Humans and other mammals sense elevated heat and other painful stimuli via a sensory ion channel protein called TRPV1. Ion channels create pores in the outer membrane of cells and act as gates that open and close to regulate the flow of ions into and out of cells. This flow of ions generates electrical signals that sensory neurons use to communicate information about the environment to the brain. The TRPV1 channel is opened by heat, but also by venom toxins, acid and the active ingredient in hot chilli peppers.

The fluid that surrounds animal cells often contains high levels of sodium ions, and these ions flow through TRPV1 channels when they are open. Also, when sodium ions are removed from the fluid surrounding a cell, TRPV1 channels will spontaneously open. It is not known why this happens, but it suggests that sodium ions help to regulate the activity of the TRPV1 channel, and despite having been extensively studied, it remains unclear how this channel senses temperature and responds to other harmful stimuli.

Jara-Oseguera et al. have now investigated the role of sodium ions in activating TRPV1 in cells grown in the laboratory. The experiments involved a technique that records the movement of ions through TRPV1 channels in the cell surface membrane while exposing the cell to various stimuli that open or close the channels. The results showed that TRPV1 contains an important site on its outer surface that binds sodium ions and fine-tunes the channel’s properties. This enables the channel to be activated by potentially damaging temperatures. Further analysis revealed that TRPV1 undergoes at least two distinct physical changes in response to heat and that the binding of sodium ions to the channel’s outer structure regulates one of these changes.

Jara-Oseguera et al. went on to discover that the binding of a tarantula toxin to the outer surface of TRPV1 renders the channel insensitive to changes in temperature. These findings suggest that structural changes to the external part of this channel protein are closely associated with its ability to sense temperature, possibly because this region of the protein is directly responsible for detecting changes in temperature.

These findings shed light on how this channel detects and responds to dangerously high temperatures, as well to other harmful stimuli. An aim for future studies is to pinpoint the specific regions of the protein that form the sodium-binding sites and to test the hypothesis that temperature sensors are located on the outer surface of the protein.

Transient receptor potential (TRP) channels belong to a large and diverse superfamily of tetrameric cation channels that contain six transmembrane helices per subunit (Liao et al., 2013; Hellmich and Gaudet, 2014), similar to voltage-activated ion channels (Long et al., 2007), ryanodine receptors (Yan et al., 2015) and IP₃ receptors (Fan et al., 2015) (Figure 1A). Several TRP channels are exquisitely temperature-dependent, enabling them to serve as biological thermosensors throughout the animal kingdom (Vay et al., 2012). The vanilloid-sensitive TRPV1 channel is a weakly voltage-dependent and cation-selective channel that is expressed in nociceptive sensory neurons, where it acts as a sensor for noxious stimuli (Caterina and Julius, 2001) including heat, extracellular acidic pH, vanilloid compounds (e.g capsaicin), and venom toxins (e.g. double-knot toxin or DkTx) (see [Hilton et al., 2015] for review). The sensitivity of TRPV1 to diverse modulators is fascinating because many stimuli are capable of influencing the responses of TRPV1 to other signals. For example, protons can sensitize the channel to activation by heat (Jordt et al., 2000) and capsaicin (Jordt et al., 2000; Ryu et al., 2003), and both capsaicin (Voets et al., 2004; Matta and Ahern, 2007; Gregorio-Teruel et al., 2014) and protons (Lee and Zheng, 2015) can sensitize the channel to activation by voltage. Conceptually, these observations can be explained by allosteric models wherein distinct stimulus-sensing domains are coupled to each other (Latorre et al., 2007), and to an internal gate within the pore that opens and closes the ion permeation pathway (Salazar et al., 2009; Cao et al., 2013) (Figure 1A).

