BPS2026 – Diffracted X-ray tracking reveals ligand- and temperature-dependent C-terminal dynamics of TRPM8

Transient receptor potential melastatin type 8 (TRPM8) is a cold-sensing ion channel. Various studies demonstrated that the gating of TRPM8 is allosterically controlled by the intracellular sensor domains. By using purified human TRPM8 proteins, we quantified sub-millisecond motions of the cytoplasmic C-terminal domain by the diffracted X-ray tracking (DXT). TRPM8 proteins were immobilized to the basement plate via an N-terminally tagged FLAG sequence using anti-FLAG antibodies. DXT experiments were performed at SPring-8 BL40XU beamline using quasi-monochromatic X-rays (Δ E / E ∼ 0.1). The C-terminal motion of TRPM8 was obtained by analyzing Laue diffraction spot trajectories from nanocrystals. At DXT, time-resolved diffraction images were collected at 0.1 ms intervals for 500 frames. A diffusion coefficient, D (mrad 2 /ms), was derived from the initial slope of the mean-squared displacement (MSD) of the nanocrystal’s angular motion. To assess temperature effects, we recorded DXT movies of apo TRPM8 at 16, 25, and 37 °C and quantified C-terminal angular mobility as the diffusion coefficient D (mrad 2 /ms) from the initial MSD slope. Relative to apo 25 °C ( D = 0.94), cooling to 16 °C increased mobility 1.42-fold ( D = 1.34), whereas warming to 37 °C reduced it to 0.87-fold ( D = 0.82). To assess ligand effects under isothermal conditions, we compared the motion of apo vs. icilin (10 μM) treated TRPM8 at 25 °C; icilin increased C-terminal mobility 2.43-fold ( D = 2.28 vs. 0.94). Thus, cooling and icilin both accelerate C-terminal motion while warming suppresses it. Icilin’s motion enhancement at 25 °C exceeds the modulation across 16–37 °C, indicating that activation signals from cooling or agonist propagate to the C-terminal domain and produce faster sub-millisecond motions consistent with TRPM8 activation.