channels.

Differential Redox Regulation of ORAI Channels: A Mechanism to Tune T-Cell Responses

Phagocytes play an essential role in host defence against pathogens by generating reactive oxygen species (ROS). Effector T helper (Th) cells migrating to sites of infection will be exposed to this highly oxidative environment. Here we show how Th-cells respond and adapt to ROS. Oxidation affects different Ca2+-signalling pathways essential for T-cell function. ORAI1 channels are inhibited with an IC50 of ∼40 μM H2O2, but ORAI3 channels are insensitive. We identify cysteine (C195) of ORAI1, absent in ORAI3, as the major redox sensor. A reduced sensitivity of effector Th-cells towards oxidation is due to upregulation of Orai3 and of cytosolic antioxidants. The differential redox regulation of ORAI channels is a novel mechanism to tune Th-cell based immune responses during clonal expansion and inflammation.