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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 self-assembles into a functional receptor channel.(a) Schematic illustration of the different tandem constructs used to study the assembly of rTRPV1 concatemers. Black squares represent wild type rTRPV1 subunit (wt) and red squares represent dominate-negative subunits (DN) mutated in positions NML676FAP. (b) Bar diagram represents the average (±SEM) amplitudes of a whole-cell, capsaicin-evoked (1 μM) currents from Xenopus laevis oocytes expressing the different tandem constructs either alone (black bars), or together with a DN construct (red bars; DN). Holding potential −60 mV. Each bar represents readings from 6–15 oocytes. The statistical significance between currents evoked with or without DN was determined using unpaired Student’s t tests, where **represents P ≤ 0.01, ***represents P ≤ 0.001. Note that no statistical significant difference in evoked currents was found between cells expressing 4wt with or without DN. (c) Western-blot analysis of HEK293T cells transiently expressing a single subunit (wt) or a tetrameric tandem (4wt) constructs, using an anti-rat TRPV1 antibody (α rTRPV1). Note that, while the expression of a single subunit wt construct yielded a band corresponded to ~90 kDa, the only specific band observed in cells expressing the 4wt construct corresponded to a ~400 kDa size (indicated with arrows).
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f2: TRPV1 tetrameric concatemer self-assembles into a functional receptor channel.(a) Schematic illustration of the different tandem constructs used to study the assembly of rTRPV1 concatemers. Black squares represent wild type rTRPV1 subunit (wt) and red squares represent dominate-negative subunits (DN) mutated in positions NML676FAP. (b) Bar diagram represents the average (±SEM) amplitudes of a whole-cell, capsaicin-evoked (1 μM) currents from Xenopus laevis oocytes expressing the different tandem constructs either alone (black bars), or together with a DN construct (red bars; DN). Holding potential −60 mV. Each bar represents readings from 6–15 oocytes. The statistical significance between currents evoked with or without DN was determined using unpaired Student’s t tests, where **represents P ≤ 0.01, ***represents P ≤ 0.001. Note that no statistical significant difference in evoked currents was found between cells expressing 4wt with or without DN. (c) Western-blot analysis of HEK293T cells transiently expressing a single subunit (wt) or a tetrameric tandem (4wt) constructs, using an anti-rat TRPV1 antibody (α rTRPV1). Note that, while the expression of a single subunit wt construct yielded a band corresponded to ~90 kDa, the only specific band observed in cells expressing the 4wt construct corresponded to a ~400 kDa size (indicated with arrows).

Mentions: To verify the functionality of the tetrameric TRPV1 concatemer, we analyzed its response to its known agonists: capsaicin, protons and heat (Fig. 1)1925. HEK293T cells transiently expressing the native rTRPV1 (wt) or tetrameric rTRPV1 concatemeric construct (four subunits, “4wt”) were continuously perfused with the standard extracellular solution when protons (pH 5.5) were applied for 15 s, followed by a minute wash, and finally capsaicin (1 μM). To avoid contamination and due to its hydrophobic nature, applications of high capsaicin concentrations (>0.2 μM) were restricted to the end of the experiments throughout the study. The current-voltage relationship was determined using whole-cell patch-clamp recordings. As shown in Fig. 1b, both extracellular protons (H+; cyan line) and capsaicin (CAP; orange line) elicited robust, outwardly rectifying currents in cells expressing either rTRPV1 (wt) or tetrameric rTRPV1 concatemer (4wt) construct. We further determined the current-voltage relationships in response to heat (temperature range of 22°C–50°C) or capsaicin by applying the two electrodes voltage clamp (TEVC) technique in Xenopus laevis oocytes expressing wt or 4wt concatemer (Figs 1c and 2; average currents at +80 mV). In both heterologous systems expressing the 4wt concatemeric receptor, the current amplitude and kinetics were comparable to those previously reported for wt rTRPV1 (average current amplitude (±SEM): HEK293T: wt ICAP+80mV = 8.3 ± 1.2 nA and 4wt ICAP+80mV = 7.8 ± 1.8 nA; oocytes: wt ICAP+80mV = 5.6 ± 0.3 μA and 4wt ICAP+80mV = 4.7 ± 0.2 μA; average temperature threshold (±SEM): wt: 42 ± 1.2 °C; 4wt: 43 ± 1.6 °C, and average temperature sensitivity (±SEM): wt: Q10 = 27 ± 3; 4wt: Q10 = 25 ± 2; Figs 1 and 2)1012262728.


The pain receptor TRPV1 displays agonist-dependent activation stoichiometry.

