Velisetty P et al. (2017), Expression and Purification of the Pain Recepto...

XB-ART-55903

Sci Rep 2017 Aug 29;71:9861. doi: 10.1038/s41598-017-10426-7.

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Expression and Purification of the Pain Receptor TRPV1 for Spectroscopic Analysis.

Velisetty P , Stein RA , Sierra-Valdez FJ , Vásquez V , Cordero-Morales JF .

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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.

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Species referenced: Xenopus
Genes referenced: adm aopep dtl mbp trpv1

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	Figure 1. Characterization of a functional minimal rat cysteine-less TRPV1 construct (eTRPV1). (a) Schematic representation of eTRPV1 depicting the regions deleted (N- and C-termini and S5-pore helix loop) over the cysteine-less (cl) TRPV1 frame. (b) Heat-evoked currents of eTRPV1 extracted from voltage-clamp ramp protocols at +80 mV. (c) Temperature-response profile (+80 mV) of wt TRPV1 (black) and eTRPV1 (red); I/I 49 °C: Currents at each temperature/current at 49 °C. (d) Current-voltage relationship of eTRPV1 challenged with pH 5. (e) pH dose–response profiles (+80 mV) of wt TRPV1 and eTRPV1. I/I max: current at each pH/current at pH 4.5. n = 6. Squares are mean ± s.d. (f–g) Vanilloid-evoked currents of eTRPV1 challenged with 10 µM of resiniferatoxin (RTX) and capsaicin (Cap). (h) pH 5, Cap, and RTX-evoked currents of wt TRPV1 (top) and eTRPV1 (bottom) extracted from voltage-clamp ramp protocols at +80 mV. (i) Temperature-response profile (+80 mV) of eTRPV1 at pH 6.0 (blue) and 7.4 (red). I/I 48 °C: Current at each temperature/current at 48 °C. Background currents (bkgrd).
	Figure 2. Purification of a stable minimal cysteine-less rat TRPV1 construct (eTRPV1). Top: schematic representation of the eTRPV1 construct used for insect cell expression. Bottom: size-exclusion chromatography profile of DDM-solubilized eTRPV1 protein after expression and purification from Sf9 cells. Inset: Stained protein on the SDS-PAGE gel corresponds to the size of the purified MBP-eTRPV1 monomer.
	Figure 3. The S5-pore loop influences vanilloid sensitivity and protein stability. (a) Amino acid sequence highlighting the changes made in the S5-pore loop of eTRPV1 to generate e1-TRPV1 (green). (b–d) Representative current-voltage relationships of e1-TRPV1 challenged with capsaicin (Cap, 10 µM), resiniferatoxin (RTX, 10 µM), and pH 5. (e) pH 5-, Cap-, and RTX-evoked currents of e1-TRPV1, extracted from voltage-clamp ramp protocols at +80 mV. Note the increase in Cap and RTX responses as compared to pH. (f) Capsaicin dose-response profile of wt TRPV1 and e1-TRPV1 n = 5. Squares are mean ± s.d. (g) Heat-evoked currents of e1-TRPV1 extracted from voltage-clamp ramp protocols at +80 mV. (h) Size-exclusion chromatography profile of DDM-solubilized e1-TRPV1 protein after expression and purification from Sf9 cells. Note that large amounts of e1-TRPV1 elute in the void volume (red arrow).
	Figure 4. Asparagine residues at the S5-pore loop affect protein stability. (a) Amino acid sequence highlighting the changes made in the S5-pore loop of eTRPV1 to generate e2-TRPV1 (green and blue residues). e2-TRPV1 contains two mutations over the frame of the e1-TRPV1: (1) the glycosylation site was mutated to threonine (N604T) and (2) the neighboring residue mutated from asparagine to glutamine (N605Q). (b) Size-exclusion chromatography profile of DDM-solubilized e2-TRPV1 protein after expression and purification from Sf9 cells. Note that large amounts of e2-TRPV1 elute predominantly as a monodisperse peak (~13 ml). (c) Representative current-voltage relationships of e2-TRPV1 challenged with capsaicin (Cap, 10 µM). (d) pH 5-, Cap-, and RTX-evoked currents of e2-TRPV1, extracted from voltage-clamp ramp protocols at +80 mV. (e) Icap/IpH (I max): maximal currents of capsaicin/maximal current at pH 4.5 (+80 mV) of wt TRPV1, eTRPV1, e1-TRPV1, and e2-TRPV1. (f) Capsaicin dose-response profiles of wt TRPV1 and e2-TRPV1. n = 5. Squares are mean ± s.d. Charged residues at the S5-pore loop impact pH sensitivity of e1- and e2-TRPV1 constructs. (g) Heat-evoked currents of e2-TRPV1 extracted from voltage-clamp ramp protocols at +80 mV. (h) Representative current-voltage relationships of e2-TRPV1 challenged with pH 5. (i) pH dose–response profiles (+80 mV) of wt TRPV1, e1-TRPV1, and e2-TRPV1. I/I max: current at each pH/current at pH 5.5. n = 4–6. Squares are mean ± s.d. Kruskall-Wallis and Dunn’s multiple comparisons tests were used in (e). *p < 0.05.
	Figure 5. Functional analysis of eTRPV1 single-cysteine mutants evaluated in HEK293 cells by Ca2+ imaging and in Xenopus oocytes by TEVC. (a) One subunit (shown for clarity) of TRPV1 tetramer structure highlighting residues (colored spheres) substituted for single-cysteines. (b) HEK293 cell-expressing mutants (loaded with the Ca2+-sensitive Fluo-4-AM; 1 µM) were analyzed for capsaicin (10 μM)-evoked responses using fluorescence imaging (red denotes non-functional mutants in Ca2+ imaging). Right inset: representative capsaicin-evoked responses of control and wt TRPV1 transfected cells. (c) Current-voltage relationships of individual eTRPV1 single-cysteine mutants (I672C and G683C) challenged with pH 5 evaluated by TEVC. Background currents (bkgrd). Bars are mean ± s.d.
	Figure 6. Purification of functional eTRPV1 single-cysteine mutants. (a) One subunit (shown for clarity) of TRPV1 tetramer structure highlighting single-cysteine residues (red spheres) introduced along the channel sequence and selected for purification. (b) Current-voltage relationships of eTRPV1 and individual eTRPV1 single-cysteine mutants (E651C, M677C, I679C, A690C, A702C, and the native Cys715) challenged with pH 5. (c) Size-exclusion chromatography profiles of DDM-solubilized eTRPV1 and eTRPV1 single-cysteine mutants after expression and purification from Sf9 cells (each color represents a different mutant). (d) Current-voltage relationships determined by TEVC from Xenopus oocytes microinjected with A702C-spin labeled-containing proteoliposomes challenged with pH 5 (red) and blocked by capsazepine (CPZ, blue: 40 µM). Background currents (bkgrd).
	Figure 7. Distances and mobilities of spin-labeled eTRPV1 mutants in detergent. (a) Two subunits (shown for clarity) of TRPV1 tetramer structure highlighting the amino acid residues (red spheres) probed through site-directed spin labeling DEER and EPR spectroscopies. (b) DEER echo (left) and distance distribution (right) of spin-labeled mutant E651C. A sum of Gaussians was fit to the DEER data. (c) First-derivative of continuous-wave EPR spectra of spin-labeled cysteine mutants in DDM-solution. ΔHo −1 denotes the magnitude of the mobility parameter. The blue and red dotted lines highlight the immobile and mobile components of the spin-label spectra, respectively. EPR spectra were normalized to the total number of spin labels.
	Figure 8. EPR spectra and mobilities of eTRPV1 spin-labeled cysteine mutants reconstituted in asolectin liposomes. Spectra were obtained at pH 7.4 (closed state). ΔHo −1 denotes the magnitude of the mobility parameter. The blue and red arrows highlight the immobile and mobile components of the spin-label spectra, respectively. EPR spectra were normalized to the total number of spin labels.

References [+] :

Bae, Structural insights into the mechanism of activation of the TRPV1 channel by a membrane-bound tarantula toxin. 2016, Pubmed