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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 maximal activation by protons requires all four proton-binding subunits.(a) Illustration of the different tetrameric concatemeric constructs used to study the stoichiometry for TRPV1 activation by protons. Black squares represent proton-binding intact subunits (wt) and grey squares represent subunits mutated in their proton-binding site in both positions E600Q and E648A (qa). (b) Whole-cell current traces (blue) in response to protons (pH 4; grey bar) of HEK293T cells transiently expressing the different mutant protons-binding concatemeric constructs, as indicated. Similar TRPV1 expression levels were estimated from the current evoked by saturating capsaicin concentration. Inset: representative whole-cell current traces (orange) in response to capsaicin (1 μM; light grey bar) of the 4wt and 4qa constructs. Note that the capsaicin response was unaffected by the mutation in the proton-binding sites. Holding potential of −40 mV. (c) Bar diagram representing the average ratio (±SEM) between protons (pH 4) - and capsaicin (at saturating concentration; 1 μM) - evoked amplitudes, normalized to the ratio obtained for the 4wt construct. Each bar represents 12–14 HEK293T cells transiently expressing the indicated concatemeric constructs. The statistical significance between the 4wt and the different proton-binding mutant constructs was determined with unpaired Student’s t test, where * represents P ≤ 0.05, **represents P ≤ 0.01 and ***represents P ≤ 0.001. Note a reduction in protons to capsaicin ratio with the inclusion of a single subunit mutated in its proton-binding sites (3wt/qa). (d) Normalized concentration-response relationships for protons of the 4wt and 3wt/qa concatemeric constructs. Each point represents the average (±SEM) response of 5–8 HEK293T cells transiently expressing the indicated concatemeric constructs. Solid lines are fit to the Hill equation (see Eq. (2)): 4wt (full circles, cyan line; nH = 1.8; EC50 = pH 5.23 ± 0.24), 3wt/qa (empty circle, blue line; nH = 1.6; EC50 = pH 5.20 ± 0.32). Holding potential of −40 mV.
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f7: TRPV1 maximal activation by protons requires all four proton-binding subunits.(a) Illustration of the different tetrameric concatemeric constructs used to study the stoichiometry for TRPV1 activation by protons. Black squares represent proton-binding intact subunits (wt) and grey squares represent subunits mutated in their proton-binding site in both positions E600Q and E648A (qa). (b) Whole-cell current traces (blue) in response to protons (pH 4; grey bar) of HEK293T cells transiently expressing the different mutant protons-binding concatemeric constructs, as indicated. Similar TRPV1 expression levels were estimated from the current evoked by saturating capsaicin concentration. Inset: representative whole-cell current traces (orange) in response to capsaicin (1 μM; light grey bar) of the 4wt and 4qa constructs. Note that the capsaicin response was unaffected by the mutation in the proton-binding sites. Holding potential of −40 mV. (c) Bar diagram representing the average ratio (±SEM) between protons (pH 4) - and capsaicin (at saturating concentration; 1 μM) - evoked amplitudes, normalized to the ratio obtained for the 4wt construct. Each bar represents 12–14 HEK293T cells transiently expressing the indicated concatemeric constructs. The statistical significance between the 4wt and the different proton-binding mutant constructs was determined with unpaired Student’s t test, where * represents P ≤ 0.05, **represents P ≤ 0.01 and ***represents P ≤ 0.001. Note a reduction in protons to capsaicin ratio with the inclusion of a single subunit mutated in its proton-binding sites (3wt/qa). (d) Normalized concentration-response relationships for protons of the 4wt and 3wt/qa concatemeric constructs. Each point represents the average (±SEM) response of 5–8 HEK293T cells transiently expressing the indicated concatemeric constructs. Solid lines are fit to the Hill equation (see Eq. (2)): 4wt (full circles, cyan line; nH = 1.8; EC50 = pH 5.23 ± 0.24), 3wt/qa (empty circle, blue line; nH = 1.6; EC50 = pH 5.20 ± 0.32). Holding potential of −40 mV.

