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An external sodium ion binding site controls allosteric gating in TRPV1 channels.

Jara-Oseguera A, Bae C, Swartz KJ - Elife (2016)

Bottom Line: Here, we show that external sodium ions stabilize the TRPV1 channel in a closed state, such that removing the external ion leads to channel activation.The binding of a tarantula toxin to the external pore also exerts control over temperature-sensor activation, whereas binding of vanilloids influences temperature-sensitivity by largely affecting the open/closed equilibrium.Our results reveal a fundamental role of the external pore in the allosteric control of TRPV1 channel gating and provide essential constraints for understanding how these channels can be tuned by diverse stimuli.

View Article: PubMed Central - PubMed

Affiliation: Molecular Physiology and Biophysics Section, Porter Neuroscience Research Center, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, United States.

ABSTRACT
TRPV1 channels in sensory neurons are integrators of painful stimuli and heat, yet how they integrate diverse stimuli and sense temperature remains elusive. Here, we show that external sodium ions stabilize the TRPV1 channel in a closed state, such that removing the external ion leads to channel activation. In studying the underlying mechanism, we find that the temperature sensors in TRPV1 activate in two steps to favor opening, and that the binding of sodium to an extracellular site exerts allosteric control over temperature-sensor activation and opening of the pore. The binding of a tarantula toxin to the external pore also exerts control over temperature-sensor activation, whereas binding of vanilloids influences temperature-sensitivity by largely affecting the open/closed equilibrium. Our results reveal a fundamental role of the external pore in the allosteric control of TRPV1 channel gating and provide essential constraints for understanding how these channels can be tuned by diverse stimuli.

No MeSH data available.


Related in: MedlinePlus

Influence of the pore turret of TRPV1 on temperature-dependent gating and modulation by sodium.(A) External Na+ dose-response relations for TRPV1 Δ604–626 (open circles, mean ± SEM, n = 4) measured at +90 mV from voltage-ramps in the whole-cell configuration. Closed circles are data for WT TRPV1 at +90 mV. The grey and black continuous curves are the predicted normalized dose-response relations generated with model i (Figure 7A and 8B with capsaicin) for TRPV1 Δ604–626 (parameters in Figure 7—source data 2E) or WT TRPV1 (parameters in Figure 7—source data 2A), respectively. (B) Mean Po-T relation (large open circles, mean ± SEM, n = 12, data from Figure 9C) for TRPV1 Δ604–626 at +90 mV. The Po-T relations for all individual cells are shown as continuous colored curves (each cell has a different color). The open squares are the mean Po ± SEM (n = 4–5 for each temperature-range) obtained from noise analysis at different temperatures (see Materials and methods and Figure 5—figure supplement 2D–G) for TRPV1 Δ604–626, after averaging Po-data obtained at similar temperatures in the presence of 130 Nao + 10 µM caps. (grey open squares) or 0 Nao +10 µM caps. (green open squares). The continuous black and light-green curves are predictions of model i for 130 Nao + 10 µM caps. (black) and 0 Nao +10 µM caps. (light green) using parameters in Figure 7—source data 2E.DOI:http://dx.doi.org/10.7554/eLife.13356.032
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fig9s1: Influence of the pore turret of TRPV1 on temperature-dependent gating and modulation by sodium.(A) External Na+ dose-response relations for TRPV1 Δ604–626 (open circles, mean ± SEM, n = 4) measured at +90 mV from voltage-ramps in the whole-cell configuration. Closed circles are data for WT TRPV1 at +90 mV. The grey and black continuous curves are the predicted normalized dose-response relations generated with model i (Figure 7A and 8B with capsaicin) for TRPV1 Δ604–626 (parameters in Figure 7—source data 2E) or WT TRPV1 (parameters in Figure 7—source data 2A), respectively. (B) Mean Po-T relation (large open circles, mean ± SEM, n = 12, data from Figure 9C) for TRPV1 Δ604–626 at +90 mV. The Po-T relations for all individual cells are shown as continuous colored curves (each cell has a different color). The open squares are the mean Po ± SEM (n = 4–5 for each temperature-range) obtained from noise analysis at different temperatures (see Materials and methods and Figure 5—figure supplement 2D–G) for TRPV1 Δ604–626, after averaging Po-data obtained at similar temperatures in the presence of 130 Nao + 10 µM caps. (grey open squares) or 0 Nao +10 µM caps. (green open squares). The continuous black and light-green curves are predictions of model i for 130 Nao + 10 µM caps. (black) and 0 Nao +10 µM caps. (light green) using parameters in Figure 7—source data 2E.DOI:http://dx.doi.org/10.7554/eLife.13356.032

