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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

Activation of rat TRPV1 channel orthologues by substituting external Na+ with NMDG+.Representative current families recorded from outside-out patches containing TRPV1 channels from different species (mouse, human and chicken) at room temperature. Currents were elicited by voltage steps of 100 ms duration going from -120 to +140 mV in 10-mV increments, and different solutions were applied using the fast solution-exchange system. The red-dotted lines denote the zero-current level.DOI:http://dx.doi.org/10.7554/eLife.13356.005
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fig1s2: Activation of rat TRPV1 channel orthologues by substituting external Na+ with NMDG+.Representative current families recorded from outside-out patches containing TRPV1 channels from different species (mouse, human and chicken) at room temperature. Currents were elicited by voltage steps of 100 ms duration going from -120 to +140 mV in 10-mV increments, and different solutions were applied using the fast solution-exchange system. The red-dotted lines denote the zero-current level.DOI:http://dx.doi.org/10.7554/eLife.13356.005

Mentions: To investigate the influence of external Na+ on TRPV1 channels, we obtained whole-cell patch clamp recordings of cells expressing rat TRPV1 using internal and external solutions containing Na+ (130 mM) as the sole charge carrier, and then exchanged the external Na+ solution with one containing N-methyl-D-glucamine (NMDG+; 130 mM) (Figure 1B). At room temperature, this simple manipulation produced a rapid and reversible increase in outward currents at positive voltages (Figure 1B), consistent with previous results (Ohta et al., 2008). Subsequent application of a saturating concentration of capsaicin further increased the outward currents independently of whether external Na+ or NMDG+ was present (Figure 1B). At moderate TRPV1 expression levels, where outward Na+ currents could be accurately measured, the inward currents with external NMDG+ were negligible even in the presence of capsaicin (Figure 1B), indicating that TRPV1 has a relatively low permeability to large cations. Although large outward currents induced by exchanging external ions were observed in all cells in which capsaicin evoked measurable currents, this manipulation also produced a variable and irreversible rundown of the channel that increased with time in Na+-free solutions (Figure 1—figure supplement 1). Exchanging external Na+ with NMDG+ also produced large outward currents in human, mouse and chicken TRPV1 orthologues (Figure 1—figure supplement 2), which together with the previous report on porcine TRPV1 (Ohta et al., 2008), suggest that this phenomenon is a conserved feature of TRPV1 channels. Given the robust effects of exchanging external Na+ with NMDG+, we set out to explore the underlying mechanism.


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

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

Activation of rat TRPV1 channel orthologues by substituting external Na+ with NMDG+.Representative current families recorded from outside-out patches containing TRPV1 channels from different species (mouse, human and chicken) at room temperature. Currents were elicited by voltage steps of 100 ms duration going from -120 to +140 mV in 10-mV increments, and different solutions were applied using the fast solution-exchange system. The red-dotted lines denote the zero-current level.DOI:http://dx.doi.org/10.7554/eLife.13356.005
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Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4764576&req=5

fig1s2: Activation of rat TRPV1 channel orthologues by substituting external Na+ with NMDG+.Representative current families recorded from outside-out patches containing TRPV1 channels from different species (mouse, human and chicken) at room temperature. Currents were elicited by voltage steps of 100 ms duration going from -120 to +140 mV in 10-mV increments, and different solutions were applied using the fast solution-exchange system. The red-dotted lines denote the zero-current level.DOI:http://dx.doi.org/10.7554/eLife.13356.005
Mentions: To investigate the influence of external Na+ on TRPV1 channels, we obtained whole-cell patch clamp recordings of cells expressing rat TRPV1 using internal and external solutions containing Na+ (130 mM) as the sole charge carrier, and then exchanged the external Na+ solution with one containing N-methyl-D-glucamine (NMDG+; 130 mM) (Figure 1B). At room temperature, this simple manipulation produced a rapid and reversible increase in outward currents at positive voltages (Figure 1B), consistent with previous results (Ohta et al., 2008). Subsequent application of a saturating concentration of capsaicin further increased the outward currents independently of whether external Na+ or NMDG+ was present (Figure 1B). At moderate TRPV1 expression levels, where outward Na+ currents could be accurately measured, the inward currents with external NMDG+ were negligible even in the presence of capsaicin (Figure 1B), indicating that TRPV1 has a relatively low permeability to large cations. Although large outward currents induced by exchanging external ions were observed in all cells in which capsaicin evoked measurable currents, this manipulation also produced a variable and irreversible rundown of the channel that increased with time in Na+-free solutions (Figure 1—figure supplement 1). Exchanging external Na+ with NMDG+ also produced large outward currents in human, mouse and chicken TRPV1 orthologues (Figure 1—figure supplement 2), which together with the previous report on porcine TRPV1 (Ohta et al., 2008), suggest that this phenomenon is a conserved feature of TRPV1 channels. Given the robust effects of exchanging external Na+ with NMDG+, we set out to explore the underlying mechanism.

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