Transient Receptor Potential Melastatin 3 (TRPM3) is a non-selective, Ca2+-permeable ion channel that plays a pivotal role in peripheral thermosensation and nociception. Moreover, gain-of-function variants in TRPM3 underlie a spectrum of neurodevelopmental and epileptic disorders in humans, indicating an important role of TRPM3 in the central nervous system. Oxidative stress contributes to various neurological disorders of both the central and peripheral nervous system, but it is unknown whether TRPM3 activity is altered by the cellular redox state. Here, we report a direct, bidirectional modification of TRPM3 channel activity by oxidizing and reducing agents. Our data demonstrate a profound effect of the redox state on the channel properties of TRPM3, including a robust shift in the response profile to pharmacology and temperature sensitivity. In addition, we identified two cysteine residues in the extracellular pore loop of TRPM3 that underlie the redox-control of the channel, due to the reversible formation of intra-subunit cysteine bridges. Taken together, these observations raise the hypothesis that TRPM3 could be modulated through an alternative mechanism, potentially affecting pathways involved in pain and neurological function. Transient Receptor Potential Melastatin 3 (TRPM3) is a non-selective, Ca2+-permeable ion channel that plays a pivotal role in the peripheral and central nervous systems; however, the effect of cellular redox state on the TRPM3 activity is unknown. Here, the authors reveal that TRPM3 channel activity is bidirectionally modulated by oxidizing and reducing agents, and identify cysteine residues in the extracellular pore loop of TRPM3 that carry the redox-control of the channel.

Transient receptor potential (TRP) cation channels play diverse roles in cellular Ca2+ signaling. First, as Ca2+-permeable channels that respond to a variety of stimuli, TRP channels can directly initiate cellular Ca2+ signals. Second, as nonselective cation channels, TRP channel activation leads to membrane depolarization, influencing Ca2+ influx via voltage-gated and store-operated Ca2+ channels. Finally, Ca2+ modulates the activity of most TRP channels, allowing them to function as molecular effectors downstream from intracellular Ca2+ signals. The past decade has seen a transformation of the TRP field. Once lacking high-resolution structures, we now have cryogenic electron microscopy (cryo-EM) structures across all seven TRP subfamilies, including multiple ligand-bound, lipid-bound, Ca2+-bound, and disease-mutant states. The rapid expansion of cryo-EM structures across all TRP subfamilies has transformed our understanding of Ca2+ permeation, selectivity, and Ca2+-dependent gating. These structures, together with functional and computational approaches, reveal how TRP channels coordinate Ca2+, how Ca2+ binding modulates pore opening or inactivation, and why some TRPs are highly Ca2+ selective while others are Ca2+ impermeable. This structural framework now underpins efforts to develop targeted therapies for a wide range of TRP-related diseases.

The transient receptor potential melastatin 3 (TRPM3) channel is a key mediator of peripheral pain signaling, and pathogenic mutations in TRPM3 are linked to neurodevelopmental delay and epilepsy. Despite the therapeutic promise of TRPM3 modulators, the molecular mechanisms by which ligands modulate channel gating remain poorly understood. Here, we combine cryo-electron microscopy (cryo-EM) with functional analyses to characterize a promiscuous ligand-binding pocket formed by transmembrane helices S1-S4. This pocket accommodates several chemically diverse plant-derived and synthetic agonists and antagonists. We show stereoselectivity of TRPM3 for the (R)-enantiomer of the flavonoid antagonist isosakuranetin and the (R)-enantiomer of the synthetic agonist CIM0216. Mutations within this pocket-including variants identified in patients -alter ligand affinity and, in some cases, invert the functional outcome of ligand binding. These findings reveal the stereoselectivity and functional plasticity of the TRPM3 ligand-binding pocket, highlighting how subtle changes in the molecular interactions can produce divergent effects on channel gating, with important ramifications for TRPM3-targeted drug development and therapy.

