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The pain receptor TRPV1 displays agonist-dependent activation stoichiometry.

Hazan A, Kumar R, Matzner H, Priel A - Sci Rep (2015)

Bottom Line: Although its physiological role as a chemosensor has been described in detail, the stoichiometry of TRPV1 activation by its different ligands remains unknown.We show that, while a single capsaicin-bound subunit was sufficient to achieve a maximal open-channel lifetime, all four proton-binding sites were required.Thus, our results demonstrate a distinct stoichiometry of TRPV1 activation through two of its different agonist-binding domains.

View Article: PubMed Central - PubMed

Affiliation: The Institute for Drug Research (IDR), School of Pharmacy, Faculty of Medicine, The Hebrew University of Jerusalem, Jerusalem 91120, Israel.

ABSTRACT
The receptor channel TRPV1 (Transient Receptor Potential Vanilloid 1) is expressed by primary afferent sensory neurons of the pain pathway, where it functions as a sensor of noxious heat and various chemicals, including eicosanoids, capsaicin, protons and peptide toxins. Comprised of four identical subunits that organize into a non-selective cationic permeable channel, this receptor has a variety of binding sites responsible for detecting their respective agonists. Although its physiological role as a chemosensor has been described in detail, the stoichiometry of TRPV1 activation by its different ligands remains unknown. Here, we combined the use of concatemeric constructs harboring mutated binding sites with patch-clamp recordings in order to determine the stoichiometry for TRPV1 activation through the vanilloid binding site and the outer-pore domain by capsaicin and protons, respectively. We show that, while a single capsaicin-bound subunit was sufficient to achieve a maximal open-channel lifetime, all four proton-binding sites were required. Thus, our results demonstrate a distinct stoichiometry of TRPV1 activation through two of its different agonist-binding domains.

No MeSH data available.


Related in: MedlinePlus

TRPV1 tetrameric concatemer maintains sensitivity to capsaicin and protons.Normalized concentration-response relationships for capsaicin (left) and protons (right) of rTRPV1 (wt; empty circles) and the tetrameric concatemeric construct (4wt; full circles) transiently expressed in HEK293 cells. Each point represents the average (±SEM) response of 6–12 cells. Solid lines (black for both agonists of wt; orange for capsaicin, and cyan for protons of 4wt) are fit to the Hill equation (see Eq. (2)) with EC50 and nH for capsaicin of 0.24 ± 0.02 μM and 1.3 for the wt receptor and 0.15 ± 0.02 μM and 1.4 for the 4wt construct, respectively. The EC50 and nH for protons are pH 5.27 ± 0.28 and 1.5 for the wt receptor and pH 5.23 ± 0.24 and 1.8 for the 4wt construct, respectively.
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f3: TRPV1 tetrameric concatemer maintains sensitivity to capsaicin and protons.Normalized concentration-response relationships for capsaicin (left) and protons (right) of rTRPV1 (wt; empty circles) and the tetrameric concatemeric construct (4wt; full circles) transiently expressed in HEK293 cells. Each point represents the average (±SEM) response of 6–12 cells. Solid lines (black for both agonists of wt; orange for capsaicin, and cyan for protons of 4wt) are fit to the Hill equation (see Eq. (2)) with EC50 and nH for capsaicin of 0.24 ± 0.02 μM and 1.3 for the wt receptor and 0.15 ± 0.02 μM and 1.4 for the 4wt construct, respectively. The EC50 and nH for protons are pH 5.27 ± 0.28 and 1.5 for the wt receptor and pH 5.23 ± 0.24 and 1.8 for the 4wt construct, respectively.

Mentions: In order to verify that our tetrameric construct maintained the sensitivity of the receptor channel to its different activators, we tested capsaicin and protons concentration-dependent activation of wt and 4wt rTRPV1 (Fig. 3). Using the whole-cell configuration of the patch clamp technique at a holding potential of −40 mV, each cell was exposed to increasing concentrations of the activator (capsaicin or protons) with a 20–30 s wash between each application, when each activator was analyzed separately. Based on the concentration-response relationship fitted by the Hill equation for the effect of capsaicin and protons (Fig. 3), the estimated EC50 and Hill coefficient (n) for the 4wt TRPV1 construct were comparable to these estimated for the wt construct (for capsaicin: EC50 = 0.24 ± 0.02 μM and nH = 1.3 for wt and EC50 = 0.15 ± 0.02 μM and nH = 1.4 for 4wt; For protons: EC50 = pH 5.27 ± 0.28 and nH = 1.5 for wt and EC50 = pH 5.23 ± 0.24 and nH = 1.8 for 4wt; Fig. 3). Therefore, our results indicate that the tetrameric concatemeric construct mimics the activation pattern and agonist sensitivity of TRPV1 to its different activators.


