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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

Individual Po-T relations in the presence of external Na+ measured over a wide range of temperatures uncover the presence of multiple temperature-dependent components in the gating mechanism of the TRPV1 channel.(A) Normalized I-T relation (mean ± SEM, grey circles) obtained from data in the presence of 130 mM external Na+ (Figure 4B) at +90 mV with superimposed I-T relations from individual cells (colored continuous curves, n = 14). (B) The inward currents at -90 mV measured in the same experiments as the data at +90 mV shown in (A) were used to construct I-T relations at -90 mV. Each individual I-T relation at -90 mV was normalized to the current at 22°C from the I-T relation at +90 mV obtained from the same cell. Of the 14 cells included in (A), only those that exhibited a substantial mono-exponential increase in the inward currents (at -90 mV) at higher temperatures were analyzed and are included in the figure. Those cells that were not included had inward currents that were still too small as compared to the leak in the measured range of temperatures. The resulting normalized mean I-T relation (mean ± SEM, n = 5) is shown as grey triangles. The individual I-T relations are shown as colored continuous curves with data from each cell colored the same as in (A). The dotted line denotes the zero-current level. (C) Mean Po-T relation (grey circles) obtained from the data in (A) with superimposed Po-T relations from individual cells (colored continuous curves, same coloring for each cell as in (A)). The continuous black curve is the prediction from model i (Figure 7A) calculated with the parameters in Figure 7—source data 2A. (D) Mean Po-T relation shown in (C) with superimposed fits of Equation 1 (see Materials and methods) to each individual Po-T relation, with each fit extending over the temperature-range in which it was constrained during the fitting procedure. The color of the fits matches the color of their corresponding Po-T curves in (C). (E) Individual apparent enthalpy values for data at +90 mV corresponding to the fits in (D) as indicated by matching colors. The mean enthalpy for the fits at higher temperatures (mean ± SEM, n = 10, fits colored in green, yellow and red) is shown as an open square. The mean enthalpy from fits at low temperatures (mean ± SEM, n = 4, fits colored in blue) is shown as a closed square. The apparent enthalpy values for data at -90 mV were obtained from fits of Equation 1 to the I-T relations in (B), followed by subtracting the enthalpy associated with ion conduction (9 kcal/mol, Figure 4—figure supplement 1B–E).DOI:http://dx.doi.org/10.7554/eLife.13356.014
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fig4s2: Individual Po-T relations in the presence of external Na+ measured over a wide range of temperatures uncover the presence of multiple temperature-dependent components in the gating mechanism of the TRPV1 channel.(A) Normalized I-T relation (mean ± SEM, grey circles) obtained from data in the presence of 130 mM external Na+ (Figure 4B) at +90 mV with superimposed I-T relations from individual cells (colored continuous curves, n = 14). (B) The inward currents at -90 mV measured in the same experiments as the data at +90 mV shown in (A) were used to construct I-T relations at -90 mV. Each individual I-T relation at -90 mV was normalized to the current at 22°C from the I-T relation at +90 mV obtained from the same cell. Of the 14 cells included in (A), only those that exhibited a substantial mono-exponential increase in the inward currents (at -90 mV) at higher temperatures were analyzed and are included in the figure. Those cells that were not included had inward currents that were still too small as compared to the leak in the measured range of temperatures. The resulting normalized mean I-T relation (mean ± SEM, n = 5) is shown as grey triangles. The individual I-T relations are shown as colored continuous curves with data from each cell colored the same as in (A). The dotted line denotes the zero-current level. (C) Mean Po-T relation (grey circles) obtained from the data in (A) with superimposed Po-T relations from individual cells (colored continuous curves, same coloring for each cell as in (A)). The continuous black curve is the prediction from model i (Figure 7A) calculated with the parameters in Figure 7—source data 2A. (D) Mean Po-T relation shown in (C) with superimposed fits of Equation 1 (see Materials and methods) to each individual Po-T relation, with each fit extending over the temperature-range in which it was constrained during the fitting procedure. The color of the fits matches the color of their corresponding Po-T curves in (C). (E) Individual apparent enthalpy values for data at +90 mV corresponding to the fits in (D) as indicated by matching colors. The mean enthalpy for the fits at higher temperatures (mean ± SEM, n = 10, fits colored in green, yellow and red) is shown as an open square. The mean enthalpy from fits at low temperatures (mean ± SEM, n = 4, fits colored in blue) is shown as a closed square. The apparent enthalpy values for data at -90 mV were obtained from fits of Equation 1 to the I-T relations in (B), followed by subtracting the enthalpy associated with ion conduction (9 kcal/mol, Figure 4—figure supplement 1B–E).DOI:http://dx.doi.org/10.7554/eLife.13356.014