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The regions of TRPV1 targeted by some activating stimuli have been identified, including the vanilloid binding site within internal regions of the S1-S4 domain (Jordt and Julius, 2002; Cao et al., 2013), and both proton- (Jordt et al., 2000) and DkTx-binding sites within the external pore (Bohlen et al., 2010; Cao et al., 2013; Bae et al., 2016) (Figure 1A). However, we currently do not understand the structural basis by which the binding of protons, capsaicin or DkTx promotes opening of the internal pore. Moreover, there is no consensus about the site and mechanism of temperature sensitivity. Mutations throughout TRPV1 alter the temperature sensitivity of the channel (Feng, 2014), and distinct models have been proposed to explain temperature sensitivity in these channels (Voets, 2012). For example, in the conventional allosteric model, a specialized temperature-sensitive transition is coupled to a temperature- and voltage-insensitive opening transition (Brauchi et al., 2004; Matta and Ahern, 2007). In contrast, others have suggested that temperature sensitivity arises from a weakly voltage-sensitive opening transition (Voets et al., 2004). Indeed, one recent study on voltage-activated potassium (Kv) channels suggests that the opening transition in these related channels can be made steeply temperature-dependent when the coupling between the voltage-sensing and pore domains is weakened (Yang et al., 2014). It has also been suggested that TRP channels may not contain localized temperature-sensors, but that their steep temperature-dependence results from changes in solvation of hydrophobic residues throughout the protein (Clapham and Miller, 2011).

Recent landmark near-atomic resolution structures of the TRPV1 channel confirm the presence of an internal gate related to that present in Kv channels (Liu et al., 1997; del Camino and Yellen, 2001). In addition, the external pore and selectivity filter change conformation in the combined presence of DkTx and the vanilloid resiniferatoxin (RTx) (Cao et al., 2013; Liao et al., 2013). Given our limited understanding of the temperature-sensitivity of TRPV1, it is unclear whether these structures provide insight into the mechanisms of heat activation. However, the structural rearrangements within the external pore are intriguing because mutations in this region have been reported to alter activation of the channel by protons (Jordt et al., 2000) and heat (Grandl et al., 2010; Cui et al., 2012; Yang et al., 2014). In addition, external Na⁺ ions have been reported to inhibit the TRPV1 channel at room temperature (Ohta et al., 2008), raising the possibility that an external cation binding site may regulate these channels. In the present study, we demonstrate the presence of a critical external Na⁺ ion-binding site that must be occupied for the channel to remain closed at room temperature, and to be available for opening by noxious heat. Our results also show that multiple steeply temperature-dependent transitions are allosterically coupled to an opening transition, and that binding of Na⁺ or DkTx to the external pore strongly influences the activity of the temperature-sensor and opening of the pore.

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 Na⁺ site stabilizes a closed state of TRPV1

To investigate the extent to which the large outward currents in external NMDG⁺ result from a change in driving force when exchanging external solutions, we obtained current-voltage (I-V) relations using rapid voltage ramps (Figure 1C) or voltage steps in cells exhibiting minimal rundown at room temperature (Figure 1—figure supplement 3). I-V relations for fully activated channels (10 µM capsaicin) in the presence of external Na⁺ or NMDG⁺ superimpose at positive voltages (Figure 1C and Figure 1—figure supplement 3), suggesting that the unitary conductance of TRPV1 channels is similar under these conditions. In addition, theoretical I-V relations calculated from the Goldman-Hodgkin-Katz equation predict little change in unitary conductance at positive voltages (Figure 1—figure supplement 4), and we confirmed that exchanging external solutions does not affect the single-channel current amplitude (i) at positive voltages in outside-out patches containing a few channels (Figure 2A). From these results, we conclude that exchanging external Na⁺ with NMDG⁺ leads to spontaneous opening of the TRPV1 channel at room temperature.