Mentions: To determine TRPV1 activation mechanism through an alternative ligand binding site, we analyzed the subunit stoichiometry of proton activation. The E600Q (EQ) and E648A (EA) substitutions located on the outer-pore domain of rTRPV1 substantially decrease its activation by low pH8404142. In order to assess the proton-mediated TRPV1 activation mechanism, we generated a series of concatemeric constructs containing subunits that harbor either an intact or a mutated (containing the E600Q/E648A dual substitutions; qa) protons binding site (Fig. 7a). We recorded the currents evoked by protons and capsaicin from HEK293T cells transiently expressing the different constructs using the whole-cell configuration of the patch clamp technique (Fig. 7b). The analyzed cell was continuously perfused with the standard extracellular solution. Concurrently, the cell was exposed initially to protons (pH 4) for 15 s, followed by minute wash, and finally capsaicin (1 μM). A high proton concentration (pH 4) was used in order to saturate all binding sites, while higher proton concentrations (pH < 4) were excluded from analysis due to patch instability and leak currents. Throughout all the experiments, the extracellular solution and bath solution were supplemented with 50 μM Amiloride to block ASIC1a activity, which is endogenously expressed by HEK293T cells124243. Membrane currents in response to protons (pH 4) were observed in concatemeric constructs having at least one proton-binding subunit (4wt, wt/3qa, 2wt/2qa and 3wt/qa; Fig. 7b). Representative traces shown in Fig. 7b were extracted from cells with comparable expression level, as estimated by the current amplitude for saturating capsaicin concentration (1 μM, 1.5-1.8 nA at −40 mV; see inset). The ratio between proton (pH 4) - and capsaicin (1 μM) - mediated activation of the different constructs was normalized to the protons/capsaicin ratio of the 4wt construct (Fig. 7c). In contrast to the VBS, elimination of the proton-binding site in a single subunit (3wt/qa construct) led to a dramatic reduction in proton-mediated TRPV1 activation (Figs 4 and 7). Moreover, a receptor lacking three of its four protons-putative subunits (wt/3qa) nearly lost its sensitivity to protons (Fig. 7b,c).


The pain receptor TRPV1 displays agonist-dependent activation stoichiometry.

