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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

Activation of TRPV1 by capsaicin displays channel symmetry.Bar diagram representing the average (±SEM) amplitude of the whole-cell currents for each indicated group of tandem constructs evoked by the relative capsaicin concentration (3wt/ya group: 0.5 μM, left bars; 2wt/2ya group: 1 μM, middle bars; wt/3ya group: 3 μM, right bars) normalized to the current amplitude of 30 μM capsaicin (I30μM CAP). Tested conditions were used according to the dose response determined in Fig. 4d. Below each bar diagram are schematic Illustrations of the different tandem constructs used to study the symmetry of the capsaicin response. Black squares represent wt subunits (wt) and empty circles represent subunits with a mutated VBS in position Y511A (ya). Holding potential −40 mV. Each bar represents 4–7 HEK293T cells transiently expressing the respective construct. Statistical significance was determined with the unpaired Student’s t tests. No statically significant differences were found.
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f5: Activation of TRPV1 by capsaicin displays channel symmetry.Bar diagram representing the average (±SEM) amplitude of the whole-cell currents for each indicated group of tandem constructs evoked by the relative capsaicin concentration (3wt/ya group: 0.5 μM, left bars; 2wt/2ya group: 1 μM, middle bars; wt/3ya group: 3 μM, right bars) normalized to the current amplitude of 30 μM capsaicin (I30μM CAP). Tested conditions were used according to the dose response determined in Fig. 4d. Below each bar diagram are schematic Illustrations of the different tandem constructs used to study the symmetry of the capsaicin response. Black squares represent wt subunits (wt) and empty circles represent subunits with a mutated VBS in position Y511A (ya). Holding potential −40 mV. Each bar represents 4–7 HEK293T cells transiently expressing the respective construct. Statistical significance was determined with the unpaired Student’s t tests. No statically significant differences were found.

Mentions: In order to determine whether the arrangement of intact and mutated subunit(s) affects channel activity, we generated all the tetrameric concatemer combinations for the 3wt/ya, wt/3ya and the 2wt/2ya, and analyzed their sensitivity to capsaicin by whole cell recordings (Fig. 5). If the location of the mutated subunit(s) affects channel sensitivity, the various combinations within each group are expected to have different response. As shown in Fig. 5, within each group, the different constructs showed comparable sensitivity to capsaicin, regardless of the location of the mutated subunit(s), demonstrating symmetry of the channel.


The pain receptor TRPV1 displays agonist-dependent activation stoichiometry.

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

Activation of TRPV1 by capsaicin displays channel symmetry.Bar diagram representing the average (±SEM) amplitude of the whole-cell currents for each indicated group of tandem constructs evoked by the relative capsaicin concentration (3wt/ya group: 0.5 μM, left bars; 2wt/2ya group: 1 μM, middle bars; wt/3ya group: 3 μM, right bars) normalized to the current amplitude of 30 μM capsaicin (I30μM CAP). Tested conditions were used according to the dose response determined in Fig. 4d. Below each bar diagram are schematic Illustrations of the different tandem constructs used to study the symmetry of the capsaicin response. Black squares represent wt subunits (wt) and empty circles represent subunits with a mutated VBS in position Y511A (ya). Holding potential −40 mV. Each bar represents 4–7 HEK293T cells transiently expressing the respective construct. Statistical significance was determined with the unpaired Student’s t tests. No statically significant differences were found.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f5: Activation of TRPV1 by capsaicin displays channel symmetry.Bar diagram representing the average (±SEM) amplitude of the whole-cell currents for each indicated group of tandem constructs evoked by the relative capsaicin concentration (3wt/ya group: 0.5 μM, left bars; 2wt/2ya group: 1 μM, middle bars; wt/3ya group: 3 μM, right bars) normalized to the current amplitude of 30 μM capsaicin (I30μM CAP). Tested conditions were used according to the dose response determined in Fig. 4d. Below each bar diagram are schematic Illustrations of the different tandem constructs used to study the symmetry of the capsaicin response. Black squares represent wt subunits (wt) and empty circles represent subunits with a mutated VBS in position Y511A (ya). Holding potential −40 mV. Each bar represents 4–7 HEK293T cells transiently expressing the respective construct. Statistical significance was determined with the unpaired Student’s t tests. No statically significant differences were found.
Mentions: In order to determine whether the arrangement of intact and mutated subunit(s) affects channel activity, we generated all the tetrameric concatemer combinations for the 3wt/ya, wt/3ya and the 2wt/2ya, and analyzed their sensitivity to capsaicin by whole cell recordings (Fig. 5). If the location of the mutated subunit(s) affects channel sensitivity, the various combinations within each group are expected to have different response. As shown in Fig. 5, within each group, the different constructs showed comparable sensitivity to capsaicin, regardless of the location of the mutated subunit(s), demonstrating symmetry of 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