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To distinguish whether channel activation results from the removal of Na⁺ or the addition of NMDG⁺, we obtained voltage ramps in 130 mM NMDG_o in the additional presence of 130 mM Na_o (with 260 mM internal Na⁺) and observed only small outward currents at positive voltages that superimposed with those obtained in the presence 260 mM Na_o (Figure 2B). This result indicates that NMDG⁺ does not activate the channel in the presence of Na⁺, but rather removal of external Na⁺ leads to channel activation. Removal of internal Na⁺ in recordings using inside-out patches produced no channel activation (Figure 2C, left panel), whereas removing external Na⁺ using outside-out patches led to robust activation (Figure 2C, right panel), demonstrating that the inhibitory influence of Na⁺ is limited to the external side of the TRPV1 channel. We also measured the concentration-dependence for external Na⁺ inhibition over a range of voltages (Figure 2D) and found that inhibition was only weakly voltage-dependent, with an extrapolated apparent affinity (K_1/2) at 0 mV of 2 mM (Figure 2E). These results suggest that external Na⁺ stabilizes a closed state of the TRPV1 channel by binding to a site on the extracellular side of the protein. This ion-binding site is not highly selective for Na⁺, as external K⁺ can stabilize the closed state almost as effectively as Na⁺, followed by Rb⁺, Tris⁺, Cs⁺ and Li⁺ (Figure 2—figure supplement 1).

External Na⁺, protons and DkTx modulate the TRPV1 through overlapping mechanisms

An external Na⁺ binding site that stabilizes TRPV1 in a closed state is interesting because titratable acidic residues are commonly found in such sites (Lev et al., 2013) and acid is a physiological stimulus for this channel, with mildly acidic conditions sensitizing the channel to either capsaicin or heat, and strongly acidic conditions causing channel activation (Jordt et al., 2000;Ryu et al., 2003). To explore a possible mechanistic link between the external Na⁺ site and acid regulation, we changed external pH from 7.4 to 6 and observed a pronounced shift in the concentration-dependence for Na⁺ inhibition to higher Na⁺ concentrations (Figure 3A). Moreover, activation of the channel by pH 6 could be completely overcome by raising the Na⁺ concentration to 300 mM. These results are consistent with the expectation that protonation of acidic residues forming the Na⁺ binding site would lower the affinity of the metal ion, suggesting that the inhibitory Na⁺ binding site is intimately involved in the regulation of the TRPV1 channel by acid. However, acidic solutions further stimulated channel activation in the absence of external Na⁺ (Figure 3A), indicating that protons also activate TRPV1 channels through a Na⁺-independent mechanism. These findings support the idea that proton-mediated channel activation and proton-dependent potentiation of responses to other stimuli have distinct mechanisms (Jordt et al., 2000; Ryu et al., 2007).

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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.

The unambiguous identification of the Na⁺-binding site would require a high resolution structure of TRPV1. However, a glutamate at position 600 near the top of S5 (Figure 3—figure supplement 1A) has been previously associated with the inhibition of TRPV1 by external Na⁺ (Ohta et al., 2008), and has also been shown to be involved in the proton-dependent potentiation of TRPV1 responses to heat and capsaicin (Jordt et al., 2000). To confirm a key role of this residue in modulation of TRPV1 by external Na⁺, we investigated the effect of removing external Na⁺ on E600Q mutant channels, and found that the removal of external Na⁺ no longer produced constitutive activation even though the mutant remained responsive to capsaicin (Figure 3D). Consistent with DkTx and external Na⁺ having convergent mechanisms for modulating TRPV1, E600Q channels were not responsive to a concentration of DkTx that is saturating in WT channels (Figure 3D). These results demonstrate an important role for E600 in mediating channel inhibition by external Na⁺ and activation by DkTx, and suggest that this residue may be a part of the Na⁺ binding site.