Mentions: (A) Mean normalized I-V relations at room temperature obtained in the whole-cell configuration using voltage ramps in the presence of extracellular solutions containing different Na+ concentrations or capsaicin. The darker thin curves are the mean, the lighter envelopes the SEM (n = 7–9). (B) Mean normalized I-V relations obtained as in (A) from cells with very high levels of expression (mean ± SEM, n = 6). (C) I-V relations from (A) and (B) (same color codes) scaled and plotted together on a log scale. The dotted line denotes that all curves were scaled relative to the current value at saturating capsaicin at +90 mV. The numbers on the right are the corresponding Po for each condition at room temperature if the Po for saturating capsaicin at +90 mV is 0.9 (see Materials and methods). (D) Representative WT TRPV1 variance (σ2I) vs mean current (Imean) plot obtained from noise analysis at ~3°C and +90 mV in the whole-cell configuration using the temperature-controlled chamber (Figure 4—figure supplement 1A). Data points on the left of the plot (yellow dots) were calculated from recordings in the absence of external Na+, whereas data on the right (black dots) were calculated from recordings in the same cell in the presence of 130 Nao + 10 µM capsaicin. Colored curves are fits to Equation 2 (, N is the number of channels in the cell, i is the single-channel current at +90 mV) with parameters: green fit (unconstrained fitting), N = 1064, i = 1.08 pA; red fit (i was constrained based on single-channel recordings, see Figure 4—figure supplement 1C–E), N = 439, i = 2.15 pA. The Po values shown at the top of the graph were calculated from the steady-state mean current at saturating capsaicin (Imean,ss) and the parameters from the fits of Equation 2 (constrained in red or unconstrained in green) with Equation 3: (E) Representative Imean from the experiment in (D) in the absence of external Na+ (yellow) or in the presence of 130 mM external Na+ and 10 µM capsaicin (black) at 3°C and +90 mV. The dotted red line denotes the zero-current level. (F) WT TRPV1 single-channel current amplitudes (+90 mV) at different temperatures estimated from recordings from outside-out patches expressing a few channels (yellow and grey circles with red envelopes, data from Figure 4—figure supplement 1E shown as mean ± SEM) or from noise analysis (green circles, individual symbols correspond to estimates from independent cells) from free-parameter fits of Equation 2 to σ2I vs Imean relations as in (E). Estimates of i for TRPV1 channels lacking the extracellular pore turret (TRPV1 Δ604–626, see Figure 9—figure supplement 1) also obtained from noise analysis are shown as green triangles. Noise analysis systematically underestimated i by a factor of ~2, as illustrated by i-values obtained from noise analysis after multiplication by 2 (blue symbols). The red and green curves are fits of Equation 1 with ΔH≠ = 9 kcal/mol, corresponding to the temperature-dependence of ion conduction through an open channel estimated from macroscopic I-T relations in saturating capsaicin (Figure 4—figure supplement 1B). (G) Po-T relations for 0 Nao and 130 Nao + 10 µM capsaicin obtained from noise analysis at different temperatures using two different methods and Equation 3. Po values calculated from unconstrained fits of Equation 2 to σ2I vs Imean relations (e.g. green fit in (D)) are shown as circles with green envelopes. Po values for the same cells calculated by constraining i in Equation 2 to the values obtained from direct single-channel recordings (e.g. red fit in (D)) are shown as circles with a red envelope. Yellow circles correspond to Po-values obtained from Equation 3 and Imean,ss in the absence of external Na+ (e.g. yellow trace in (E)), whereas black circles were calculated from Imean,ss from data in the presence of external Na+ and saturating capsaicin. Black and yellow dotted lines denote Po values of 0.9 and 0.2, respectively.


