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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 bound subunit evokes full channel activation.(a) Representative current records from an excised outside-out membrane patch of capsaicin-exposed Flp-in TREX HEK293 cells stably transfected with 4wt and wt/3ya TRPV1 concatemers. Upward (outward) currents indicate channel opening (grey dash line). Shown are representative channel activities upon exposing the patches to saturating capsaicin concentration (2 μM CAP; orange line) of 4wt (top trace) and wt/3ya (middle trace) concatemers. Also shown is a representative channel activity of the same wt/3ya patch exposed to 30 μM capsaicin (30 μM CAP; red line; bottom trace). Holding potential at +60 mV sampled at 50 kHz and filtered at 1 kHz for display. Note the similar channel activation for the relative saturating capsaicin concentration of the two constructs. (b, c) Bar diagram representing the average (±SEM) amplitude (b) and open probability (P(open); (c) of the single-channel current activated by capsaicin at 2 μM (grey bars) and saturating concentration (Max. CAP; black bars) of the various concatemeric constructs. Each bar represents an average of 3–5 patches. The statistical significance between the 2 μM and the relative saturating concentration of capsaicin was determined using paired Student’s t test, where **represents P ≤ 0.01 and ***represents P ≤ 0.001. Note that all the different VBS-mutated concatemeric constructs reached similar open probability to that reached by the 4wt construct at saturating capsaicin concentrations.
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f6: A single VBS bound subunit evokes full channel activation.(a) Representative current records from an excised outside-out membrane patch of capsaicin-exposed Flp-in TREX HEK293 cells stably transfected with 4wt and wt/3ya TRPV1 concatemers. Upward (outward) currents indicate channel opening (grey dash line). Shown are representative channel activities upon exposing the patches to saturating capsaicin concentration (2 μM CAP; orange line) of 4wt (top trace) and wt/3ya (middle trace) concatemers. Also shown is a representative channel activity of the same wt/3ya patch exposed to 30 μM capsaicin (30 μM CAP; red line; bottom trace). Holding potential at +60 mV sampled at 50 kHz and filtered at 1 kHz for display. Note the similar channel activation for the relative saturating capsaicin concentration of the two constructs. (b, c) Bar diagram representing the average (±SEM) amplitude (b) and open probability (P(open); (c) of the single-channel current activated by capsaicin at 2 μM (grey bars) and saturating concentration (Max. CAP; black bars) of the various concatemeric constructs. Each bar represents an average of 3–5 patches. The statistical significance between the 2 μM and the relative saturating concentration of capsaicin was determined using paired Student’s t test, where **represents P ≤ 0.01 and ***represents P ≤ 0.001. Note that all the different VBS-mutated concatemeric constructs reached similar open probability to that reached by the 4wt construct at saturating capsaicin concentrations.

Mentions: Our results are consistent with a concert model of capsaicin-mediated TRPV1 activation. In order to uncover the mechanisms by which the sensitivity to capsaicin increases in direct relation to the number of VBS-containing subunits (Fig. 4d), single-channel currents were measured in the outside-out configuration of the patch-clamp technique (Fig. 6)36. In order to facilitate recording from a single channel, we used an inducible system, Flp-in TREX, which allows doxycycline-controlled construct expression levels. This system was shown to be highly useful in obtaining membrane patches with a single channel, which is challenging to achieve in transiently transfected cells due to high TRPV1 expression levels1237. We therefore stably transfected Flp-in TREX HEK293 cells with the capsaicin sensitive concatemeric constructs for single channel analyses. The membrane potential was held at +60 mV considering that the conductance of TRPV1 receptor channels is higher at positive potentials123839. The pipette contained standard intracellular patch solution, and the patch of membrane was continuously perfused with standard extracellular solution. First, we selected outside out patches containing a functional, single channel by exposing them to capsaicin (2–30 μM). Bursts of single channel activity were detected in 74 of 354 (~21%) tested patches, but, in 58 of them, channel activity was lost within 20 s and did not recover following several minutes of wash; therefore, we used the remaining 16 single channels for further analysis. Representative current traces recorded in the presence of 2 μM capsaicin for 4wt and wt/3ya channels are shown in Fig. 6a (top traces). In the 4wt receptor at 2 μM capsaicin (saturated concentration for this construct; Fig. 3), a flickering burst of activity was evoked with an average amplitude of 5.2 ± 0.4 pA (corresponding to unitary conductance of 86 ± 3 pS) and open probability of 0.89 ± 0.10 (Fig. 6a, top trace). However, in the presence of only a single wt subunit (wt/3ya), the same capsaicin concentration evoked short openings with similar average amplitude of 4.8 ± 0.5 pA (corresponding to unitary conductance of 80 ± 5 pS) but with an open probability of 0.05 ± 0.02 (Fig. 6a, middle trace). When we exposed the wt/3ya construct patch to 30 μM capsaicin (a saturating concentration for this construct; Fig. 4d), flickering bursts of activity was observed, with an average amplitude of 4.9 ± 0.4 pA (corresponding to unitary conductance of 82 ± 3 pS) and an open probability of 0.73 ± 0.10 (Fig. 6a, bottom trace). Fig. 6b,c summarize the average amplitude and the open probability of the analyzed capsaicin sensitive constructs in 2 μM and the relative saturating capsaicin concentration (as determined in Fig. 4d). Our results show that, while the unitary conductance remains same regardless of agonist concentration (Fig. 6b), the open probability was strongly correlated with the number of putative subunits (at 2 μM; Fig. 6c, grey bars). Nevertheless, at relative saturating concentration for each of the constructs, similar high open probability bursts of activity were obtained. Thus, our results indicate that a single active VBS is sufficient to achieve a maximal open-channel lifetime.


The pain receptor TRPV1 displays agonist-dependent activation stoichiometry.