Another interesting region within the outer pore of TRPV1 is the pore turret (Δ604–626, Figure 3—figure supplement 1B), a stretch of 22 residues that was removed in the construct of TRPV1 recently used for structure determination (Liao et al., 2013) and that is located near to E600 and to the binding site for DkTx (Figure 3—figure supplement 1A). We therefore explored whether the deletion of the pore turret retains modulation by external Na⁺. Initially, we found that the removal of external Na⁺ produced robust channel activation at depolarized potentials in channels lacking the pore turret (Figure 3E), and that the addition of 10 µM capsaicin in the presence of external Na⁺ further activated the channels, similar to WT TRPV1. Interestingly, the removal of the pore turret increased the apparent affinity of the channel for Na⁺ (Figure 3F), indicating that the pore turret does not form the Na⁺ binding site, but influences the cation-binding site through an allosteric mechanism. However, we also made the surprising discovery that the removal of external Na⁺ in the presence of 10 µM capsaicin resulted in enhanced channel activation at positive voltages relative to 10 µM capsaicin in the presence of 130 mM external Na⁺ (Figure 3E), in stark contrast with WT channels where I-V relations under these two conditions superimpose at depolarized potentials due to maximal channel activation (Figure 1C and Figure 3C). Increasing the concentration of capsaicin from 10 to 40 µM did not further activate Δ604–626 TRPV1 channels in the presence of external Na⁺ (Figure 3E), indicating that 10 µM is a saturating concentration and that capsaicin is a partial agonist for this construct. This reveals that the pore turret is mechanistically associated with the modulation of the channel not only by stimuli acting on the extracellular pore (e.g. external Na⁺), but also by capsaicin that binds near the intracellular part of the protein (Figure 1A).

Finally, we were interested in determining whether external Mg²⁺ ions activate the TRPV1 channel directly, as was suggested in two previous studies that analyzed the effect of substituting external Na⁺ with Mg²⁺ without considering the inhibitory effects of external Na⁺ (Cao et al., 2014; Yang et al., 2014). We tested the ability of 130 mM external MgCl₂ to activate the TRPV1 channel in the presence of 130 mM external NaCl (and 260 mM intracellular NaCl), as we did previously in Figure 2B to determine whether NMDG⁺ was an activator of TRPV1. Contrary to what we observed for NMDG⁺, Mg²⁺ ions produced robust activation of the TRPV1 channel in the presence of 130 mM external Na⁺ (Figure 3—figure supplement 1C), indicating that Mg²⁺ can activate the TRPV1 channel directly.

Collectively, our results thus far highlight a central role of the outer pore in the allosteric control of TRPV1 channel gating. Furthermore, they suggest that the binding of DkTx, protons and external Na⁺ to the extracellular pore of TRPV1 modulate channel function through at least partially overlapping mechanisms. We next set out to determine whether the external Na⁺ site has an influence in the mechanism of temperature-sensitivity of the TRPV1 channel, as protons have been shown to potentiate responses of the channel to heat (Jordt et al., 2000), and the outer pore has been associated with temperature-sensitivity (Myers et al., 2008; Grandl et al., 2010; Cui et al., 2012; Cao et al., 2014; Yang et al., 2014).

External Na⁺ is required for activation of TRPV1 by heat

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 (P_o) 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 P_o, 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 P_o, we estimated the temperature-dependence of ion conduction (Figure 4—figure supplement 1B–E) and then calculated P_o-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 P_o-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 (ΔH_app) values (see Figure 4—figure supplement 2C–E). At temperatures > 35°C, we observed steep temperature-dependence to P_o 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 P_o (Figure 4C and D, Figure 4—figure supplement 2C–E). However, the entire P_o-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.

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Having found that TRPV1 remains temperature-sensitive at room temperature and below in the presence of external Na⁺, we measured macroscopic current-temperature (I-T) relations in the absence of external Na⁺ (Figure 5A and B) and constructed P_o-T relations (Figure 5C). Remarkably, both I-T and P_o-T relations displayed greatly diminished slopes and readily detectable channel activity at the lowest experimentally accessible temperatures (Figure 5A–C). In addition, removal of external Na⁺ produced marked temperature-dependent inactivation of TRPV1 channels between 25 and 38°C (Figure 5B, see yellow arrow), a process that typically occurs after repetitive cycles of heating and cooling in the presence of extracellular Na⁺ (Joseph et al., 2013) and at temperatures above ~50°C in single heat-activation experiments (Figure 5B, see purple arrow). Notably, temperature-inactivated channels could not be subsequently activated by heating, but were partially responsive to saturating capsaicin (Figure 5—figure supplement 1A) (Cao et al., 2014). This facilitation of temperature-dependent inactivation in the absence of external Na⁺ can explain why removing Na⁺ also leads to progressive rundown of the TRPV1 channel at room temperature (Figure 1—figure supplement 1). We also observed that capsaicin diminishes rundown at room temperature when external Na⁺ was removed (Figure 5—figure supplement 1B), consistent with a mechanistic link between temperature-dependent inactivation and channel rundown in the absence of external Na⁺ at room temperature.