The transient receptor potential melastatin 3 (TRPM3) channel is a key mediator of peripheral pain signaling, and pathogenic mutations in TRPM3 are linked to neurodevelopmental delay and epilepsy. Despite the therapeutic promise of TRPM3 modulators, the molecular mechanisms by which ligands modulate channel gating remain poorly understood. Here, we combine cryo-electron microscopy (cryo-EM) with functional analyses to characterize a promiscuous ligand-binding pocket formed by transmembrane helices S1–S4. This pocket accommodates several chemically diverse plant-derived and synthetic agonists and antagonists. We uncover unanticipated stereoselectivity of TRPM3 for the non-natural enantiomer of the flavonoid antagonist isosakuranetin and the (R) -enantiomer of the synthetic agonist CIM0216. Mutations within this pocket—including newly identified patient variants—alter ligand affinity and, in some cases, invert the functional outcome of ligand binding. These findings reveal the stereoselectivity and functional plasticity of the TRPM3 ligand-binding pocket, highlighting how subtle changes in the molecular interactions can produce divergent effects on channel gating, with important ramifications for TRPM3-targeted drug development and therapy. ### Competing Interest Statement The authors have declared no competing interest. Research Foundation - Flanders, https://ror.org/03qtxy027, G0B9520N, G0B7620N, G055124N KU Leuven, C24M/21/028 Queen Elisabeth Medical Foundation for Neurosciences Vlaams Instituut voor Biotechnologie, https://ror.org/03xrhmk39

Rapamycin (sirolimus), a macrolide compound isolated from the bacterium Streptomyces hygroscopicus, is widely used as oral medication for the prevention of transplant rejection and the treatment of lymphangioleiomyomatosis. It is also incorporated in coronary stent coatings to prevent restenosis and in topical preparations for the treatment of skin disorders. Rapamycin's in vivo activities are generally ascribed to its binding to the protein FKBP12, leading to potent inhibition of the mechanistic target of rapamycin kinase (mTOR) by the FKBP12-rapamycin complex. The specific rapamycin-induced interaction between domains from mTOR and FKBP12 is also frequently employed in cell biological research, for rapid chemically-induced protein dimerization strategies. Here, we show that rapamycin activates TRPM8, a cation channel expressed in sensory nerve endings that serves as the primary cold sensor in mammals. Using a combination of electrophysiology, Saturation Transfer Triple-Difference (STTD) NMR spectroscopy, and molecular docking-based targeted mutagenesis, we demonstrate that rapamycin directly binds to human TRPM8. We identify a rapamycin-binding site in the groove between voltage sensor-like domain and the pore domain, distinct from the interaction sites of cooling agents and known TRPM8 agonists menthol and icilin. Related macrolide immunosuppressants act as partial TRPM8 agonists, competing with rapamycin for the same binding site. These findings identify a novel molecular target for rapamycin and provide new insights into the mechanisms of TRPM8 activation, which may assist in the development of therapies targeting this ion channel. Moreover, our findings also indicate that caution is needed when using molecular approaches based on rapamycin-induced dimerization to study ion channel regulation.

Background: and purpose: Phenazopyridine (PAP) is an over-the-counter drug widely used to provide symp-tomatic relief of bladder pain in conditions such as cystitis or bladder pain syndrome (BPS). Whereas the analgesic effect of PAP has been attributed to a local effect on the mucosa of the lower urinary tract (LUT), the molecular targets of PAP remain unknown. We investigated the effect of PAP on pain-related Transient Receptor Potential (TRP) channels expressed in sensory neurons that innervate the bladder wall.Experimental approach: The effects of PAP on the relevant TRP channels (TRPV1, TRPA1, TRPM8, TRPM3) expressed in HEK293 or CHO cells was investigated using Fura-2-based calcium measurements and whole-cell patch-clamp recordings. Activity of PAP on TRPM8 was further analysed using Fura-2-based calcium imaging on sensory neurons isolated from lumbosacral dorsal root ganglia (DRG) of mice.Key results: PAP rapidly and reversibly inhibits responses of TRPM8 expressed in HEK293 cells to cold and menthol, with IC50 values between 2 and 10 mu M. It acts by shifting the voltage dependence of channel activation towards positive potentials, opposite to the effect of menthol. PAP also inhibits TRPM8-mediated, menthol -evoked calcium responses in lumbosacral DRG neurons. At a concentration of 10 mu M, PAP did not significantly affect TRPA1, TRPV1, or TRPM3.Conclusion and implications: PAP inhibits TRPM8 in a concentration range consistent with PAP levels in the urine of treated patients. Since TRPM8 is expressed in bladder afferent neurons and upregulated in patients with painful bladder disorders, TRPM8 inhibition may underlie the analgesic activity of PAP.