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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

A single VBS-containing subunit is sufficient to evoke capsaicin sensitive current.(a) Schematic representation of the different tetrameric concatemeric constructs used to study the stoichiometry for TRPV1 activation by capsaicin. Black squares represent wt subunits while empty circles represent subunits with an Y511A mutated VBS (ya). (b, c) Current-voltage relationship traces in HEK293 cells transiently expressing the 4ya (b) and wt/3ya (c) constructs in response to capsaicin (CAP; 100 μM; orange line) and protons (H+; pH 5.5; cyan line). Currents were recorded using whole-cell patch-clamp recording (in 1 s−1 voltage ramps between −80 and +80 mV). Mutations in all VBS nearly eliminated the capsaicin response, while the proton response was intact (b). A single intact VBS was sufficient to produce a robust capsaicin response (c). (d) Normalized concentration-response relationships for capsaicin of the different concatemers. Each point represents the average (±SEM) response of 10–14 HEK293 cells transiently expressing the respective construct. Solid lines are fit to the Hill equation (see Eq. (2)): 4wt (full circles, dark red line; nH = 1.4; EC50 = 0.15 ± 0.02 μM), 3wt/ya (empty squares, red line; nH = 1.1; EC50 = 0.42 ± 0.07 μM), 2wt/2ya (empty triangle; orange line; nH = 1.2; EC50 = 1.09 ± 0.08 μM) and wt/3ya (full diamonds, yellow line; nH = 1.1; EC50 = 3.10 ± 0.47 μM). Reduction in the number of subunits containing an intact VBS leads to a shift in the affinity of capsaicin.
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f4: A single VBS-containing subunit is sufficient to evoke capsaicin sensitive current.(a) Schematic representation of the different tetrameric concatemeric constructs used to study the stoichiometry for TRPV1 activation by capsaicin. Black squares represent wt subunits while empty circles represent subunits with an Y511A mutated VBS (ya). (b, c) Current-voltage relationship traces in HEK293 cells transiently expressing the 4ya (b) and wt/3ya (c) constructs in response to capsaicin (CAP; 100 μM; orange line) and protons (H+; pH 5.5; cyan line). Currents were recorded using whole-cell patch-clamp recording (in 1 s−1 voltage ramps between −80 and +80 mV). Mutations in all VBS nearly eliminated the capsaicin response, while the proton response was intact (b). A single intact VBS was sufficient to produce a robust capsaicin response (c). (d) Normalized concentration-response relationships for capsaicin of the different concatemers. Each point represents the average (±SEM) response of 10–14 HEK293 cells transiently expressing the respective construct. Solid lines are fit to the Hill equation (see Eq. (2)): 4wt (full circles, dark red line; nH = 1.4; EC50 = 0.15 ± 0.02 μM), 3wt/ya (empty squares, red line; nH = 1.1; EC50 = 0.42 ± 0.07 μM), 2wt/2ya (empty triangle; orange line; nH = 1.2; EC50 = 1.09 ± 0.08 μM) and wt/3ya (full diamonds, yellow line; nH = 1.1; EC50 = 3.10 ± 0.47 μM). Reduction in the number of subunits containing an intact VBS leads to a shift in the affinity of capsaicin.

Mentions: Activation of TRPV1 by exo-vanilloids (such as capsaicin) and endo-vanilloids (such as 12-(S)- and 15-(S)-hydroperoxyeicosatetraenoic acid (12- and 15-HPETE)) has been studied extensively in order to better understand pain physiology and behavior52530; however, the stoichiometry and allosteric regulatory mechanisms through which they activate TRPV1 remain unknown. The Y511A (YA) substitution, located in the vanilloid binding site (VBS), was shown by mutagenesis and biochemical studies to substantially decrease the sensitivity of TRPV1 to capsaicin9313233. In order to determine the number of subunits necessary for capsaicin-mediated TRPV1 activation, we generated a series of concatemeric constructs composed of subunits that harbor either a wt (wt) or a mutated VBS (containing the YA substitution; ya) in various subunit combinations (Fig. 4a). We transiently expressed different concatemeric constructs containing various ratios of wt and mutated subunits in HEK293T cells and recorded the currents evoked by capsaicin and protons using whole-cell recordings. As expected, the 4ya construct (in which VBS is mutated in all subunits) was insensitive to capsaicin, even when the agonist was applied at a maximal concentration (100 μM due to solubility; three orders of magnitude above the EC50 of the wt and 4wt, Fig. 3), while maintaining its sensitivity to protons (Fig. 4b). Interestingly, the inclusion of a single wt subunit (wt/3ya) was sufficient to induce a robust outward-rectifying current in response to a high capsaicin concentration (100 μM; Fig. 4c). Therefore, our results indicate that a single VBS subunit is sufficient to activate the channel.