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

TRPV1 tetrameric concatemer self-assembles into a functional receptor channel.(a) Schematic illustration of the different tandem constructs used to study the assembly of rTRPV1 concatemers. Black squares represent wild type rTRPV1 subunit (wt) and red squares represent dominate-negative subunits (DN) mutated in positions NML676FAP. (b) Bar diagram represents the average (±SEM) amplitudes of a whole-cell, capsaicin-evoked (1 μM) currents from Xenopus laevis oocytes expressing the different tandem constructs either alone (black bars), or together with a DN construct (red bars; DN). Holding potential −60 mV. Each bar represents readings from 6–15 oocytes. The statistical significance between currents evoked with or without DN was determined using unpaired Student’s t tests, where **represents P ≤ 0.01, ***represents P ≤ 0.001. Note that no statistical significant difference in evoked currents was found between cells expressing 4wt with or without DN. (c) Western-blot analysis of HEK293T cells transiently expressing a single subunit (wt) or a tetrameric tandem (4wt) constructs, using an anti-rat TRPV1 antibody (α rTRPV1). Note that, while the expression of a single subunit wt construct yielded a band corresponded to ~90 kDa, the only specific band observed in cells expressing the 4wt construct corresponded to a ~400 kDa size (indicated with arrows).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2: TRPV1 tetrameric concatemer self-assembles into a functional receptor channel.(a) Schematic illustration of the different tandem constructs used to study the assembly of rTRPV1 concatemers. Black squares represent wild type rTRPV1 subunit (wt) and red squares represent dominate-negative subunits (DN) mutated in positions NML676FAP. (b) Bar diagram represents the average (±SEM) amplitudes of a whole-cell, capsaicin-evoked (1 μM) currents from Xenopus laevis oocytes expressing the different tandem constructs either alone (black bars), or together with a DN construct (red bars; DN). Holding potential −60 mV. Each bar represents readings from 6–15 oocytes. The statistical significance between currents evoked with or without DN was determined using unpaired Student’s t tests, where **represents P ≤ 0.01, ***represents P ≤ 0.001. Note that no statistical significant difference in evoked currents was found between cells expressing 4wt with or without DN. (c) Western-blot analysis of HEK293T cells transiently expressing a single subunit (wt) or a tetrameric tandem (4wt) constructs, using an anti-rat TRPV1 antibody (α rTRPV1). Note that, while the expression of a single subunit wt construct yielded a band corresponded to ~90 kDa, the only specific band observed in cells expressing the 4wt construct corresponded to a ~400 kDa size (indicated with arrows).
Mentions: To verify the functionality of the tetrameric TRPV1 concatemer, we analyzed its response to its known agonists: capsaicin, protons and heat (Fig. 1)1925. HEK293T cells transiently expressing the native rTRPV1 (wt) or tetrameric rTRPV1 concatemeric construct (four subunits, “4wt”) were continuously perfused with the standard extracellular solution when protons (pH 5.5) were applied for 15 s, followed by a minute wash, and finally capsaicin (1 μM). To avoid contamination and due to its hydrophobic nature, applications of high capsaicin concentrations (>0.2 μM) were restricted to the end of the experiments throughout the study. The current-voltage relationship was determined using whole-cell patch-clamp recordings. As shown in Fig. 1b, both extracellular protons (H+; cyan line) and capsaicin (CAP; orange line) elicited robust, outwardly rectifying currents in cells expressing either rTRPV1 (wt) or tetrameric rTRPV1 concatemer (4wt) construct. We further determined the current-voltage relationships in response to heat (temperature range of 22°C–50°C) or capsaicin by applying the two electrodes voltage clamp (TEVC) technique in Xenopus laevis oocytes expressing wt or 4wt concatemer (Figs 1c and 2; average currents at +80 mV). In both heterologous systems expressing the 4wt concatemeric receptor, the current amplitude and kinetics were comparable to those previously reported for wt rTRPV1 (average current amplitude (±SEM): HEK293T: wt ICAP+80mV = 8.3 ± 1.2 nA and 4wt ICAP+80mV = 7.8 ± 1.8 nA; oocytes: wt ICAP+80mV = 5.6 ± 0.3 μA and 4wt ICAP+80mV = 4.7 ± 0.2 μA; average temperature threshold (±SEM): wt: 42 ± 1.2 °C; 4wt: 43 ± 1.6 °C, and average temperature sensitivity (±SEM): wt: Q10 = 27 ± 3; 4wt: Q10 = 25 ± 2; Figs 1 and 2)1012262728.

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