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We next explored why the TRPV1 channel lacks steep temperature-dependent activation in the absence of external Na⁺. One possibility is that the mechanism of heat activation remains intact, but that increases in P_o upon heating cannot be seen because channels run down after opening during temperature ramps (see Figure 7—figure supplement 5 and Materials and methods for an illustration using an allosteric model for channel gating). If this is the case, increasing the rate at which temperature is changed in the absence of external Na⁺ should diminish the effects of rundown and reveal the presence of heat activation. To examine this possibility, we measured I-T relations in either the presence (Figure 5D, large grey circles) or absence (Figure 5D, large orange circles) of external Na⁺ by rapidly jumping between solutions at different temperatures (Figure 5—figure supplement 3), and observed that I-T relations in the absence of external Na⁺ obtained from temperature-ramps and from jumps agreed up to ~25°C, after which they deviated markedly (Figure 5D, note deviation between orange circles and small yellow circles as highlighted by the blue lines). Additionally, the rate of temperature-dependent inactivation in the absence of external Na⁺ at T > 45°C became so fast that the speed of our temperature-jumps was not sufficient to visualize the increase in P_o before significant inactivation occurred (note the last orange circle in Figure 5D). These results suggest that temperature-dependent activation has been greatly perturbed in the absence of external Na⁺ at temperatures below 25°C, but that activation at higher temperatures appears largely intact (Figure 5D, compare orange and grey large circles). Taken together, these findings suggest that the Na⁺ regulatory site is strongly coupled to a temperature-dependent transition that governs TRPV1 activation at low temperature, and confirm the presence of multiple temperature-dependent transitions.

External Na⁺ tunes a temperature-dependent transition and stabilizes a closed state of the channel

The results obtained from measurements of P_o-T relations are depicted schematically in Figure 5E, where dotted horizontal lines represent plateau regions where steep temperature-dependence cannot be readily observed (ΔH_App~0), and arrows represent regions where P_o increases steeply with temperature (ΔH_App>> 0). The vertical dotted grey line indicates the lowest temperature that we can achieve experimentally, with the yellow arrow crossing that temperature representing a hypothetical temperature-sensitive transition in the absence of external Na⁺. We include this transition because the P_o-T relation in the absence of Na⁺ shows some temperature-dependence to P_o at T < 10°C (Figure 5C and 6A). To verify the existence of this transition in the absence of external Na⁺, we tested two manipulations that should shift the temperature range over which that transition operates. Capsazepine has been proposed to shift heat activation of TRPV1 to higher temperatures (Matta and Ahern, 2007), and we therefore tested whether this allosteric inhibitor might produce a rightward shift in the P_o-T relation, making the temperature- sensitive transition more readily detectable in the absence of external Na⁺. Indeed, the addition of capsazepine resulted in clear temperature-dependence of P_o at low temperatures in the absence of external Na⁺ (Figure 6A and C). Next, we tested intermediate external Na⁺ concentrations to see if temperature-dependent changes in P_o could be detected at lower temperatures. We obtained P_o-T relations at 10, 30 and 65 mM external Na⁺, and observed clear temperature-dependence at low temperatures (Figure 6B), with enthalpies close to those measured in the presence of 130 mM external Na⁺ in the same temperature range (Figure 6C). Taken together, these findings suggest that the TRPV1 channel undergoes the same conformational transitions in response to heat regardless of whether external Na⁺ is present, and thus that the enthalpy (ΔH^o) associated with Na⁺ binding is not itself responsible for temperature-dependent gating (for a more detailed discussion see Materials and methods). Rather, it seems that external Na⁺ modifies the midpoint (or entropy, △S) of the temperature-sensitive transition that operates at low temperatures. Additionally, external Na⁺ appears to stabilize a closed state of the channel independently of its effect on temperature-sensitivity, as the P_o values corresponding to the inferred plateau regions (denoted by dotted lines in Figure 6B - insert) decrease as the concentration of external Na⁺ is increased.