The pain receptor TRPV1 displays agonist-dependent activation stoichiometry.

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

A single VBS-containing subunit is sufficient to evoke capsaicin sensitive current.(a) Schematic representation of the different tetrameric concatemeric constructs used to study the stoichiometry for TRPV1 activation by capsaicin. Black squares represent wt subunits while empty circles represent subunits with an Y511A mutated VBS (ya). (b, c) Current-voltage relationship traces in HEK293 cells transiently expressing the 4ya (b) and wt/3ya (c) constructs in response to capsaicin (CAP; 100 μM; orange line) and protons (H+; pH 5.5; cyan line). Currents were recorded using whole-cell patch-clamp recording (in 1 s−1 voltage ramps between −80 and +80 mV). Mutations in all VBS nearly eliminated the capsaicin response, while the proton response was intact (b). A single intact VBS was sufficient to produce a robust capsaicin response (c). (d) Normalized concentration-response relationships for capsaicin of the different concatemers. Each point represents the average (±SEM) response of 10–14 HEK293 cells transiently expressing the respective construct. Solid lines are fit to the Hill equation (see Eq. (2)): 4wt (full circles, dark red line; nH = 1.4; EC50 = 0.15 ± 0.02 μM), 3wt/ya (empty squares, red line; nH = 1.1; EC50 = 0.42 ± 0.07 μM), 2wt/2ya (empty triangle; orange line; nH = 1.2; EC50 = 1.09 ± 0.08 μM) and wt/3ya (full diamonds, yellow line; nH = 1.1; EC50 = 3.10 ± 0.47 μM). Reduction in the number of subunits containing an intact VBS leads to a shift in the affinity of capsaicin.
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f4: A single VBS-containing subunit is sufficient to evoke capsaicin sensitive current.(a) Schematic representation of the different tetrameric concatemeric constructs used to study the stoichiometry for TRPV1 activation by capsaicin. Black squares represent wt subunits while empty circles represent subunits with an Y511A mutated VBS (ya). (b, c) Current-voltage relationship traces in HEK293 cells transiently expressing the 4ya (b) and wt/3ya (c) constructs in response to capsaicin (CAP; 100 μM; orange line) and protons (H+; pH 5.5; cyan line). Currents were recorded using whole-cell patch-clamp recording (in 1 s−1 voltage ramps between −80 and +80 mV). Mutations in all VBS nearly eliminated the capsaicin response, while the proton response was intact (b). A single intact VBS was sufficient to produce a robust capsaicin response (c). (d) Normalized concentration-response relationships for capsaicin of the different concatemers. Each point represents the average (±SEM) response of 10–14 HEK293 cells transiently expressing the respective construct. Solid lines are fit to the Hill equation (see Eq. (2)): 4wt (full circles, dark red line; nH = 1.4; EC50 = 0.15 ± 0.02 μM), 3wt/ya (empty squares, red line; nH = 1.1; EC50 = 0.42 ± 0.07 μM), 2wt/2ya (empty triangle; orange line; nH = 1.2; EC50 = 1.09 ± 0.08 μM) and wt/3ya (full diamonds, yellow line; nH = 1.1; EC50 = 3.10 ± 0.47 μM). Reduction in the number of subunits containing an intact VBS leads to a shift in the affinity of capsaicin.
Mentions: Activation of TRPV1 by exo-vanilloids (such as capsaicin) and endo-vanilloids (such as 12-(S)- and 15-(S)-hydroperoxyeicosatetraenoic acid (12- and 15-HPETE)) has been studied extensively in order to better understand pain physiology and behavior52530; however, the stoichiometry and allosteric regulatory mechanisms through which they activate TRPV1 remain unknown. The Y511A (YA) substitution, located in the vanilloid binding site (VBS), was shown by mutagenesis and biochemical studies to substantially decrease the sensitivity of TRPV1 to capsaicin9313233. In order to determine the number of subunits necessary for capsaicin-mediated TRPV1 activation, we generated a series of concatemeric constructs composed of subunits that harbor either a wt (wt) or a mutated VBS (containing the YA substitution; ya) in various subunit combinations (Fig. 4a). We transiently expressed different concatemeric constructs containing various ratios of wt and mutated subunits in HEK293T cells and recorded the currents evoked by capsaicin and protons using whole-cell recordings. As expected, the 4ya construct (in which VBS is mutated in all subunits) was insensitive to capsaicin, even when the agonist was applied at a maximal concentration (100 μM due to solubility; three orders of magnitude above the EC50 of the wt and 4wt, Fig. 3), while maintaining its sensitivity to protons (Fig. 4b). Interestingly, the inclusion of a single wt subunit (wt/3ya) was sufficient to induce a robust outward-rectifying current in response to a high capsaicin concentration (100 μM; Fig. 4c). Therefore, our results indicate that a single VBS subunit is sufficient to activate the channel.

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