Thermosensitive transient receptor potential (TRP) ion channels detect changes in ambient temperature to regulate body temperature and temperature-dependent cellular activity. Rodent orthologs of TRP vanilloid 2 (TRPV2) are activated by nonphysiological heat exceeding 50 °C, and human TRPV2 is heat-insensitive. TRPV2 is required for phagocytic activity of macrophages which are rarely exposed to excessive heat, but what activates TRPV2 in vivo remains elusive. Here we describe the molecular mechanism of an oxidation-induced temperature-dependent gating of TRPV2. While high concentrations of H₂O₂ induce a modest sensitization of heat-induced inward currents, the oxidant chloramine-T (ChT), ultraviolet A light, and photosensitizing agents producing reactive oxygen species (ROS) activate and sensitize TRPV2. This oxidation-induced activation also occurs in excised inside-out membrane patches, indicating a direct effect on TRPV2. The reducing agent dithiothreitol (DTT) in combination with methionine sulfoxide reductase partially reverses ChT-induced sensitization, and the substitution of the methionine (M) residues M528 and M607 to isoleucine almost abolishes oxidation-induced gating of rat TRPV2. Mass spectrometry on purified rat TRPV2 protein confirms oxidation of these residues. Finally, macrophages generate TRPV2-like heat-induced inward currents upon oxidation and exhibit reduced phagocytosis when exposed to the TRP channel inhibitor ruthenium red (RR) or to DTT. In summary, our data reveal a methionine-dependent redox sensitivity of TRPV2 which may be an important endogenous mechanism for regulation of TRPV2 activity and account for its pivotal role for phagocytosis in macrophages. Significance Redox sensitivity is a common property of temperature-sensitive transient receptor potential (TRP) ion channels. Here we show that oxidation sensitizes and activates rodent TRPV2 orthologs known to have a high temperature threshold (>50 °C), but also the heat-insensitive human TRPV2. This oxidation-induced channel gating is intact in cell-free membrane patches, cysteine-independent but reduced upon replacement of the methionine residues M528 and M607. Blocking of TRPV2 and the reducing agent DTT reduce phagocytosis in macrophages which also generate heat-induced membrane currents following oxidation. These data reveal a methionine-dependent redox sensitivity of TRPV2 which may resemble a decisive endogenous mechanism enabling channel activation.

TRPV1 mediates pain occurring during sickling episodes in sickle cell disease (SCD). We examined if hemin, a porphyrin released during intravascular hemolysis modulates TRPV1. Calcium imaging and patch clamp were employed to examine effects of hemin on mouse dorsal root ganglion (DRG) neurons and HEK293t cells expressing TRPV1 and TRPA1. Hemin induced a concentration-dependent calcium influx in DRG neurons which was abolished by the unspecific TRP-channel inhibitor ruthenium red. The selective TRPV1-inhibitor BCTC or genetic deletion of TRPV1 only marginally impaired hemin-induced calcium influx in DRG neurons. While hTRPV1 expressed in HEK293 cells mediated a hemin-induced calcium influx which was blocked by BCTC, patch clamp recordings only showed potentiated proton- and heat-evoked currents. This effect was abolished by the PKC-inhibitor chelerythrine chloride and in protein kinase C (PKC)-insensitive TRPV1-mutants. Hemin-induced calcium influx through TRPV1 was only partly PKC-sensitive, but it was abolished by the reducing agent dithiothreitol (DTT). In contrast, hemin-induced potentiation of inward currents was not reduced by DTT. Hemin also induced a redox-dependent calcium influx, but not inward currents on hTRPA1. Our data suggest that hemin induces a PKC-mediated sensitization of TRPV1. However, it also acts as a photosensitizer when exposed to UVA-light used for calcium imaging. The resulting activation of redox-sensitive ion channels such as TRPV1 and TRPA1 may be an in vitro artifact with limited physiological relevance.

Propacetamol (PPCM) is a prodrug of paracetamol (PCM) which was generated to increase water solubility of PCM for intravenous delivery. PPCM is rapidly hydrolyzed by plasma esterases to PCM and diethylglycine, and it shares some structural and metabolic properties with lidocaine. While PPCM is considered to be comparable to PCM regarding its analgesic properties, injection pain is a common side effect only described for PPCM. Injection pain is a frequent and unpleasant side effect of numerous drugs in clinical use, and previous reports have indicated that the ligand gated ion channels TRPA1 and TRPV1 on sensory neurons can mediate this effect. The aim of this paper was to investigate molecular mechanisms by which PPCM, in contrast to PCM, causes injection pain. Therefor human TRPV1 and TRPA1 receptors were expressed in HEK 293 cells and investigated by means of whole cell patch clamp and ratiometric calcium imaging. PPCM, but not PCM activated TRPV1, sensitized heat-induced currents and caused an increase in intracellular calcium. In TRPA1 expressing cells however, PPCM and PCM both evoked calcium responses but failed to induce inward currents. Intracutaneous injection of PPCM, but not of PCM, in human volunteers induced an intense and short-lasting pain and an increase in superficial blood flow indicating activation of nociceptive C-fibers and subsequent neuropeptide release. In conclusion, activation of human TRPV1 by propacetamol seems to be a relevant mechanism for induction of pain upon intracutaneous injection and thus also for pain reported as an adverse side effect upon intravenous administration.

The activation of TRPV1 and TRPA1 by UVA light is a complex process involving channel modulation by reactive oxygen species (ROS). The present study describes staurosporine and midostaurin, two protein kinase inhibitors, as photosensitizers that can modulate the activity of TRPV1 and TRPA1 in a UVA light-dependent manner. Patch-clamp and calcium imaging were used to investigate effects of staurosporine and midostaurin on recombinant human (h) TRPV1 and TRPA1 in HEK 293T cells and on native mouse dorsal root ganglion (DRG) cells. Staurosporine applied alone did not induce channel activation, but co-application with UVA light activated both TRPV1 and TRPA1. Staurosporine with UVA light also potentiated TRPV1-mediated membrane currents induced by heat and protons. Midostaurin induced the UVA light-independent activation and sensitization of TRPV1 and TRPA1, and this effect was strongly potentiated by UVA light. Effects induced by both staurosporine and midostaurin were reversed by the reducing agent dithiothreitol (DTT). Midostaurin induced a calcium influx in TRPA1-expressing DRG neurons. Our results show that staurosporine and midostaurin modulate the activity of TRPV1 and TRPA1 channels in the presence of UVA light. These photosensitizing properties can be relevant when staurosporine is used for in vitro experiments, and they may account for the phototoxic side effects of midostaurin.