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An allosteric framework for the gating of TRPV1 in response to temperature and external Na⁺

These features of our results can be depicted in a simple allosteric model wherein two temperature-sensitive transitions are allosterically coupled to a temperature-insensitive opening transition, with Na⁺ regulating both the midpoint (△S) of the temperature-sensitive transition operating at low temperatures and the open/closed equilibrium (Figure 7A, see Materials and methods for an in-depth discussion on the choice of model and the inherent assumptions). In the presence of Na⁺, the two temperature-sensitive transitions have similar midpoints, such that the plateau between the two is barely noticeable in the P_o-T relation (Figure 6B insert, grey arrows). Upon Na⁺ removal, the midpoint of the first temperature-sensitive transition shifts to lower temperatures, effectively extending the middle plateau where P_o is temperature-independent (Figure 6B insert, yellow lines and arrows) and the channel mostly occupies the open/closed equilibrium highlighted with orange squares in Figure 7A. Additionally, external Na⁺ also negatively impacts the opening transition through allosteric coupling, which results in the intermediate plateau between the two temperature-dependent transitions in P_o-T relations having a lower P_o in the presence of external Na⁺. This simple model can qualitatively reproduce our results at intermediate Na⁺ concentrations (Figure 7B and Figure 6—figure supplement 1). One important feature of the model is that the opening transition is relatively temperature-insensitive, and that the channel can open even when the temperature sensor is in the deactivated state (Figure 7A, C – O transitions with blue squares). Although we would not rule out measurable changes in ΔH^o during opening, as shown by an alternate model that also satisfactorily reproduced our experimental results (Figure 7—figure supplement 1A), such a mechanism would require divergent temperature-sensitive transitions depending on whether external Na⁺ is bound to the channel, which we consider to be more conceptually complex than the allosteric model proposed here (see Materials and methods for a more detailed discussion). Additionally, in the alternate model ii, external Na⁺ influences the temperature-dependent transition that occurs at lower temperatures in the same way as in Model i (Figure 7—figure supplement 1A). We also cannot rule out the possibility that the temperature sensors have to activate for the channel to open (i.e., occupancy of the open states with blue squares in Figure 7A is negligible), as the gating of TRPV1 channels remains temperature-sensitive even at the lowest temperatures examined. However, in the absence of constraining experimental data, we have opted for the more general mechanism depicted in Figure 7A.

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Figure 7—source data 1

Analytical expressions for P_o in different models.

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Figure 7—source data 2

Parameters for the mathematical gating models.

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Voltage is also known to influence temperature-sensitivity of TRPV1 (Figure 4B and D) (Voets et al., 2004; Matta and Ahern, 2007; Yao et al., 2010). However, the low permeability of NMDG⁺ precluded examination of the influence of external Na⁺ on temperature-sensitivity of TRPV1 at negative voltages. Nonetheless, our data show that at the highly depolarized potentials where we performed our experimental studies and modeling, voltage does not have a predominant influence on temperature-sensitivity of this channel (see Figure 7—figure supplement 3 and Materials and methods for a more detailed discussion), and therefore we did not include voltage in our modeling.