Mentions: TRPV1 is widely agreed to be activated by noxious heat above 35°C in the absence of other stimuli (Liu et al., 2003; Yao et al., 2010), but whether the channel responds to lower temperatures has not been carefully examined because the open probability (Po) in this temperature range is very low (Oseguera et al., 2007). If TRPV1 remains sensitive to changes in temperature below 35°C, it is possible that the spontaneous activation that we observed upon removing external Na+ at room temperature might be caused by an alteration in the temperature-dependence for channel activation. We therefore began by investigating TRPV1 channel activity over a broad temperature range in the presence of external Na+ using cells with widely varying expression levels of the channel. We measured macroscopic current-temperature (I-T) relations between ~8°C and 45°C using slow temperature ramps and observed steep temperature-dependent activation over the entire range of temperatures explored (Figure 4A and B). Although these I-T relations largely reflect temperature-dependent changes in Po, the rate of ion conduction through open channels is also weakly temperature-dependent and thus will contribute to the slope of I-T relations. To directly evaluate temperature-dependent changes in Po, we estimated the temperature-dependence of ion conduction (Figure 4—figure supplement 1B–E) and then calculated Po-T relations from the macroscopic I-T relations (Figure 4C and Figure 4—figure supplement 1F, see Materials and methods). To qualitatively compare the slopes of Po-T relations between cells and conditions, we fit a single exponential function (Equation 1, see Materials and methods) to the data over defined temperature ranges to obtain apparent enthalpy (ΔHapp) values (see Figure 4—figure supplement 2C–E). At temperatures > 35°C, we observed steep temperature-dependence to Po at positive membrane voltages (Figure 4C and D), which became even steeper at negative membrane voltages (Figure 4D, Figure 4—figure supplement 2B and E), consistent with previous studies (Voets et al., 2004; Yao et al., 2010). At temperatures between 8 and 25°C, a range in which the temperature-sensitivity of TRPV1 has not been previously investigated, we also observed steep temperature-dependence to Po (Figure 4C and D, Figure 4—figure supplement 2C–E). However, the entire Po-T relationship could not be described by a single temperature-dependent transition due to the presence of an apparent plateau near 22°C, raising the possibility that multiple temperature-dependent transitions are involved in the gating mechanism of TRPV1.10.7554/eLife.13356.012Figure 4.Temperature-dependent gating of TRPV1 in the presence of external Na+.


