TRPM3, part of the transient receptor potential (TRP) family, is a key thermosensor in sensory neurons. It activates around 35 °C, with sharply increased activity between 40 °C and 45 °C, key temperatures associated with pain.
TRPM3 is also sensitive to chemical ligands, including the neurosteroid pregnenolone sulfate (PregS), synthetic superagonist CIM0216, and the epilepsy drug primidone.
In mice, removing TRPM3 reduces avoidance of noxious heat and impairs inflammatory thermal hyperalgesia, underlining its role in pain signaling. Gain-of-function mutations in TRPM3 are also linked to epilepsy.
However, despite its significance, the molecular mechanisms by which TRPM3 is activated or inhibited have remained poorly understood.
Investigating How TRPM3 Activates
To probe how TRPM3 responds to temperature and ligands, the authors used cryo-electron microscopy (cryo-EM) and electrophysiology to analyze rabbit TRPM3 channels under different conditions: in the absence of stimuli (apo), in the presence of CIM0216 and primidone, and at low (18 °C) and elevated (37 °C) temperatures.
TRPM3 was expressed in tsA201 and Sf9 cells and purified for structural studies.
Because heat lacks a defined binding site, the team used CIM0216, a potent agonist known to stabilize the active conformation, to model TRPM3 activation.
By comparing structures under different conditions, they captured conformational transitions that underlie both thermal and chemical gating.
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A Shared Structural Switch
Heat and CIM0216 both induce similar rearrangements in TRPM3’s intracellular domain (ICD), shifting the channel toward an active state.
The key event here is the dissociation of the MHR1/2 domain from the adjacent rib helix. This conserved structural transition relaxes the intracellular gating mechanism, enabling cells to receive signals.
Importantly, TRPM3 doesn't simply flip between off and on. Instead, it exists in a dynamic equilibrium between resting and activated states, even in the absence of stimulation.
External cues like heat or ligands shift this balance, a mechanism consistent with the conformational selection model.
At 18 °C, most TRPM3 subunits are at rest. At 37 °C, a temperature chosen to elicit partial activation, the proportion of activated subunits rises significantly, but full channel opening likely requires three or more subunits to be in the active conformation.
This agrees with previous research, which suggests that heat-induced TRPM3 currents are relatively weak unless combined with other factors, such as membrane depolarization.
Mutations That Amplify Activation
To test the gating mechanism, the researchers introduced mutations that weakened electrostatic interactions between the rib helix and MHR1/2 domain.
These mutants showed enhanced sensitivity to both heat and agonists, confirming that disrupting this interaction lowers the energetic barrier for activation.
Some mutations also altered voltage sensitivity and produced large inward currents not observed in the wild-type channel, emphasizing the delicate, precise tuning of TRPM3’s activation threshold.
Inhibition by Primidone
Despite acting at the same S1-S4 binding site as CIM0216, the antagonist primidone stabilizes the inactive conformation.
While it causes some movement in the TRP helix, it doesn’t trigger the larger structural shifts needed to open the pore. Instead, it locks the channel in a nonconductive state, suppressing heat- and ligand-induced activity.
This duality, two molecules binding the same site with opposite effects, demonstrates the versatility of TRPM3’s ligand pocket and could be significant in the design of selective modulators.
Unresolved Questions
The study was unable to pinpoint the binding site for PregS, likely due to interference from cholesteryl hemisuccinate (CHS), which was used during protein purification.
However, disease-associated mutations near CHS binding sites suggest overlap with potential endogenous ligand sites.
New Therapeutics
By mapping the structural basis for TRPM3 gating, this work positions the channel as a compelling drug target for pain and epilepsy.
The findings suggest that modulating the rib helix-MHR1/2 interface, or designing ligands that bias conformational equilibrium, could result in a new wave of TRPM3-directed therapies.
Journal Reference
Kumar, S. et al. (2025). Structural basis for agonist and heat activation of nociceptor TRPM3. Nature Structural & Molecular Biology, 1-9. DOI: 10.1038/s41594-025-01692-5
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