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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 mimics the activation profile of the wild-type protein.(a) Schematic representation of wt and 4wt TRPV1 constructs. The concatemeric rat TRPV1 (rTRPV1) construct was engineered by insertion of a unique restriction enzyme site at the C terminal (without the stop codon) and to the N-terminal (without the start codon) of adjacent wt subunits (bottom; dashed line). (b) Both capsaicin (CAP; 1 μM; orange line) and extracellular protons (H+; pH 5.5; cyan line) elicited robust, outwardly rectifying currents in HEK293 cells transiently expressing either rTRPV1 (“wt”; left) or concatemeric rTRPV1 (four subunits, “4wt”; right) construct. Current-voltage relationship traces were recorded using whole-cell patch-clamp recording (in 1 s−1 voltage-ramps between −80 and +80 mV). (c) Thermal response profiles of oocytes expressing either rTRPV1 (“wt”; light grey) or concatemeric rTRPV1 (“4wt”; dark grey) construct. The current at each indicated temperature was normalized to that evoked at 50 °C. Temperature threshold for both wt and 4wt constructs was 43 °C; the Q10 was 27 for wt and 26 for 4wt rTRPV1.
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f1: TRPV1 tetrameric concatemer mimics the activation profile of the wild-type protein.(a) Schematic representation of wt and 4wt TRPV1 constructs. The concatemeric rat TRPV1 (rTRPV1) construct was engineered by insertion of a unique restriction enzyme site at the C terminal (without the stop codon) and to the N-terminal (without the start codon) of adjacent wt subunits (bottom; dashed line). (b) Both capsaicin (CAP; 1 μM; orange line) and extracellular protons (H+; pH 5.5; cyan line) elicited robust, outwardly rectifying currents in HEK293 cells transiently expressing either rTRPV1 (“wt”; left) or concatemeric rTRPV1 (four subunits, “4wt”; right) construct. Current-voltage relationship traces were recorded using whole-cell patch-clamp recording (in 1 s−1 voltage-ramps between −80 and +80 mV). (c) Thermal response profiles of oocytes expressing either rTRPV1 (“wt”; light grey) or concatemeric rTRPV1 (“4wt”; dark grey) construct. The current at each indicated temperature was normalized to that evoked at 50 °C. Temperature threshold for both wt and 4wt constructs was 43 °C; the Q10 was 27 for wt and 26 for 4wt rTRPV1.

Mentions: In order to determine the stoichiometry for activation of TRPV1 through the VBS and the outer-pore domain, we generated concatemeric TRPV1 constructs. Concatemers have been proven a useful tool to control the number and location of a mutated subunit of a homomeric protein17181920212223. TRPV1 is assembled as a homotetrameric channel with intracellular N- and C-termini624. Using this feature as guidelines, we generated concatemeric wild type (wt) rat TRPV1 (rTRPV1) constructs (illustrated in Fig. 1a).


The pain receptor TRPV1 displays agonist-dependent activation stoichiometry.

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

TRPV1 tetrameric concatemer mimics the activation profile of the wild-type protein.(a) Schematic representation of wt and 4wt TRPV1 constructs. The concatemeric rat TRPV1 (rTRPV1) construct was engineered by insertion of a unique restriction enzyme site at the C terminal (without the stop codon) and to the N-terminal (without the start codon) of adjacent wt subunits (bottom; dashed line). (b) Both capsaicin (CAP; 1 μM; orange line) and extracellular protons (H+; pH 5.5; cyan line) elicited robust, outwardly rectifying currents in HEK293 cells transiently expressing either rTRPV1 (“wt”; left) or concatemeric rTRPV1 (four subunits, “4wt”; right) construct. Current-voltage relationship traces were recorded using whole-cell patch-clamp recording (in 1 s−1 voltage-ramps between −80 and +80 mV). (c) Thermal response profiles of oocytes expressing either rTRPV1 (“wt”; light grey) or concatemeric rTRPV1 (“4wt”; dark grey) construct. The current at each indicated temperature was normalized to that evoked at 50 °C. Temperature threshold for both wt and 4wt constructs was 43 °C; the Q10 was 27 for wt and 26 for 4wt rTRPV1.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f1: TRPV1 tetrameric concatemer mimics the activation profile of the wild-type protein.(a) Schematic representation of wt and 4wt TRPV1 constructs. The concatemeric rat TRPV1 (rTRPV1) construct was engineered by insertion of a unique restriction enzyme site at the C terminal (without the stop codon) and to the N-terminal (without the start codon) of adjacent wt subunits (bottom; dashed line). (b) Both capsaicin (CAP; 1 μM; orange line) and extracellular protons (H+; pH 5.5; cyan line) elicited robust, outwardly rectifying currents in HEK293 cells transiently expressing either rTRPV1 (“wt”; left) or concatemeric rTRPV1 (four subunits, “4wt”; right) construct. Current-voltage relationship traces were recorded using whole-cell patch-clamp recording (in 1 s−1 voltage-ramps between −80 and +80 mV). (c) Thermal response profiles of oocytes expressing either rTRPV1 (“wt”; light grey) or concatemeric rTRPV1 (“4wt”; dark grey) construct. The current at each indicated temperature was normalized to that evoked at 50 °C. Temperature threshold for both wt and 4wt constructs was 43 °C; the Q10 was 27 for wt and 26 for 4wt rTRPV1.
Mentions: In order to determine the stoichiometry for activation of TRPV1 through the VBS and the outer-pore domain, we generated concatemeric TRPV1 constructs. Concatemers have been proven a useful tool to control the number and location of a mutated subunit of a homomeric protein17181920212223. TRPV1 is assembled as a homotetrameric channel with intracellular N- and C-termini624. Using this feature as guidelines, we generated concatemeric wild type (wt) rat TRPV1 (rTRPV1) constructs (illustrated in Fig. 1a).

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