Expression and Purification of the Pain Receptor TRPV1 for Spectroscopic Analysis

Phanindra Velisetty, Richard A. Stein, Francisco Sierra Valdez, Valeria Vasquez, Julio Cordero-Morales

Research output: Contribution to journalArticle

4 Citations (Scopus)

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.

Original languageEnglish (US)
Article number9861
JournalScientific reports
Volume7
Issue number1
DOIs
StatePublished - Dec 1 2017

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Nociceptors
Electrons
Electron Spin Resonance Spectroscopy
Cysteine
Protons
Hot Temperature
vanilloid receptor subtype 1
Protein Stability
Liposomes
Detergents
Spectrum Analysis
Ligands
Pain
Temperature

All Science Journal Classification (ASJC) codes

  • General

Cite this

Expression and Purification of the Pain Receptor TRPV1 for Spectroscopic Analysis. / Velisetty, Phanindra; Stein, Richard A.; Sierra Valdez, Francisco; Vasquez, Valeria; Cordero-Morales, Julio.

In: Scientific reports, Vol. 7, No. 1, 9861, 01.12.2017.

Research output: Contribution to journalArticle

Velisetty, Phanindra ; Stein, Richard A. ; Sierra Valdez, Francisco ; Vasquez, Valeria ; Cordero-Morales, Julio. / Expression and Purification of the Pain Receptor TRPV1 for Spectroscopic Analysis. In: Scientific reports. 2017 ; Vol. 7, No. 1.
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