Transient receptor potential vanilloid (TRPV) channels play a critical role in calcium homeostasis, pain sensation, immunological response, and cancer progression. TRPV channels are blocked by ruthenium red (RR), a universal pore blocker for a wide array of cation channels. Here we use cryo-electron microscopy to reveal the molecular details of RR block in TRPV2 and TRPV5, members of the two TRPV subfamilies. In TRPV2 activated by 2-aminoethoxydiphenyl borate, RR is tightly coordinated in the open selectivity filter, blocking ion flow and preventing channel inactivation. In TRPV5 activated by phosphatidylinositol 4,5-bisphosphate, RR blocks the selectivity filter and closes the lower gate through an interaction with polar residues in the pore vestibule. Together, our results provide a detailed understanding of TRPV subfamily pore block, the dynamic nature of the selectivity filter and allosteric communication between the selectivity filter and lower gate.

Transient receptor potential (TRP) ion channels are involved in the surveillance or regulation of the acid-base balance. Here, we demonstrate that weak carbonic acids, including acetic acid, lactic acid, and CO ₂ activate and sensitize TRPV2 through a mechanism requiring permeation through the cell membrane. TRPV2 channels in cell-free inside-out patches maintain weak acid-sensitivity, but protons applied on either side of the membrane do not induce channel activation or sensitization. The involvement of proton modulation sites for weak acid-sensitivity was supported by the identification of titratable extracellular (Glu495, Glu561) and intracellular (His521) residues on a cryo-EM structure of rat TRPV2 (rTRPV2) treated with acetic acid. Molecular dynamics simulations as well as patch clamp experiments on mutant rTRPV2 constructs confirmed that these residues are critical for weak acid-sensitivity. We also demonstrate that the pore residue Glu609 dictates an inhibition of weak acid-induced currents by extracellular calcium. Finally, TRPV2-expression in HEK293 cells is associated with an increased weak acid-induced cytotoxicity. Together, our data provide new insights into weak acids as endogenous modulators of TRPV2.

Transient receptor potential vanilloid 2 (TRPV2) is relevant for diseases like cancer, cardiac dysfunction, and infection, warranting drug development targeting TRPV2. However, this has been complicated by the lack of good modulators targeting TRPV2 and questions about species selectivity, so more detailed molecular insights into channel function and pharmacology are required. Two recent studies identified distinct binding sites on rat (r) and mouse (m) TRPV2 for activation by 2-aminoethoxydiphenyl borate (2-APB). Here we aimed to determine whether the mechanisms for 2-APB sensitivity of TRPV2 indeed differ among these closely related orthologs. Patch clamp electrophysiology revealed that mTRPV2 and human TRPV2 display similar sensitivities to 2-APB when compared with a considerably higher sensitivity of rTRPV2. For both mTRPV2 and rTRPV2, we observed that the exchange of putative 2-APB binding residues within the vanilloid binding pocket alters overall channel sensitivity to 3 TRPV2 pharmacological activators that bind at different sites: 2-APB, cannabidiol, and probenecid. By contrast, the exchange of putative 2-APB binding residues at the S5 binding pocket in both channels resulted in strongly reduced 2-APB sensitivies without reducing sensitivity to cannabidiol and probenecid. rTRPV2 mutants lacking key residues of both binding sites were almost completely 2-APB insensitive. These functional data suggest that the mechanisms accounting for 2-APB sensitivity are similar across mammalian TRPV2 orthologs. Except for serving as a binding site for 2-APB, the vanilloid binding pocket plays a key role in the overall function of TRPV2. These findings are relevant for the emerging framework toward an improved understanding of TRPV2. SIGNIFICANCE STATEMENT: This study resolves the conflict regarding how 2-aminoethoxydiphenyl borate binds to transient receptor potential vanilloid 2 (TRPV2), showing a shared mechanism despite sensitivity differences. These findings enhance TRPV2 modulation insights and highlight species considerations in drug design, aiding the development of selective TRPV2-targeted therapies.

