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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

The location of E600, the extracellular pore turret and the binding site for DkTx within the outer pore of TRPV1, and the role of external Mg2+ ions in TRPV1 modulation.(A) Side view of a ribbon representation of the transmembrane domain of the TRPV1 channel bound to DkTx/RTx (refined structural model for TRPV1 with the docked solution structure of DkTx) (Bae et al., 2016). The pore domains of two adjacent subunits in contact with the K1 lobe of DkTx (shown in green) are colored in teal and yellow, and their respective S1-S4 domains are colored in lighter blue and yellow, respectively. All other subunits (including that in contact with the K2 lobe of DkTx, shown in cyan) are colored in white. The DkTx molecule bound to the two subunits in the back was omitted for clarity. Residue E600 is shown in stick representation and colored in dark blue. The red coloring near E600 indicates the position from which the extracellular pore turret was deleted in the construct used for structure determination. (B) Amino acid sequence alignment corresponding to the pore region of several TRPV1 channel orthologues together with rat TRPV2, highlighting the location and sequence conservation of the extracellular pore turret denoted by the thick orange line. The green thick lines delimit the location of the S5 and S6 transmembrane regions as based on the structure of the rat TRPV1 channel (Cao et al., 2013; Liao et al., 2013). The purple line denotes the pore helix and the blue line the location of the selectivity filter. The intensity of the blue text background denotes sequence conservation between the aligned proteins, with darker coloring representing higher conservation. The vertical arrow denotes the position of E600 within the sequence. (C) Normalized I-V relations constructed from voltage-ramps measured in the whole-cell configuration with 260 mM internal Na+ (Nai) and the extracellular solutions indicated in the figure. The dark curves are the mean and the lighter-colored envelopes are the standard error (n = 4). The data without magnesium are the same as in Figure 2B, as activation by NMDG+ and Mg2+ was tested in the same experiment.DOI:http://dx.doi.org/10.7554/eLife.13356.011
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fig3s1: The location of E600, the extracellular pore turret and the binding site for DkTx within the outer pore of TRPV1, and the role of external Mg2+ ions in TRPV1 modulation.(A) Side view of a ribbon representation of the transmembrane domain of the TRPV1 channel bound to DkTx/RTx (refined structural model for TRPV1 with the docked solution structure of DkTx) (Bae et al., 2016). The pore domains of two adjacent subunits in contact with the K1 lobe of DkTx (shown in green) are colored in teal and yellow, and their respective S1-S4 domains are colored in lighter blue and yellow, respectively. All other subunits (including that in contact with the K2 lobe of DkTx, shown in cyan) are colored in white. The DkTx molecule bound to the two subunits in the back was omitted for clarity. Residue E600 is shown in stick representation and colored in dark blue. The red coloring near E600 indicates the position from which the extracellular pore turret was deleted in the construct used for structure determination. (B) Amino acid sequence alignment corresponding to the pore region of several TRPV1 channel orthologues together with rat TRPV2, highlighting the location and sequence conservation of the extracellular pore turret denoted by the thick orange line. The green thick lines delimit the location of the S5 and S6 transmembrane regions as based on the structure of the rat TRPV1 channel (Cao et al., 2013; Liao et al., 2013). The purple line denotes the pore helix and the blue line the location of the selectivity filter. The intensity of the blue text background denotes sequence conservation between the aligned proteins, with darker coloring representing higher conservation. The vertical arrow denotes the position of E600 within the sequence. (C) Normalized I-V relations constructed from voltage-ramps measured in the whole-cell configuration with 260 mM internal Na+ (Nai) and the extracellular solutions indicated in the figure. The dark curves are the mean and the lighter-colored envelopes are the standard error (n = 4). The data without magnesium are the same as in Figure 2B, as activation by NMDG+ and Mg2+ was tested in the same experiment.DOI:http://dx.doi.org/10.7554/eLife.13356.011

Mentions: We next set out to determine whether other stimuli acting on the extracellular pore activate TRPV1 through a mechanism associated with the binding of external Na+. The double-knot tarantula toxin (DkTx) is an interesting candidate as it activates the TRPV1 channel with high avidity by binding to the periphery of outer pore of the channel (Figure 3—figure supplement 1A) (Bohlen et al., 2010; Cao et al., 2013; Bae et al., 2016). We recently solved the solution structure of DkTx and performed a detailed analysis of its interactions with the TRPV1 channel, which suggested that the toxin activates TRPV1 by disrupting a cluster of hydrophobic residues at the extracellular half of the S5 and S6 segments (Bae et al., 2016). Interestingly, removal of external Na+ before application of the toxin activates TRPV1 to a similar extent when compared with a saturating concentration of DkTx (Figure 3B and C), and removal of external Na+ did not produce further activation of DkTx-bound channels. In contrast, application of a saturating concentration of capsaicin produced further activation (Figure 3C). The lack of additivity for the activation of TRPV1 by DkTx and the removal of external Na+ contrasts with our results with acidic extracellular pH in the absence of external Na+ (Figure 3A), and suggests that DkTx and external Na+ modulate the TRPV1 through convergent mechanisms.