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

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

Individual Po-T relations in the presence of external Na+ measured over a wide range of temperatures uncover the presence of multiple temperature-dependent components in the gating mechanism of the TRPV1 channel.(A) Normalized I-T relation (mean ± SEM, grey circles) obtained from data in the presence of 130 mM external Na+ (Figure 4B) at +90 mV with superimposed I-T relations from individual cells (colored continuous curves, n = 14). (B) The inward currents at -90 mV measured in the same experiments as the data at +90 mV shown in (A) were used to construct I-T relations at -90 mV. Each individual I-T relation at -90 mV was normalized to the current at 22°C from the I-T relation at +90 mV obtained from the same cell. Of the 14 cells included in (A), only those that exhibited a substantial mono-exponential increase in the inward currents (at -90 mV) at higher temperatures were analyzed and are included in the figure. Those cells that were not included had inward currents that were still too small as compared to the leak in the measured range of temperatures. The resulting normalized mean I-T relation (mean ± SEM, n = 5) is shown as grey triangles. The individual I-T relations are shown as colored continuous curves with data from each cell colored the same as in (A). The dotted line denotes the zero-current level. (C) Mean Po-T relation (grey circles) obtained from the data in (A) with superimposed Po-T relations from individual cells (colored continuous curves, same coloring for each cell as in (A)). The continuous black curve is the prediction from model i (Figure 7A) calculated with the parameters in Figure 7—source data 2A. (D) Mean Po-T relation shown in (C) with superimposed fits of Equation 1 (see Materials and methods) to each individual Po-T relation, with each fit extending over the temperature-range in which it was constrained during the fitting procedure. The color of the fits matches the color of their corresponding Po-T curves in (C). (E) Individual apparent enthalpy values for data at +90 mV corresponding to the fits in (D) as indicated by matching colors. The mean enthalpy for the fits at higher temperatures (mean ± SEM, n = 10, fits colored in green, yellow and red) is shown as an open square. The mean enthalpy from fits at low temperatures (mean ± SEM, n = 4, fits colored in blue) is shown as a closed square. The apparent enthalpy values for data at -90 mV were obtained from fits of Equation 1 to the I-T relations in (B), followed by subtracting the enthalpy associated with ion conduction (9 kcal/mol, Figure 4—figure supplement 1B–E).DOI:http://dx.doi.org/10.7554/eLife.13356.014
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fig4s2: Individual Po-T relations in the presence of external Na+ measured over a wide range of temperatures uncover the presence of multiple temperature-dependent components in the gating mechanism of the TRPV1 channel.(A) Normalized I-T relation (mean ± SEM, grey circles) obtained from data in the presence of 130 mM external Na+ (Figure 4B) at +90 mV with superimposed I-T relations from individual cells (colored continuous curves, n = 14). (B) The inward currents at -90 mV measured in the same experiments as the data at +90 mV shown in (A) were used to construct I-T relations at -90 mV. Each individual I-T relation at -90 mV was normalized to the current at 22°C from the I-T relation at +90 mV obtained from the same cell. Of the 14 cells included in (A), only those that exhibited a substantial mono-exponential increase in the inward currents (at -90 mV) at higher temperatures were analyzed and are included in the figure. Those cells that were not included had inward currents that were still too small as compared to the leak in the measured range of temperatures. The resulting normalized mean I-T relation (mean ± SEM, n = 5) is shown as grey triangles. The individual I-T relations are shown as colored continuous curves with data from each cell colored the same as in (A). The dotted line denotes the zero-current level. (C) Mean Po-T relation (grey circles) obtained from the data in (A) with superimposed Po-T relations from individual cells (colored continuous curves, same coloring for each cell as in (A)). The continuous black curve is the prediction from model i (Figure 7A) calculated with the parameters in Figure 7—source data 2A. (D) Mean Po-T relation shown in (C) with superimposed fits of Equation 1 (see Materials and methods) to each individual Po-T relation, with each fit extending over the temperature-range in which it was constrained during the fitting procedure. The color of the fits matches the color of their corresponding Po-T curves in (C). (E) Individual apparent enthalpy values for data at +90 mV corresponding to the fits in (D) as indicated by matching colors. The mean enthalpy for the fits at higher temperatures (mean ± SEM, n = 10, fits colored in green, yellow and red) is shown as an open square. The mean enthalpy from fits at low temperatures (mean ± SEM, n = 4, fits colored in blue) is shown as a closed square. The apparent enthalpy values for data at -90 mV were obtained from fits of Equation 1 to the I-T relations in (B), followed by subtracting the enthalpy associated with ion conduction (9 kcal/mol, Figure 4—figure supplement 1B–E).DOI:http://dx.doi.org/10.7554/eLife.13356.014
Mentions: TRPV1 is widely agreed to be activated by noxious heat above 35°C in the absence of other stimuli (Liu et al., 2003; Yao et al., 2010), but whether the channel responds to lower temperatures has not been carefully examined because the open probability (Po) in this temperature range is very low (Oseguera et al., 2007). If TRPV1 remains sensitive to changes in temperature below 35°C, it is possible that the spontaneous activation that we observed upon removing external Na+ at room temperature might be caused by an alteration in the temperature-dependence for channel activation. We therefore began by investigating TRPV1 channel activity over a broad temperature range in the presence of external Na+ using cells with widely varying expression levels of the channel. We measured macroscopic current-temperature (I-T) relations between ~8°C and 45°C using slow temperature ramps and observed steep temperature-dependent activation over the entire range of temperatures explored (Figure 4A and B). Although these I-T relations largely reflect temperature-dependent changes in Po, the rate of ion conduction through open channels is also weakly temperature-dependent and thus will contribute to the slope of I-T relations. To directly evaluate temperature-dependent changes in Po, we estimated the temperature-dependence of ion conduction (Figure 4—figure supplement 1B–E) and then calculated Po-T relations from the macroscopic I-T relations (Figure 4C and Figure 4—figure supplement 1F, see Materials and methods). To qualitatively compare the slopes of Po-T relations between cells and conditions, we fit a single exponential function (Equation 1, see Materials and methods) to the data over defined temperature ranges to obtain apparent enthalpy (ΔHapp) values (see Figure 4—figure supplement 2C–E). At temperatures > 35°C, we observed steep temperature-dependence to Po at positive membrane voltages (Figure 4C and D), which became even steeper at negative membrane voltages (Figure 4D, Figure 4—figure supplement 2B and E), consistent with previous studies (Voets et al., 2004; Yao et al., 2010). At temperatures between 8 and 25°C, a range in which the temperature-sensitivity of TRPV1 has not been previously investigated, we also observed steep temperature-dependence to Po (Figure 4C and D, Figure 4—figure supplement 2C–E). However, the entire Po-T relationship could not be described by a single temperature-dependent transition due to the presence of an apparent plateau near 22°C, raising the possibility that multiple temperature-dependent transitions are involved in the gating mechanism of TRPV1.10.7554/eLife.13356.012Figure 4.Temperature-dependent gating of TRPV1 in the presence of external Na+.

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