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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

DkTx binding to TRPV1.(A) Overlay of the side views of the S3-S6 segments of one TRPV1 subunit and the S5-S6 segments of an adjacent subunit in the apo (subunit 1, S3-S4 in light pink, S5-S6 in magenta; subunit 2, S5-S6 in light grey) and the DkTx/RTx bound state (subunit 1, S3-S4 in light blue, S5-S6 in teal; subunit 2, S5-S6 in light orange). The structures shown are the refined structural models of TRPV1 from (Bae et al., 2016), with the docked solution structure of DkTx (K1 in green and K2 in cyan). E600 is shown in stick representation and colored in dark blue, and the red highlight denotes the location from which the pore turret was deleted in the structure used for structure determination (Cao et al., 2013; Liao et al., 2013). (B) Mean Po-T relation (mean ± SEM, n = 9, data from Figure 10,) for WT TRPV1 obtained in the presence of external Na+ and DkTx. The Po-T relations for individual cells are shown as colored continuous curves.DOI:http://dx.doi.org/10.7554/eLife.13356.034
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fig10s1: DkTx binding to TRPV1.(A) Overlay of the side views of the S3-S6 segments of one TRPV1 subunit and the S5-S6 segments of an adjacent subunit in the apo (subunit 1, S3-S4 in light pink, S5-S6 in magenta; subunit 2, S5-S6 in light grey) and the DkTx/RTx bound state (subunit 1, S3-S4 in light blue, S5-S6 in teal; subunit 2, S5-S6 in light orange). The structures shown are the refined structural models of TRPV1 from (Bae et al., 2016), with the docked solution structure of DkTx (K1 in green and K2 in cyan). E600 is shown in stick representation and colored in dark blue, and the red highlight denotes the location from which the pore turret was deleted in the structure used for structure determination (Cao et al., 2013; Liao et al., 2013). (B) Mean Po-T relation (mean ± SEM, n = 9, data from Figure 10,) for WT TRPV1 obtained in the presence of external Na+ and DkTx. The Po-T relations for individual cells are shown as colored continuous curves.DOI:http://dx.doi.org/10.7554/eLife.13356.034

Mentions: The results thus far demonstrate that the external pore of TRPV1 contains Na+ ion binding sites that exert strong control over temperature-dependent activation and inactivation, as well as the closed/open equilibrium. Our previous results with DkTx suggest external Na+ and DkTx modulate the TRPV1 through a common mechanism (Figure 3B–D). In addition, our recent analysis of the interaction between DkTx and TRPV1 suggests that the toxin activates the channel by inducing a displacement of the S5-S6 segments relative to the S1-S4 domain (Figure 10—figure supplement 1A), which results in the disruption of a cluster of buried hydrophobic residues at the extracellular ends of the S5 and S6 segments (Bae et al., 2016). This is interesting in light of a recent demonstration in the Shaker Kv channel that movement of hydrophobic residues between buried and solvent-exposed environments imparts temperature-dependence (Chowdhury et al., 2014). We therefore measured Po-T relations in the presence of external Na+ and DkTx to test whether the binding of the toxin has an effect on temperature-sensitivity, and observed that activation of the channel by heat was effectively ablated between 5 and 35°C (Figure 10, and Figure 10—figure supplement 1B for Po-T relations from individual cells). This finding is particularly striking when considering that the Po is situated well below the theoretical upper limit of 1.0. These results demonstrate that DkTx also exerts strong control over temperature-sensor activation, which in our model can be achieved simply by trapping the channel in the intermediate plateau and preventing temperature-sensor deactivation or full activation (Figure 10, insert).10.7554/eLife.13356.033Figure 10.The binding of DkTx to the outer pore of TRPV1 effectively ablates temperature-dependent gating over a wide range of temperatures.


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

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

DkTx binding to TRPV1.(A) Overlay of the side views of the S3-S6 segments of one TRPV1 subunit and the S5-S6 segments of an adjacent subunit in the apo (subunit 1, S3-S4 in light pink, S5-S6 in magenta; subunit 2, S5-S6 in light grey) and the DkTx/RTx bound state (subunit 1, S3-S4 in light blue, S5-S6 in teal; subunit 2, S5-S6 in light orange). The structures shown are the refined structural models of TRPV1 from (Bae et al., 2016), with the docked solution structure of DkTx (K1 in green and K2 in cyan). E600 is shown in stick representation and colored in dark blue, and the red highlight denotes the location from which the pore turret was deleted in the structure used for structure determination (Cao et al., 2013; Liao et al., 2013). (B) Mean Po-T relation (mean ± SEM, n = 9, data from Figure 10,) for WT TRPV1 obtained in the presence of external Na+ and DkTx. The Po-T relations for individual cells are shown as colored continuous curves.DOI:http://dx.doi.org/10.7554/eLife.13356.034
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fig10s1: DkTx binding to TRPV1.(A) Overlay of the side views of the S3-S6 segments of one TRPV1 subunit and the S5-S6 segments of an adjacent subunit in the apo (subunit 1, S3-S4 in light pink, S5-S6 in magenta; subunit 2, S5-S6 in light grey) and the DkTx/RTx bound state (subunit 1, S3-S4 in light blue, S5-S6 in teal; subunit 2, S5-S6 in light orange). The structures shown are the refined structural models of TRPV1 from (Bae et al., 2016), with the docked solution structure of DkTx (K1 in green and K2 in cyan). E600 is shown in stick representation and colored in dark blue, and the red highlight denotes the location from which the pore turret was deleted in the structure used for structure determination (Cao et al., 2013; Liao et al., 2013). (B) Mean Po-T relation (mean ± SEM, n = 9, data from Figure 10,) for WT TRPV1 obtained in the presence of external Na+ and DkTx. The Po-T relations for individual cells are shown as colored continuous curves.DOI:http://dx.doi.org/10.7554/eLife.13356.034
Mentions: The results thus far demonstrate that the external pore of TRPV1 contains Na+ ion binding sites that exert strong control over temperature-dependent activation and inactivation, as well as the closed/open equilibrium. Our previous results with DkTx suggest external Na+ and DkTx modulate the TRPV1 through a common mechanism (Figure 3B–D). In addition, our recent analysis of the interaction between DkTx and TRPV1 suggests that the toxin activates the channel by inducing a displacement of the S5-S6 segments relative to the S1-S4 domain (Figure 10—figure supplement 1A), which results in the disruption of a cluster of buried hydrophobic residues at the extracellular ends of the S5 and S6 segments (Bae et al., 2016). This is interesting in light of a recent demonstration in the Shaker Kv channel that movement of hydrophobic residues between buried and solvent-exposed environments imparts temperature-dependence (Chowdhury et al., 2014). We therefore measured Po-T relations in the presence of external Na+ and DkTx to test whether the binding of the toxin has an effect on temperature-sensitivity, and observed that activation of the channel by heat was effectively ablated between 5 and 35°C (Figure 10, and Figure 10—figure supplement 1B for Po-T relations from individual cells). This finding is particularly striking when considering that the Po is situated well below the theoretical upper limit of 1.0. These results demonstrate that DkTx also exerts strong control over temperature-sensor activation, which in our model can be achieved simply by trapping the channel in the intermediate plateau and preventing temperature-sensor deactivation or full activation (Figure 10, insert).10.7554/eLife.13356.033Figure 10.The binding of DkTx to the outer pore of TRPV1 effectively ablates temperature-dependent gating over a wide range of temperatures.

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