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

A single VBS bound subunit evokes full channel activation.(a) Representative current records from an excised outside-out membrane patch of capsaicin-exposed Flp-in TREX HEK293 cells stably transfected with 4wt and wt/3ya TRPV1 concatemers. Upward (outward) currents indicate channel opening (grey dash line). Shown are representative channel activities upon exposing the patches to saturating capsaicin concentration (2 μM CAP; orange line) of 4wt (top trace) and wt/3ya (middle trace) concatemers. Also shown is a representative channel activity of the same wt/3ya patch exposed to 30 μM capsaicin (30 μM CAP; red line; bottom trace). Holding potential at +60 mV sampled at 50 kHz and filtered at 1 kHz for display. Note the similar channel activation for the relative saturating capsaicin concentration of the two constructs. (b, c) Bar diagram representing the average (±SEM) amplitude (b) and open probability (P(open); (c) of the single-channel current activated by capsaicin at 2 μM (grey bars) and saturating concentration (Max. CAP; black bars) of the various concatemeric constructs. Each bar represents an average of 3–5 patches. The statistical significance between the 2 μM and the relative saturating concentration of capsaicin was determined using paired Student’s t test, where **represents P ≤ 0.01 and ***represents P ≤ 0.001. Note that all the different VBS-mutated concatemeric constructs reached similar open probability to that reached by the 4wt construct at saturating capsaicin concentrations.
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Related In: Results  -  Collection

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

f6: A single VBS bound subunit evokes full channel activation.(a) Representative current records from an excised outside-out membrane patch of capsaicin-exposed Flp-in TREX HEK293 cells stably transfected with 4wt and wt/3ya TRPV1 concatemers. Upward (outward) currents indicate channel opening (grey dash line). Shown are representative channel activities upon exposing the patches to saturating capsaicin concentration (2 μM CAP; orange line) of 4wt (top trace) and wt/3ya (middle trace) concatemers. Also shown is a representative channel activity of the same wt/3ya patch exposed to 30 μM capsaicin (30 μM CAP; red line; bottom trace). Holding potential at +60 mV sampled at 50 kHz and filtered at 1 kHz for display. Note the similar channel activation for the relative saturating capsaicin concentration of the two constructs. (b, c) Bar diagram representing the average (±SEM) amplitude (b) and open probability (P(open); (c) of the single-channel current activated by capsaicin at 2 μM (grey bars) and saturating concentration (Max. CAP; black bars) of the various concatemeric constructs. Each bar represents an average of 3–5 patches. The statistical significance between the 2 μM and the relative saturating concentration of capsaicin was determined using paired Student’s t test, where **represents P ≤ 0.01 and ***represents P ≤ 0.001. Note that all the different VBS-mutated concatemeric constructs reached similar open probability to that reached by the 4wt construct at saturating capsaicin concentrations.
Mentions: Our results are consistent with a concert model of capsaicin-mediated TRPV1 activation. In order to uncover the mechanisms by which the sensitivity to capsaicin increases in direct relation to the number of VBS-containing subunits (Fig. 4d), single-channel currents were measured in the outside-out configuration of the patch-clamp technique (Fig. 6)36. In order to facilitate recording from a single channel, we used an inducible system, Flp-in TREX, which allows doxycycline-controlled construct expression levels. This system was shown to be highly useful in obtaining membrane patches with a single channel, which is challenging to achieve in transiently transfected cells due to high TRPV1 expression levels1237. We therefore stably transfected Flp-in TREX HEK293 cells with the capsaicin sensitive concatemeric constructs for single channel analyses. The membrane potential was held at +60 mV considering that the conductance of TRPV1 receptor channels is higher at positive potentials123839. The pipette contained standard intracellular patch solution, and the patch of membrane was continuously perfused with standard extracellular solution. First, we selected outside out patches containing a functional, single channel by exposing them to capsaicin (2–30 μM). Bursts of single channel activity were detected in 74 of 354 (~21%) tested patches, but, in 58 of them, channel activity was lost within 20 s and did not recover following several minutes of wash; therefore, we used the remaining 16 single channels for further analysis. Representative current traces recorded in the presence of 2 μM capsaicin for 4wt and wt/3ya channels are shown in Fig. 6a (top traces). In the 4wt receptor at 2 μM capsaicin (saturated concentration for this construct; Fig. 3), a flickering burst of activity was evoked with an average amplitude of 5.2 ± 0.4 pA (corresponding to unitary conductance of 86 ± 3 pS) and open probability of 0.89 ± 0.10 (Fig. 6a, top trace). However, in the presence of only a single wt subunit (wt/3ya), the same capsaicin concentration evoked short openings with similar average amplitude of 4.8 ± 0.5 pA (corresponding to unitary conductance of 80 ± 5 pS) but with an open probability of 0.05 ± 0.02 (Fig. 6a, middle trace). When we exposed the wt/3ya construct patch to 30 μM capsaicin (a saturating concentration for this construct; Fig. 4d), flickering bursts of activity was observed, with an average amplitude of 4.9 ± 0.4 pA (corresponding to unitary conductance of 82 ± 3 pS) and an open probability of 0.73 ± 0.10 (Fig. 6a, bottom trace). Fig. 6b,c summarize the average amplitude and the open probability of the analyzed capsaicin sensitive constructs in 2 μM and the relative saturating capsaicin concentration (as determined in Fig. 4d). Our results show that, while the unitary conductance remains same regardless of agonist concentration (Fig. 6b), the open probability was strongly correlated with the number of putative subunits (at 2 μM; Fig. 6c, grey bars). Nevertheless, at relative saturating concentration for each of the constructs, similar high open probability bursts of activity were obtained. Thus, our results indicate that a single active VBS is sufficient to achieve a maximal open-channel lifetime.

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