Abstract
The transient receptor potential vanilloid 1 (TRPV1) channel is an essential component of the cellular mechanism through which noxious stimuli evoke pain. Functional and structural characterizations of TRPV1 shed light on vanilloid activation, yet the mechanisms for temperature and proton gating remain largely unknown. Spectroscopic approaches are needed to understand the mechanisms by which TRPV1 translates diverse stimuli into channel opening. Here, we have engineered a minimal cysteine-less rat TRPV1 construct (eTRPV1) that can be stably purified and reconstituted for spectroscopic studies. Biophysical analyses of TRPV1 constructs reveal that the S5-pore helix loop influences protein stability and vanilloid and proton responses, but not thermal sensitivity. Cysteine mutants retain function and stability for double electron-electron resonance (DEER) and electron paramagnetic resonance (EPR) spectroscopies. DEER measurements in the closed state demonstrate that eTRPV1 reports distances in the extracellular vestibule, equivalent to those observed in the apo TRPV1 structure. EPR measurements show a distinct pattern of mobilities and spectral features, in detergent and liposomes, for residues at the pore domain that agree with their location in the TRPV1 structure. Our results set the stage for a systematic characterization of TRPV1 using spectroscopic approaches to reveal conformational changes compatible with thermal- and ligand-dependent gating.
Highlights
The detection of painful stimuli involves ion channels that depolarize sensory neurons to elicit or intensify inflammatory pain[1]
We found that engineered a minimal cysteine-less rat TRPV1 construct (eTRPV1) recapitulates the thermosensitivity observed in wt transient receptor potential vanilloid 1 (TRPV1) (Fig. 1b,c); this construct can faithfully report the conformational changes that occur during thermal gating
ETRPV1 can be gated by RTX and capsaicin (Fig. 1f–g), we observed a significant decrease in the response to vanilloids when compared to wt TRPV1 (Fig. 1h, black arrow, and Supplementary S1a,b)
Summary
The detection of painful stimuli involves ion channels that depolarize sensory neurons to elicit or intensify inflammatory pain[1]. Even though the proton binding site is known, the pH-dependent dynamic conformational rearrangements that couple the extracellular vestibule with the opening of the hydrophobic plug (lower gate) remain largely unknown. This mechanism can be further addressed by monitoring the allosteric conformational changes of TRPV1 using spectroscopic approaches. Since heat sensitivity is an intrinsic property of TRPV128, the stage is set for an in-depth analysis of its dynamic conformational changes during thermal gating using spectroscopic techniques such as metal ion fluorescence resonance energy transfer[29] and electron paramagnetic resonance (EPR) spectroscopy. Functional and biochemical analyses of minimal cysteine-less rat TRPV1 constructs reveal that the S5-pore helix loop influences protein stability and vanilloid and proton responses, but not thermal sensitivity. Further insights into TRPV1 gating mechanisms could be achieved by monitoring dynamic conformational changes at different temperatures and pHs
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