Structural Insights into the TRPV1 Pain Channel Using Magnetic Resonance Spectroscopy

Abstract

Transient receptor potential vanilloid 1 (TRPV1) is a pain-sensing ion channel activated by noxious heat, low pH, and capsaicin—the active component in chili peppers. As a polymodal sensor involved in inflammatory and neuropathic pain, TRPV1 has emerged as a promising target for therapeutic intervention. However, its dynamic conformational changes and membrane-embedded nature make it challenging to study using conventional structural biology methods. 

In this project, we investigate the truncated version of TRPV1, specifically focusing on the voltage-sensing-like domain (VSLD), which spans transmembrane helices S1 to S4. This domain plays a key role in modulating channel gating and allosteric regulation. To probe the structural dynamics of the VSLD in a membrane-mimicking environment, we employ site-directed spin labeling (SDSL) combined with continuous-wave electron paramagnetic resonance (CW EPR) spectroscopy. 

Using single-cysteine TRPV1 mutants, we map the local mobility and membrane topology of individual residues. Our findings reveal distinct spectral differences between transmembrane and solvent-exposed sites, validating the predicted topology and demonstrating the sensitivity of CW EPR to environmental changes. By narrowing our focus to the VSLD region, this work provides foundational insights into domain-specific dynamics, which may inform the broader mechanisms of TRPV1 activation and guide future drug development efforts targeting pain pathways.

Methodology 

• Introduced single-cysteine TRPV1 mutants via site-directed mutagenesis PCR using mutation-specific forward and reverse primers
  
  

• Treated PCR products with DpnI to digest the methylated, non-mutated parental plasmid
  
  

• Verified successful amplification via agarose gel electrophoresis
  
  

• Transformed mutated plasmids into E. coli XL-Gold competent cells and plated on kanamycin-containing LB agar
  
  

• Selected individual colonies and cultured overnight in small-scale LB with kanamycin, then used to inoculate larger LB cultures
  
  

• Induced TRPV1 expression by adding IPTG when cultures reached an OD₆₀₀ of 0.4–0.6
  
  

• Harvested cells by centrifugation, lysed in buffer, and sonicated to release the protein
  
  

• Solubilized TRPV1 using detergent and purified it by nickel affinity chromatography, eluting with imidazole
  
  

• Measured protein concentration using a Nanodrop spectrophotometer
  
  

• Treated samples with DTT to maintain cysteine residues in a reduced state before spin labeling
  
  

• Labeled proteins with MTSL, which binds selectively to thiol (-SH) groups
  
  

• Removed excess spin label through buffer washing
  
  

• Assessed labeling efficiency and protein purity via SDS-PAGE
  
  

• Reconstituted labeled TRPV1 into lipid vesicles for CW EPR spectroscopy
  
  

• Used a cysteine-free TRPV1 variant as a negative control to confirm specific labeling

Figure 1 presents the continuous-wave (CW) EPR spectrum of the A452C-labeled TRPV1 mutant. The spectrum exhibits a broad lineshape, indicative of restricted spin label mobility. This pattern is consistent with A452C being situated within the hydrophobic core of the lipid bilayer, where molecular motion is more constrained due to the dense, ordered environment. In contrast, the CW EPR spectrum of L534C shown in Figure 2 reveals sharper, more defined peaks, reflecting increased spin label mobility and faster rotational diffusion. This is characteristic of residues located in more dynamic, aqueous environments, suggesting that L534C resides outside the membrane at a solvent-exposed interface. These spectral differences not only support the predicted topology of TRPV1 but also demonstrate the strength of site-directed spin labeling (SDSL) combined with CW EPR in resolving subtle differences in local environment and protein dynamics. 

Unlike classical structural techniques such as X-ray crystallography or Cryo-EM, which provide static, high-resolution snapshots of macromolecular structures, CW EPR offers real-time insight into the dynamic behavior of membrane-embedded proteins in native-like environments. Nuclear magnetic resonance (NMR) spectroscopy, while also capable of capturing dynamic information, is often limited by molecular size and requires high protein concentrations and isotopic labeling, which can be challenging for large membrane proteins like TRPV1.

In contrast, EPR is particularly well-suited for studying membrane proteins under physiologically relevant conditions, such as in lipid vesicles or bilayers. Its sensitivity to molecular motion, polarity, and local accessibility allows for precise mapping of membrane topology and conformational changes. The successful differentiation between A452C and L534C in this study highlights EPR’s utility in distinguishing membrane-embedded regions from solvent-exposed domains—key information for understanding the structural and functional mechanisms of TRPV1. 

Overall, the results validate the application of SDSL and CW EPR as powerful tools for probing the conformational landscape of membrane proteins, offering complementary insight to traditional structure-determining methods and guiding future studies aimed at elucidating TRPV1’s gating mechanisms and ligand interactions.