The pain receptor TRPV1 displays agonist-dependent activation stoichiometry.

Hazan A, Kumar R, Matzner H, Priel A - Sci Rep (2015)

TRPV1 tetrameric concatemer maintains sensitivity to capsaicin and protons.Normalized concentration-response relationships for capsaicin (left) and protons (right) of rTRPV1 (wt; empty circles) and the tetrameric concatemeric construct (4wt; full circles) transiently expressed in HEK293 cells. Each point represents the average (±SEM) response of 6–12 cells. Solid lines (black for both agonists of wt; orange for capsaicin, and cyan for protons of 4wt) are fit to the Hill equation (see Eq. (2)) with EC50 and nH for capsaicin of 0.24 ± 0.02 μM and 1.3 for the wt receptor and 0.15 ± 0.02 μM and 1.4 for the 4wt construct, respectively. The EC50 and nH for protons are pH 5.27 ± 0.28 and 1.5 for the wt receptor and pH 5.23 ± 0.24 and 1.8 for the 4wt construct, respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4508619&req=5

f3: TRPV1 tetrameric concatemer maintains sensitivity to capsaicin and protons.Normalized concentration-response relationships for capsaicin (left) and protons (right) of rTRPV1 (wt; empty circles) and the tetrameric concatemeric construct (4wt; full circles) transiently expressed in HEK293 cells. Each point represents the average (±SEM) response of 6–12 cells. Solid lines (black for both agonists of wt; orange for capsaicin, and cyan for protons of 4wt) are fit to the Hill equation (see Eq. (2)) with EC50 and nH for capsaicin of 0.24 ± 0.02 μM and 1.3 for the wt receptor and 0.15 ± 0.02 μM and 1.4 for the 4wt construct, respectively. The EC50 and nH for protons are pH 5.27 ± 0.28 and 1.5 for the wt receptor and pH 5.23 ± 0.24 and 1.8 for the 4wt construct, respectively.
Mentions: In order to verify that our tetrameric construct maintained the sensitivity of the receptor channel to its different activators, we tested capsaicin and protons concentration-dependent activation of wt and 4wt rTRPV1 (Fig. 3). Using the whole-cell configuration of the patch clamp technique at a holding potential of −40 mV, each cell was exposed to increasing concentrations of the activator (capsaicin or protons) with a 20–30 s wash between each application, when each activator was analyzed separately. Based on the concentration-response relationship fitted by the Hill equation for the effect of capsaicin and protons (Fig. 3), the estimated EC50 and Hill coefficient (n) for the 4wt TRPV1 construct were comparable to these estimated for the wt construct (for capsaicin: EC50 = 0.24 ± 0.02 μM and nH = 1.3 for wt and EC50 = 0.15 ± 0.02 μM and nH = 1.4 for 4wt; For protons: EC50 = pH 5.27 ± 0.28 and nH = 1.5 for wt and EC50 = pH 5.23 ± 0.24 and nH = 1.8 for 4wt; Fig. 3). Therefore, our results indicate that the tetrameric concatemeric construct mimics the activation pattern and agonist sensitivity of TRPV1 to its different activators.

Bottom Line: Although its physiological role as a chemosensor has been described in detail, the stoichiometry of TRPV1 activation by its different ligands remains unknown.We show that, while a single capsaicin-bound subunit was sufficient to achieve a maximal open-channel lifetime, all four proton-binding sites were required.Thus, our results demonstrate a distinct stoichiometry of TRPV1 activation through two of its different agonist-binding domains.

View Article: PubMed Central - PubMed

Affiliation: The Institute for Drug Research (IDR), School of Pharmacy, Faculty of Medicine, The Hebrew University of Jerusalem, Jerusalem 91120, Israel.

ABSTRACT
The receptor channel TRPV1 (Transient Receptor Potential Vanilloid 1) is expressed by primary afferent sensory neurons of the pain pathway, where it functions as a sensor of noxious heat and various chemicals, including eicosanoids, capsaicin, protons and peptide toxins. Comprised of four identical subunits that organize into a non-selective cationic permeable channel, this receptor has a variety of binding sites responsible for detecting their respective agonists. Although its physiological role as a chemosensor has been described in detail, the stoichiometry of TRPV1 activation by its different ligands remains unknown. Here, we combined the use of concatemeric constructs harboring mutated binding sites with patch-clamp recordings in order to determine the stoichiometry for TRPV1 activation through the vanilloid binding site and the outer-pore domain by capsaicin and protons, respectively. We show that, while a single capsaicin-bound subunit was sufficient to achieve a maximal open-channel lifetime, all four proton-binding sites were required. Thus, our results demonstrate a distinct stoichiometry of TRPV1 activation through two of its different agonist-binding domains.

No MeSH data available.


Related in: MedlinePlus