BPS2026 – Probing temperature-dependent structural dynamics of the human TRPV1 voltage sensor-like domain in membrane mimetics via site directed spin labeling and CW-EPR spectroscopy

Transient receptor potential vanilloid 1 (TRPV1) is a polymodal ion channel that is activated by variations in temperature, pH levels, and chemical ligands. It plays a crucial role in thermosensation and pain signaling. The voltage-sensor-like domain (VSLD) of the TRPV1 consists of a bundle of four transmembrane helices, labeled S1 through S4. While structural studies have provided insights into the architecture of TRPV1, the dynamic behavior of its residues in a native-like environment across different thermal conditions remains poorly understood. This study aims to investigate the structural dynamics of the human TRPV1 VSLD in various membrane mimetics over a temperature gradient using site-directed spin labeling and continuous-wave electron paramagnetic resonance (CW-EPR) spectroscopy. We created TRPV1 VSLD mutants targeting the helix linker regions, as their movements are essential for thermosensation. Successfully expressed, purified, and spin-labeled mutant samples were incorporated in detergent micelles with 0.1% 1-palmitoyl-2-hydroxy-sn-glycero-3-phospho-(1′-rac-glycerol) (LPPG) and further in liposomes with a 3:1 molar ratio of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) to 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (POPG). We collected CW-EPR spectra starting at 298 K, increasing in 5 K increments up to 323 K. A progressive line sharpening was observed in both LPPG micelles and POPC/POPG as the temperature increased, indicating higher mobility and conformational flexibility at the labeled sites. These findings suggest that the linker regions of TRPV1 VSLD undergo temperature-dependent dynamic changes, reflecting functional transitions related to channel gating. Future experiments will target additional residues that represent the entire VSLD. We will also incorporate TRPV1 VSLD into styrene-maleic acid copolymer-lipid nanoparticles to investigate its dynamics in a more compact environment. Overall, this study will reshape our understanding of molecular heat detection and open new paths for targeted pain therapies.