Limitations & Future Directions

While this study successfully demonstrates the utility of site-directed spin labeling and CW EPR in probing the dynamics of the TRPV1 VSLD region, there are several limitations worth noting. First, the current work focuses on a small number of single-cysteine mutants. The generation, expression, purification, and labeling of each individual mutant is a multi-step process that typically spans two to three weeks per site. As a result, the pace of data collection is inherently slow, limiting the breadth of residues that can be investigated within a single study. 

Additionally, the use of a truncated construct encompassing only the VSLD (S1–S4) provides valuable insight into domain-specific behavior but excludes key structural regions such as the pore-forming S5–S6 helices and intracellular domains. These omitted regions are crucial for understanding full-length TRPV1 gating mechanisms and signal transduction. 

Looking ahead, I aim to expand this work by probing a broader set of residues across the VSLD and adjacent domains to develop a more comprehensive mobility map. In parallel, a major objective is to use SDSL-EPR to map conformational changes upon ligand binding. This includes identifying potential binding sites for small molecules or antagonists that modulate TRPV1 activity. Incorporating ligand titration studies and advanced EPR techniques such as power saturation or DEER will enable a deeper understanding of how dynamic shifts in the protein structure relate to functional states. 

Together, these future efforts will help clarify the structural underpinnings of TRPV1 modulation and guide the rational design of next-generation therapeutics targeting this critical pain-sensing ion channel.

References 

Sierra-Valdez, F.J., Stein, R.A., Velissety, P., Vasquez, V., Cordero-Morales, J.F. Purification and Reconstitution of TRPV1 for Spectroscopic Analysis. J. Vis. Exp. (137), e57796, doi:10.3791/57796 (2018).

Stowe, R. B. (2023). A spectroscopic and biochemical study of protein interactions and membrane mimetic systems (Doctoral dissertation, Miami University).

Cao, E., Liao, M., Cheng, Y., & Julius, D. (2013). TRPV1 structures in distinct conformations reveal activation mechanisms. Nature, 504(7478), 113-118.

Benítez-Angeles, M., Morales-Lázaro, S. L., Juárez-González, E., & Rosenbaum, T. (2020). TRPV1: structure, endogenous agonists, and mechanisms. International journal of molecular sciences, 21(10), 3421.

Tominaga, M., & Tominaga, T. (2005). Structure and function of TRPV1. Pflügers Archiv, 451, 143-150.

Goodsell, D. S. (2019). Capsaicin Receptor TRPV1. RCSB Protein Data Bank, Molecule of the Month. Retrieved from https://pdb101.rcsb.org/motm/250

Career Readiness Skills: 

• Communication: My research experience has significantly strengthened my ability to communicate in both collaborative and technical settings. I regularly interacted with other research students and occasionally with collaborators from other labs, which taught me how to communicate clearly and respectfully across teams. Presenting my research during lab meetings and preparing both oral and poster presentations helped me grow as a scientific communicator. I learned to tailor my explanations depending on the audience — whether it was fellow undergraduates, faculty, or a general audience. In addition, I maintained a detailed and well-organized lab notebook, documenting experimental procedures, observations, and results in a way that others in the lab could follow. This reinforced the importance of clarity and consistency in written scientific communication — not only for my own reference, but also for enabling reproducibility and continuity within our lab group.

• Career & Self-Development: My undergraduate research experience has been instrumental in shaping my career goals and preparing me for real-world laboratory work. From early on, I took initiative to go beyond the minimum expectations by asking questions, seeking feedback, and actively learning from both my mentor and fellow researchers. This curiosity and drive to improve helped me recognize not only my strengths, such as attention to detail and scientific reasoning, but also areas where I could grow, including troubleshooting complex experiments and managing long-term projects independently. Equally important was the opportunity to connect with graduate students, postdocs, and faculty members who served as mentors and role models. These relationships have been a valuable source of guidance, support, and continued learning. I still reach out to many of them for advice, and I’ve learned to advocate for myself in professional conversations, whether discussing next steps in my project or seeking feedback on presentations.

• Critical Thinking: Working in a research lab has played a major role in developing my critical thinking skills. One of the most impactful parts of the experience was learning to question everything, not in a skeptical way, but in a purposeful, analytical one. Rather than simply following protocols, I was encouraged to ask why each step was necessary, how each instrument worked, and what the data was really telling us. This approach helped me understand the reasoning behind every technique, from spin-labeling and sample preparation to using UV-Vis and EPR spectroscopy. It also helped me in navigating complex data sets, identifying trends or unexpected outcomes, and thinking critically about what they meant.

• Professionalism: Working in a faculty-led research lab required me to take initiative, manage my time effectively, and maintain accurate lab records. I had to conduct experiments, meet deadlines, and ensure reproducibility in my work. Whether it was preparing buffers, running gels, or analyzing data, I learned the importance of consistency, attention to detail, and integrity in the research process — all of which are key elements of professionalism.