An external sodium ion binding site controls allosteric gating in TRPV1 channels.

Jara-Oseguera A, Bae C, Swartz KJ - Elife (2016)

Influence of the pore turret of TRPV1 on temperature-dependent gating and modulation by sodium.(A) External Na+ dose-response relations for TRPV1 Δ604–626 (open circles, mean ± SEM, n = 4) measured at +90 mV from voltage-ramps in the whole-cell configuration. Closed circles are data for WT TRPV1 at +90 mV. The grey and black continuous curves are the predicted normalized dose-response relations generated with model i (Figure 7A and 8B with capsaicin) for TRPV1 Δ604–626 (parameters in Figure 7—source data 2E) or WT TRPV1 (parameters in Figure 7—source data 2A), respectively. (B) Mean Po-T relation (large open circles, mean ± SEM, n = 12, data from Figure 9C) for TRPV1 Δ604–626 at +90 mV. The Po-T relations for all individual cells are shown as continuous colored curves (each cell has a different color). The open squares are the mean Po ± SEM (n = 4–5 for each temperature-range) obtained from noise analysis at different temperatures (see Materials and methods and Figure 5—figure supplement 2D–G) for TRPV1 Δ604–626, after averaging Po-data obtained at similar temperatures in the presence of 130 Nao + 10 µM caps. (grey open squares) or 0 Nao +10 µM caps. (green open squares). The continuous black and light-green curves are predictions of model i for 130 Nao + 10 µM caps. (black) and 0 Nao +10 µM caps. (light green) using parameters in Figure 7—source data 2E.DOI:http://dx.doi.org/10.7554/eLife.13356.032
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Related In: Results  -  Collection

