Electrophiles produced during oxidative stress trigger pain responses by reacting with TRPA1 ion channels on sensory nerves. Bahia et al. show that residue C621 on TRPA1 has remarkable reactivity with electrophiles—more than cellular antioxidants—and is crucial for this sensory response.

Na+–K+–2Cl− Cotransporter (NKCC1) is a protein that aids in the active transport of sodium, potassium, and chloride ions across cell membranes. It has been shown that long-term systemic treatment with aldosterone (ALD) can enhance NKCC1 protein expression and activity in the aging cochlea resulting in improved hearing. In the present work, we used a cell line with confirmed NKCC1 expression to demonstrate that in vitro application of ALD increased outward voltage-gated potassium currents significantly, and simultaneously upregulated whole lysate and membrane portion NKCC1 protein expression. These ALD-induced changes were blocked by applying the mineralocorticoid receptor antagonist eplerenone. However, application of the NKCC1 inhibitor bumetanide or the potassium channel antagonist Tetraethyl ammonium had no effect. In addition, NKKC1 mRNA levels remained stable, indicating that ALD modulates NKCC1 protein expression via the activation of mineralocorticoid receptors and post-transcriptional modifications. Further, in vitro electrophysiology experiments, with ALD in the presence of NKCC1, K+ channel and mineralocorticoid receptor inhibitors, revealed interactions between NKCC1 and outward K+ channels, mediated by a mineralocorticoid receptor-ALD complex. These results provide evidence of the therapeutic potential of ALD for the prevention/treatment of inner ear disorders such as age-related hearing loss.

Transient Receptor Potential Ankyrin 1 (TRPA1) is a tetrameric, nonselective cation channel expressed on nociceptive sensory nerves whose activation elicits nocifensive responses (e.g. pain). TRPA1 is activated by electrophiles found in foods and pollution, or produced during inflammation and oxidative stress, via covalent modification of reactive cysteines, but the mechanism underlying electrophilic activation of TRPA1 is poorly understood. Here we studied TRPA1 activation by the irreversible electrophiles iodoacetamide and N-ethylmaleimide (NEM) following transient expression in HEK293 cells. We found that in Ca 2+ imaging studies C621 is critical for electrophile-induced TRPA1 activation, but the role of C665 in TRPA1 activation is dependent on the size of the electrophile. We identified slower TRPA1 activation in whole-cell recordings compared to studies with intact cells, which is rescued by pipette solution supplementation with the antioxidant glutathione. Single-channel recordings identified two distinct electrophilic-induced TRPA1 activation phases: a partial activation that, in some channels, switched to full activation with continued electrophile exposure. Full activation but not the initial activation was regulated by C665. Fitting of open time distributions suggests that full activation correlated with an additional (and long) exponential component, thus suggesting the phases are manifestations of distinct activation states. Our results suggest that distinct NEM-induced TRPA1 activation states are evoked by sequential modification of C621 then C665.

Transient Receptor Potential Ankyrin 1 (TRPA1) is a non‐selective cation channel expressed largely on nociceptive sensory nerves. TRPA1 is rapidly activated by electrophiles, such as iodoacetamide, through covalent modification of intracellular cysteine (Cys) residues. We have previously shown that, at physiologically relevant exposures, iodoacetamide rapidly labels four Cys residues at positions 273, 621, 655, 1085. Of these, Cys residue at position 621 (C621) appears to be crucial for binding and activation of the human channel (hTRPA1). Interestingly, when comparing hTRPA1 with the electrophile‐insensitive rattlesnake homolog, while the amino acids lysine K620 and C621 are conserved the proline at P622 is not. We hypothesized that this rapid binding of C621 residue in human TRPA1 is mediated by neighboring amino acids K620 and P622 rendering it more sensitive to electrophilic adduction. We expressed V5‐His tagged hTRPA1 constructs in HEK293 cells including the wild‐type and the point mutants C621A, K620A and P622A. We used pulse‐chase experiments to label TRPA1 protein with iodoacetamide‐alkyne (IA‐alk), then used click chemistry to label with a fluorescent tag. Binding was measured using immunoprecipitated TRPA1 separated by SDS‐polyacrylamide gel electrophoresis and western blotting. TRPA1 channel activation was measured using ratiometric calcium imaging. The K620A mutant had the effect of greatly reducing electrophilic binding and activation of the channel. Modeling of deprotonation energies suggests that K620 contributes to C621 reactivity by reducing its pKa. The K620 mutant therefore likely reduces the probability that C621 will be thiolate and therefore able to rapidly react with electrophiles. The P622A mutant was also largely insensitive to electrophilic adduction and activation. IA‐alk binding was ~10% of that seen with the wild‐type channel. We hypothesize this is because, in the wild‐type hTRPA1, P622 induces a kink in the protein resulting in the positively charged K620 being closer to C621 compared with the nearby negatively charged glutamates, E625 and E681. The work described here highlights the importance of the amino acids K620 and P622 flanking C621. Our data demonstrate how the integrity of the local environment is essential to TRPA1 function as an electrophilic sensor.Support or Funding InformationThis work was supported by the National Heart Lung and Blood Institute in Bethesda, USA(R01‐HL119802‐S1)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Transient Receptor Potential Ankyrin 1 (TRPA1) is a tetrameric, non‐selective, cationic channel that is expressed in the dorsal root ganglia, trigeminal ganglia, and vagal ganglia and is activated by harmful chemicals including pollutants and endogenous irritants produced during inflammation and oxidative stress. Many irritants activate TRPA1 via electrophilic modification of highly reactive cysteine residues located on the cytosolic side of the channel, however the underlying processes that precede activation are poorly understood. Our lab has identified four highly reactive cysteines on TRPA1 (thus 16 highly reactive cysteines per channel) that rapidly bind electrophiles: C273, C621, C665 and C1085. C621 was the most reactive and point mutation studies show that both C621 and C665 are required for activation. In this study, we use cell‐attached single‐channel patch clamp analyses of transiently expressed human TRPA1 (hTRPA1) in HEK293 cells to determine the relationship between irreversible electrophilic binding and channel activity. We recorded stochastic gating events of single channels in response to N‐ethylmaleimide (NEM) treatment. Our open probability (Po) analysis indicates that NEM evoke two distinct hTRPA1 activation profiles: (1) weak activation (low Po) throughout NEM treatment which does not increase with time; (2) weak activation which then suddenly and spontaneously progresses to full activation (near 100% Po). Open time histogram analyses suggest that the weak activation state evoked by NEM has similar open times to spontaneous hTRPA1 activation in control conditions; whereas the full activation state has significantly longer open times. We conclude that the weak and full hTRPA1 activation states by NEM are due to differences in progressive cysteine modification. Furthermore, we suggest that full activation is not the summation of multiple weak states, but is instead a separate activation state that can only be evoked by a particular complement of cysteine modifications. Support or Funding Information This research is funded by the National Heart, Lung and Blood Institute (5R01HL119802‐S1). This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.