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

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

The location of E600, the extracellular pore turret and the binding site for DkTx within the outer pore of TRPV1, and the role of external Mg2+ ions in TRPV1 modulation.(A) Side view of a ribbon representation of the transmembrane domain of the TRPV1 channel bound to DkTx/RTx (refined structural model for TRPV1 with the docked solution structure of DkTx) (Bae et al., 2016). The pore domains of two adjacent subunits in contact with the K1 lobe of DkTx (shown in green) are colored in teal and yellow, and their respective S1-S4 domains are colored in lighter blue and yellow, respectively. All other subunits (including that in contact with the K2 lobe of DkTx, shown in cyan) are colored in white. The DkTx molecule bound to the two subunits in the back was omitted for clarity. Residue E600 is shown in stick representation and colored in dark blue. The red coloring near E600 indicates the position from which the extracellular pore turret was deleted in the construct used for structure determination. (B) Amino acid sequence alignment corresponding to the pore region of several TRPV1 channel orthologues together with rat TRPV2, highlighting the location and sequence conservation of the extracellular pore turret denoted by the thick orange line. The green thick lines delimit the location of the S5 and S6 transmembrane regions as based on the structure of the rat TRPV1 channel (Cao et al., 2013; Liao et al., 2013). The purple line denotes the pore helix and the blue line the location of the selectivity filter. The intensity of the blue text background denotes sequence conservation between the aligned proteins, with darker coloring representing higher conservation. The vertical arrow denotes the position of E600 within the sequence. (C) Normalized I-V relations constructed from voltage-ramps measured in the whole-cell configuration with 260 mM internal Na+ (Nai) and the extracellular solutions indicated in the figure. The dark curves are the mean and the lighter-colored envelopes are the standard error (n = 4). The data without magnesium are the same as in Figure 2B, as activation by NMDG+ and Mg2+ was tested in the same experiment.DOI:http://dx.doi.org/10.7554/eLife.13356.011
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Related In: Results  -  Collection

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fig3s1: The location of E600, the extracellular pore turret and the binding site for DkTx within the outer pore of TRPV1, and the role of external Mg2+ ions in TRPV1 modulation.(A) Side view of a ribbon representation of the transmembrane domain of the TRPV1 channel bound to DkTx/RTx (refined structural model for TRPV1 with the docked solution structure of DkTx) (Bae et al., 2016). The pore domains of two adjacent subunits in contact with the K1 lobe of DkTx (shown in green) are colored in teal and yellow, and their respective S1-S4 domains are colored in lighter blue and yellow, respectively. All other subunits (including that in contact with the K2 lobe of DkTx, shown in cyan) are colored in white. The DkTx molecule bound to the two subunits in the back was omitted for clarity. Residue E600 is shown in stick representation and colored in dark blue. The red coloring near E600 indicates the position from which the extracellular pore turret was deleted in the construct used for structure determination. (B) Amino acid sequence alignment corresponding to the pore region of several TRPV1 channel orthologues together with rat TRPV2, highlighting the location and sequence conservation of the extracellular pore turret denoted by the thick orange line. The green thick lines delimit the location of the S5 and S6 transmembrane regions as based on the structure of the rat TRPV1 channel (Cao et al., 2013; Liao et al., 2013). The purple line denotes the pore helix and the blue line the location of the selectivity filter. The intensity of the blue text background denotes sequence conservation between the aligned proteins, with darker coloring representing higher conservation. The vertical arrow denotes the position of E600 within the sequence. (C) Normalized I-V relations constructed from voltage-ramps measured in the whole-cell configuration with 260 mM internal Na+ (Nai) and the extracellular solutions indicated in the figure. The dark curves are the mean and the lighter-colored envelopes are the standard error (n = 4). The data without magnesium are the same as in Figure 2B, as activation by NMDG+ and Mg2+ was tested in the same experiment.DOI:http://dx.doi.org/10.7554/eLife.13356.011
Mentions: We next set out to determine whether other stimuli acting on the extracellular pore activate TRPV1 through a mechanism associated with the binding of external Na+. The double-knot tarantula toxin (DkTx) is an interesting candidate as it activates the TRPV1 channel with high avidity by binding to the periphery of outer pore of the channel (Figure 3—figure supplement 1A) (Bohlen et al., 2010; Cao et al., 2013; Bae et al., 2016). We recently solved the solution structure of DkTx and performed a detailed analysis of its interactions with the TRPV1 channel, which suggested that the toxin activates TRPV1 by disrupting a cluster of hydrophobic residues at the extracellular half of the S5 and S6 segments (Bae et al., 2016). Interestingly, removal of external Na+ before application of the toxin activates TRPV1 to a similar extent when compared with a saturating concentration of DkTx (Figure 3B and C), and removal of external Na+ did not produce further activation of DkTx-bound channels. In contrast, application of a saturating concentration of capsaicin produced further activation (Figure 3C). The lack of additivity for the activation of TRPV1 by DkTx and the removal of external Na+ contrasts with our results with acidic extracellular pH in the absence of external Na+ (Figure 3A), and suggests that DkTx and external Na+ modulate the TRPV1 through convergent mechanisms.

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