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

TRPV1 maximal activation by protons requires all four proton-binding subunits.(a) Illustration of the different tetrameric concatemeric constructs used to study the stoichiometry for TRPV1 activation by protons. Black squares represent proton-binding intact subunits (wt) and grey squares represent subunits mutated in their proton-binding site in both positions E600Q and E648A (qa). (b) Whole-cell current traces (blue) in response to protons (pH 4; grey bar) of HEK293T cells transiently expressing the different mutant protons-binding concatemeric constructs, as indicated. Similar TRPV1 expression levels were estimated from the current evoked by saturating capsaicin concentration. Inset: representative whole-cell current traces (orange) in response to capsaicin (1 μM; light grey bar) of the 4wt and 4qa constructs. Note that the capsaicin response was unaffected by the mutation in the proton-binding sites. Holding potential of −40 mV. (c) Bar diagram representing the average ratio (±SEM) between protons (pH 4) - and capsaicin (at saturating concentration; 1 μM) - evoked amplitudes, normalized to the ratio obtained for the 4wt construct. Each bar represents 12–14 HEK293T cells transiently expressing the indicated concatemeric constructs. The statistical significance between the 4wt and the different proton-binding mutant constructs was determined with unpaired Student’s t test, where * represents P ≤ 0.05, **represents P ≤ 0.01 and ***represents P ≤ 0.001. Note a reduction in protons to capsaicin ratio with the inclusion of a single subunit mutated in its proton-binding sites (3wt/qa). (d) Normalized concentration-response relationships for protons of the 4wt and 3wt/qa concatemeric constructs. Each point represents the average (±SEM) response of 5–8 HEK293T cells transiently expressing the indicated concatemeric constructs. Solid lines are fit to the Hill equation (see Eq. (2)): 4wt (full circles, cyan line; nH = 1.8; EC50 = pH 5.23 ± 0.24), 3wt/qa (empty circle, blue line; nH = 1.6; EC50 = pH 5.20 ± 0.32). Holding potential of −40 mV.
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f7: TRPV1 maximal activation by protons requires all four proton-binding subunits.(a) Illustration of the different tetrameric concatemeric constructs used to study the stoichiometry for TRPV1 activation by protons. Black squares represent proton-binding intact subunits (wt) and grey squares represent subunits mutated in their proton-binding site in both positions E600Q and E648A (qa). (b) Whole-cell current traces (blue) in response to protons (pH 4; grey bar) of HEK293T cells transiently expressing the different mutant protons-binding concatemeric constructs, as indicated. Similar TRPV1 expression levels were estimated from the current evoked by saturating capsaicin concentration. Inset: representative whole-cell current traces (orange) in response to capsaicin (1 μM; light grey bar) of the 4wt and 4qa constructs. Note that the capsaicin response was unaffected by the mutation in the proton-binding sites. Holding potential of −40 mV. (c) Bar diagram representing the average ratio (±SEM) between protons (pH 4) - and capsaicin (at saturating concentration; 1 μM) - evoked amplitudes, normalized to the ratio obtained for the 4wt construct. Each bar represents 12–14 HEK293T cells transiently expressing the indicated concatemeric constructs. The statistical significance between the 4wt and the different proton-binding mutant constructs was determined with unpaired Student’s t test, where * represents P ≤ 0.05, **represents P ≤ 0.01 and ***represents P ≤ 0.001. Note a reduction in protons to capsaicin ratio with the inclusion of a single subunit mutated in its proton-binding sites (3wt/qa). (d) Normalized concentration-response relationships for protons of the 4wt and 3wt/qa concatemeric constructs. Each point represents the average (±SEM) response of 5–8 HEK293T cells transiently expressing the indicated concatemeric constructs. Solid lines are fit to the Hill equation (see Eq. (2)): 4wt (full circles, cyan line; nH = 1.8; EC50 = pH 5.23 ± 0.24), 3wt/qa (empty circle, blue line; nH = 1.6; EC50 = pH 5.20 ± 0.32). Holding potential of −40 mV.
Mentions: To determine TRPV1 activation mechanism through an alternative ligand binding site, we analyzed the subunit stoichiometry of proton activation. The E600Q (EQ) and E648A (EA) substitutions located on the outer-pore domain of rTRPV1 substantially decrease its activation by low pH8404142. In order to assess the proton-mediated TRPV1 activation mechanism, we generated a series of concatemeric constructs containing subunits that harbor either an intact or a mutated (containing the E600Q/E648A dual substitutions; qa) protons binding site (Fig. 7a). We recorded the currents evoked by protons and capsaicin from HEK293T cells transiently expressing the different constructs using the whole-cell configuration of the patch clamp technique (Fig. 7b). The analyzed cell was continuously perfused with the standard extracellular solution. Concurrently, the cell was exposed initially to protons (pH 4) for 15 s, followed by minute wash, and finally capsaicin (1 μM). A high proton concentration (pH 4) was used in order to saturate all binding sites, while higher proton concentrations (pH < 4) were excluded from analysis due to patch instability and leak currents. Throughout all the experiments, the extracellular solution and bath solution were supplemented with 50 μM Amiloride to block ASIC1a activity, which is endogenously expressed by HEK293T cells124243. Membrane currents in response to protons (pH 4) were observed in concatemeric constructs having at least one proton-binding subunit (4wt, wt/3qa, 2wt/2qa and 3wt/qa; Fig. 7b). Representative traces shown in Fig. 7b were extracted from cells with comparable expression level, as estimated by the current amplitude for saturating capsaicin concentration (1 μM, 1.5-1.8 nA at −40 mV; see inset). The ratio between proton (pH 4) - and capsaicin (1 μM) - mediated activation of the different constructs was normalized to the protons/capsaicin ratio of the 4wt construct (Fig. 7c). In contrast to the VBS, elimination of the proton-binding site in a single subunit (3wt/qa construct) led to a dramatic reduction in proton-mediated TRPV1 activation (Figs 4 and 7). Moreover, a receptor lacking three of its four protons-putative subunits (wt/3qa) nearly lost its sensitivity to protons (Fig. 7b,c).

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