Background and Purpose: Itch is associated with several pathologies and is a common drug‐induced side effect. Chloroquine (CQ) is reported to induce itch by activating the Mas‐related G protein‐coupled receptor MrgprA3 and subsequently TRPA1. In this study, we demonstrate that CQ employs at least two MrgprA3‐independent mechanisms to activate or sensitize TRPA1 and TRPV1.Experimental Approach: Patch clamp and calcium imaging were utilized to examine effects of CQ on TRPA1 and TRPV1 expressed in HEK 293T cells.Key Results: In calcium imaging, CQ induces a concentration‐dependent but MrgprA3‐independent activation of TRPA1 and TRPV1. Although CQ itself inhibits TRPA1 and TRPV1 in patch clamp recordings, co‐application of CQ and ultraviolet A (UVA) light evokes membrane currents through both channels. This effect is inhibited by the reducing agent dithiothreitol (DTT) and is reduced on mutants lacking cysteine residues accounting for reactive oxygen species (ROS) sensitivity. The combination of CQ and UVA light triggers an accumulation of intracellular ROS, removes fast inactivation of voltage‐gated sodium currents and activates TRPV2. On the other hand, CQ is a weak base and induces intracellular alkalosis. Intracellular alkalosis can activate TRPA1 and TRPV1, and CQ applied at alkaline pH values indeed activates both channels.Conclusion and Implications: Our data reveal novel pharmacological properties of CQ, allowing activation of TRPA1 and TRPV1 via photosensitization as well as intracellular alkalosis. These findings add more complexity to the commonly accepted dogma that CQ‐induced itch is specifically mediated by MrgprA3 coupling to TRPA1.

Background: A large number of patients suffer from neuropathic pain, and systemic therapy often remains ineffective while inducing severe side effects. Topical therapy with the TRPV1-agonist capsaicin is an established alternative, and the identification of co-therapeutics that modulate TRPV1 may be a promising approach to reduce the dose of capsaicin while maintaining efficacy. Here, we aimed to determine if the antidepressant amitriptyline displays properties rendering it a potential co-therapeutic agent. Methods: We performed patch clamp and calcium imaging experiments on HEK293T cells expressing human (h) TRPV1 as well as on dorsal root ganglion (DRG) neurons from adult mice. Results: Amitriptyline induced an increase in intracellular calcium in both HEK293T and mouse DRG neurons expressing TRPV1. Patch clamp experiments revealed a concentration-dependent activation of hTRPV1 by amitriptyline that was also evident in cell-free inside-out patches. When hTRPV1 was fully activated by capsaicin, amitriptyline induced concentration-dependent and partly reversible current inhibition. In contrast, amitriptyline potentiated small responses to capsaicin, heat and protons. We also found that amitriptyline desensitized hTRPV1 to capsaicin. This effect was reduced by the intracellular application of the strong calcium chelator BAPTA. Furthermore, the non-desensitizing mutant hTRPV1-Y672K displayed a reduced amitriptyline-induced desensitization. Conclusions: Our data showed that amitriptyline can activate, sensitize, desensitize and even inhibit TRPV1. Together with its property as a strong local anesthetic, our data suggest that amitriptyline may be a promising adjunct to topical capsaicin.

Transient receptor potential vanilloid 2 (TRPV2) is a non-selective cation channel involved in diverse physiological and pathological processes. Tranilast has frequently been described and used as a rather specific inhibitor of TRPV2. However, the molecular basis of this inhibition was never been studied in detail. Here, we investigated whether tranilast indeed directly inhibits TRPV2. Rat TRPV2 was expressed in human embryonic kidney (HEK293) cells, and channel function was assessed using whole-cell electrophysiology and calcium imaging in response to established agonists. In parallel, we conducted phagocytosis assays in rat basophilic leukemia (RBL) cells, including a CRISPR/Cas9-generated TRPV2-knockout cell line. Tranilast up to 1 mM did not inhibit TRPV2-mediated currents or calcium influx induced by any agonist. However, when co-applied with the oxidant chloramine T, tranilast diminished oxidation-induced activation of TRPV2. This effect may indicate a general interference of tranilast with redox signaling. Accordingly, tranilast also reduced chloramine T-induced activation of TRPA1 as well as the development of non-inactivating currents of voltage-gated Na⁺ channels. Furthermore, tranilast decreased phagocytic activity in both wildtype and TRPV2-knockout RBL cells. However, the reduction was less pronounced in TRPV2-knockout cells. These findings demonstrate that tranilast does not directly inhibit TRPV2. Instead, tranilast seems to indirectly suppress channel activation by reducing reactive oxygen species (ROS). This refined understanding of how tranilast modulates TRPV2 has important implications for the interpretation of prior and future pharmacological studies targeting TRPV2.