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fig9s1: Influence of the pore turret of TRPV1 on temperature-dependent gating and modulation by sodium.(A) External Na+ dose-response relations for TRPV1 Δ604–626 (open circles, mean ± SEM, n = 4) measured at +90 mV from voltage-ramps in the whole-cell configuration. Closed circles are data for WT TRPV1 at +90 mV. The grey and black continuous curves are the predicted normalized dose-response relations generated with model i (Figure 7A and 8B with capsaicin) for TRPV1 Δ604–626 (parameters in Figure 7—source data 2E) or WT TRPV1 (parameters in Figure 7—source data 2A), respectively. (B) Mean Po-T relation (large open circles, mean ± SEM, n = 12, data from Figure 9C) for TRPV1 Δ604–626 at +90 mV. The Po-T relations for all individual cells are shown as continuous colored curves (each cell has a different color). The open squares are the mean Po ± SEM (n = 4–5 for each temperature-range) obtained from noise analysis at different temperatures (see Materials and methods and Figure 5—figure supplement 2D–G) for TRPV1 Δ604–626, after averaging Po-data obtained at similar temperatures in the presence of 130 Nao + 10 µM caps. (grey open squares) or 0 Nao +10 µM caps. (green open squares). The continuous black and light-green curves are predictions of model i for 130 Nao + 10 µM caps. (black) and 0 Nao +10 µM caps. (light green) using parameters in Figure 7—source data 2E.DOI:http://dx.doi.org/10.7554/eLife.13356.032
Mentions: (A) Mean normalized I-V relations at room temperature obtained in the whole-cell configuration using voltage ramps in the presence of extracellular solutions containing different Na+ concentrations or capsaicin. The darker thin curves are the mean, the lighter envelopes the SEM (n = 7–9). (B) Mean normalized I-V relations obtained as in (A) from cells with very high levels of expression (mean ± SEM, n = 6). (C) I-V relations from (A) and (B) (same color codes) scaled and plotted together on a log scale. The dotted line denotes that all curves were scaled relative to the current value at saturating capsaicin at +90 mV. The numbers on the right are the corresponding Po for each condition at room temperature if the Po for saturating capsaicin at +90 mV is 0.9 (see Materials and methods). (D) Representative WT TRPV1 variance (σ2I) vs mean current (Imean) plot obtained from noise analysis at ~3°C and +90 mV in the whole-cell configuration using the temperature-controlled chamber (Figure 4—figure supplement 1A). Data points on the left of the plot (yellow dots) were calculated from recordings in the absence of external Na+, whereas data on the right (black dots) were calculated from recordings in the same cell in the presence of 130 Nao + 10 µM capsaicin. Colored curves are fits to Equation 2 (, N is the number of channels in the cell, i is the single-channel current at +90 mV) with parameters: green fit (unconstrained fitting), N = 1064, i = 1.08 pA; red fit (i was constrained based on single-channel recordings, see Figure 4—figure supplement 1C–E), N = 439, i = 2.15 pA. The Po values shown at the top of the graph were calculated from the steady-state mean current at saturating capsaicin (Imean,ss) and the parameters from the fits of Equation 2 (constrained in red or unconstrained in green) with Equation 3: (E) Representative Imean from the experiment in (D) in the absence of external Na+ (yellow) or in the presence of 130 mM external Na+ and 10 µM capsaicin (black) at 3°C and +90 mV. The dotted red line denotes the zero-current level. (F) WT TRPV1 single-channel current amplitudes (+90 mV) at different temperatures estimated from recordings from outside-out patches expressing a few channels (yellow and grey circles with red envelopes, data from Figure 4—figure supplement 1E shown as mean ± SEM) or from noise analysis (green circles, individual symbols correspond to estimates from independent cells) from free-parameter fits of Equation 2 to σ2I vs Imean relations as in (E). Estimates of i for TRPV1 channels lacking the extracellular pore turret (TRPV1 Δ604–626, see Figure 9—figure supplement 1) also obtained from noise analysis are shown as green triangles. Noise analysis systematically underestimated i by a factor of ~2, as illustrated by i-values obtained from noise analysis after multiplication by 2 (blue symbols). The red and green curves are fits of Equation 1 with ΔH≠ = 9 kcal/mol, corresponding to the temperature-dependence of ion conduction through an open channel estimated from macroscopic I-T relations in saturating capsaicin (Figure 4—figure supplement 1B). (G) Po-T relations for 0 Nao and 130 Nao + 10 µM capsaicin obtained from noise analysis at different temperatures using two different methods and Equation 3. Po values calculated from unconstrained fits of Equation 2 to σ2I vs Imean relations (e.g. green fit in (D)) are shown as circles with green envelopes. Po values for the same cells calculated by constraining i in Equation 2 to the values obtained from direct single-channel recordings (e.g. red fit in (D)) are shown as circles with a red envelope. Yellow circles correspond to Po-values obtained from Equation 3 and Imean,ss in the absence of external Na+ (e.g. yellow trace in (E)), whereas black circles were calculated from Imean,ss from data in the presence of external Na+ and saturating capsaicin. Black and yellow dotted lines denote Po values of 0.9 and 0.2, respectively.

Bottom Line: Here, we show that external sodium ions stabilize the TRPV1 channel in a closed state, such that removing the external ion leads to channel activation.The binding of a tarantula toxin to the external pore also exerts control over temperature-sensor activation, whereas binding of vanilloids influences temperature-sensitivity by largely affecting the open/closed equilibrium.Our results reveal a fundamental role of the external pore in the allosteric control of TRPV1 channel gating and provide essential constraints for understanding how these channels can be tuned by diverse stimuli.

View Article: PubMed Central - PubMed

Affiliation: Molecular Physiology and Biophysics Section, Porter Neuroscience Research Center, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, United States.

ABSTRACT
TRPV1 channels in sensory neurons are integrators of painful stimuli and heat, yet how they integrate diverse stimuli and sense temperature remains elusive. Here, we show that external sodium ions stabilize the TRPV1 channel in a closed state, such that removing the external ion leads to channel activation. In studying the underlying mechanism, we find that the temperature sensors in TRPV1 activate in two steps to favor opening, and that the binding of sodium to an extracellular site exerts allosteric control over temperature-sensor activation and opening of the pore. The binding of a tarantula toxin to the external pore also exerts control over temperature-sensor activation, whereas binding of vanilloids influences temperature-sensitivity by largely affecting the open/closed equilibrium. Our results reveal a fundamental role of the external pore in the allosteric control of TRPV1 channel gating and provide essential constraints for understanding how these channels can be tuned by diverse stimuli.

No MeSH data available.


Related in: MedlinePlus