Oxygen (O(2)) is a prerequisite for cellular respiration in aerobic organisms but also elicits toxicity. To understand how animals cope with the ambivalent physiological nature of O(2), it is critical to elucidate the molecular mechanisms responsible for O(2) sensing. Here our systematic evaluation of transient receptor potential (TRP) cation channels using reactive disulfides with different redox potentials reveals the capability of TRPA1 to sense O(2). O(2) sensing is based upon disparate processes: whereas prolyl hydroxylases (PHDs) exert O(2)-dependent inhibition on TRPA1 activity in normoxia, direct O(2) action overrides the inhibition via the prominent sensitivity of TRPA1 to cysteine-mediated oxidation in hyperoxia. Unexpectedly, TRPA1 is activated through relief from the same PHD-mediated inhibition in hypoxia. In mice, disruption of the Trpa1 gene abolishes hyperoxia- and hypoxia-induced cationic currents in vagal and sensory neurons and thereby impedes enhancement of in vivo vagal discharges induced by hyperoxia and hypoxia. The results suggest a new O(2)-sensing mechanism mediated by TRPA1.

TRPA1 is a member of the transient receptor potential (TRP) cation channel family, and is predominantly expressed in nocicep-In TRPA1 responses to other cysteine-reactive inflammatory mediators, such as NO and H 2 O 2 , the extent of impairment by respective cysteine mutations differed from those in TRPA1 responses to 15d-PGJ 2 . Interestingly, the Cys421 mutation critically impaired the TRPA1 response to H + as well. Our findings suggest that TRPA1 channels are targeted by an array of inflammatory mediators to elicit inflammatory pain in the nervous system.

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...

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Redox sensitivity of TRP channels has been shown to mediate previously unexplained biological phenomena and is involved in various pathologies. Understanding the physiological significance and activation mechanisms of TRP channel regulation by reactive species may lead to TRP channels becoming viable pharmacological targets, and modulators of these channels may offer therapeutic options for previously untreatable diseases.

The transient receptor potential (trp) gene superfamily encodes cation channels that act as multimodal sensors for a wide variety of stimuli from outside and inside the cell. Upon sensing, they transduce electrical and Ca2+ signals via their cation channel activities. These functional features of TRP channels allow the body to react and adapt to different forms of environmental changes. Indeed, members of one class of TRP channels have emerged as sensors of gaseous messenger molecules that control various cellular processes. Nitric oxide (NO), a vasoactive gaseous molecule, regulates TRP channels directly via cysteine (Cys) S-nitrosylation or indirectly via cyclic GMP (cGMP)/protein kinase G (PKG)-dependent phosphorylation. Recent studies have revealed that changes in the availability of molecular oxygen (O2) also control the activation of TRP channels. Anoxia induced by O2-glucose deprivation and severe hypoxia (1% O2) activates TRPM7 and TRPC6, respectively, whereas TRPA1 has recently been identified as a novel sensor of hyperoxia and mild hypoxia (15% O2) in vagal and sensory neurons. TRPA1 also detects other gaseous molecules such as hydrogen sulfide (H2S) and carbon dioxide (CO2). In this review, we focus on how signaling by gaseous molecules is sensed and integrated by TRP channels.

S-Nitrosylation, the addition of a nitrosyl group to cysteine thiols, regulates various protein functions to mediate nitric oxide (NO) bioactivity. Recent studies have demonstrated that selectivity in protein S-nitrosylation signaling pathways is conferred through transnitrosylation, a transfer of the NO group, between proteins via interaction. We previously demonstrated that sensitivity to activation by synthetic NO-releasing agents via S-nitrosylation is a common feature of members of the transient receptor potential (TRP) family of Ca 21 -permeable cation channels. However, strategies to confer subtype selectivity to nitrosylating agents targeted to TRP channels are yet to be developed. Here, we show selective activation of TRPA1 channels by novel NO donors derived from the ABBH (7-azabenzobicyclo[2.2.1]heptane) N-nitrosamines, which exhibit transnitrosylation reactivity to thiols without releasing NO. The NNO-ABBH1 (N-nitroso-2-exo,3-exo-ditrifluoromethyl-7-azabenzobicyclo[2.2.1]heptane) elicits S-nitrosylation of TRPA1 proteins, and dose-dependently induces robust Ca 21 influx via both recombinant and native TRPA1 channels, but not via other NO-activated TRP channels. TRPA1 activation by NNO-ABBH1 is suppressed by specific cysteine mutations but not by NO scavenging, suggesting that cysteine transnitrosylation underlies the activation of TRPA1 by NNO-ABBH1. This is supported by the correlation of N-NO bond reactivity and TRPA1-activating potency in a congeneric series of ABBH N-nitrosamines. Interestingly, nonelectrophilic derivatives of ABBH also activate TRPA1 selectively, but less potently, compared with NNO-ABBH1. Thus, ABBH N-nitrosamines confer subtype selectivity on S-nitrosylation in TRP channels through synergetic effects of two chemical processes: cysteine transnitrosylation and molecular recognition of the nonelectrophilic moiety.

Recent advances in cryo-electron microscopy (cryo-EM) have enabled protein structure determination at atomic resolutions. Cryo-EM specimens are prepared by rapidly freezing a protein solution on a metal grid coated with a holey carbon film; this results in the formation of an ice film on each hole. The thickness of the ice film is a critical factor for high-resolution structure determination; ice that is too thick degrades the contrast of the protein image while ice that is too thin excludes the protein from the hole or denatures the protein. Therefore, trained researchers need to manually select "good" regions with appropriate ice thicknesses for imaging. To reduce the time spent on such tasks, we developed a deep learning program consisting of a "detector" and a "classifier" to identify good regions from low-magnification EM images. In our method, the holes in a low-magnification EM image are detected via a detector, and the ice image on each hole is classified as either good or bad via a classifier. The detector detected more than 95% of the holes regardless of the type of samples. The classifier was trained for different types of samples because the appropriate ice thickness varies between sample types. The accuracies of the classifiers were 93.8% for a soluble protein sample (β-galactosidase) and 95.3% for a membrane protein sample (bovine heart cytochrome c oxidase). In addition, we found that a training data set containing~2100 hole images from 300 low-magnification EM images was sufficient to obtain good accuracy, such as higher than 90%. We expect that the throughput of the cryo-EM data collection step will be greatly improved by using our method.