The influence of capsaicin on temperature-dependent gating

Capsaicin is arguably the best characterized TRPV1 agonist, activating the TRPV1 channel to a maximal P_o close to 1 (Premkumar et al., 2002; Hui et al., 2003), and has also been shown to influence temperature-sensitivity of this receptor (Voets et al., 2004; Matta and Ahern, 2007; Yao et al., 2010). We were therefore interested in determining whether it exerts a similar influence on the temperature-sensitivity of TRPV1 as external Na⁺. To explore this possibility, we measured P_o-T relations using slow temperature ramps in the presence of 130 mM external Na⁺ and different concentrations of capsaicin (Figure 8A and Figure 8A—figure supplement 1A for P_o-T relations from individual cells). Although we observed detectable temperature-dependent changes in P_o at low temperatures for subsaturating concentrations of capsaicin, the P_o values varied by more than 100-fold over the range of capsaicin concentrations, suggesting that capsaicin has a larger effect on the closed/open transition compared to the temperature-dependent transition (Figure 8A - insert). This contrasts with the effects of removing external Na⁺, which produces much larger shifts in the P_o-T relation and smaller changes in P_o (Figure 6B). Notably, incorporating these features in our model (Figure 8B) can qualitatively describe our experimental findings with capsaicin (Figure 8A and Figure 8—figure supplement 1A). The P_o-T relation in saturating capsaicin exhibited marginal temperature-dependent changes, consistent with a large influence of capsaicin on the open/closed equilibrium of the channel leading to full receptor activation at a saturating concentration of this agonist (Figure 8A).

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If capsaicin has a relatively modest influence on the activation of the temperature-sensor (as compared to its larger effects on the open/closed equilibrium), then it should be possible to observe temperature-dependent changes in saturating capsaicin in a construct where capsaicin-bound channels are not fully activated, such as the turret deletion (Figure 3E). To test this possibility, we first determined whether TRPV1 channels lacking the pore turret are temperature-sensitive, as this is a controversial issue (Yang et al., 2010; Yao et al., 2010; Cui et al., 2012; Liao et al., 2013). Interestingly, we found that the turret deletion can still be robustly activated by heat, albeit at somewhat higher temperatures compared to the WT channel (Figure 9A), confirming that the turret is not the temperature sensor.

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To look for temperature-dependent changes in P_o for the turret-deletion construct at saturating capsaicin, we initially examined the effects of lowering temperature from 22 to 8°C on capsaicin activation in the presence of external Na⁺ compared to maximal activation in the presence of saturating capsaicin with no external Na⁺ (Figure 9B). At 8°C, the fractional activation by saturating capsaicin in the presence of external Na⁺ was substantially lower than what we observed at 22°C, revealing that lowering temperature further decreased the efficacy of capsaicin (Figure 9B; 8°C colored circles; 22°C colored ramps; see black arrows). We then obtained the full P_o-T relationship in the presence of external Na⁺ and capsaicin, as before for the WT channel, but for the turret deletion we observed large temperature-dependent changes in P_o, with two components in the relation separated by a clear plateau at intermediate temperatures (Figure 9C and Figure 9—figure supplement 1B for P_o-T relations from individual cells). These findings with the turret deletion further support the allosteric framework for conceptualizing the mechanism by which Na⁺ and capsaicin influence temperature-dependent gating. Notably, we could accurately describe the P_o-T relations in saturating capsaicin and the dose-response for external Na⁺ for the turret deletion using model i (Figure 9—figure supplement 1A and B), simply by assuming that the deletion of the pore turret increased the affinity of the channel for external Na⁺ and reduced the strength of the coupling between the binding of capsaicin and the opening of the pore (Figure 7—source data 2E). Together, these results qualitatively show that capsaicin influences both the activity of the temperature-sensor and the open/closed equilibrium, but appears to have a larger influence on the latter. Even though the values of the coupling parameters in our modeling are not well-determined, we believe that the larger influence of capsaicin on the open/closed equilibrium is robust enough to support this conclusion.

Influence of DkTx on temperature-dependent gating

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 P_o-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 P_o-T relations from individual cells). This finding is particularly striking when considering that the P_o 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).

Figure 10 with 1 supplement see all

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The present results examining the regulation of temperature-dependent gating by external Na⁺ and capsaicin have four important implications for understanding the gating mechanisms of the TRPV1 channel. First, our results demonstrate that the TRPV1 channel has evolved external Na⁺ binding sites that must be occupied for the channel to remain closed at physiological body temperatures in mammals. The steeply temperature-sensitive transition that normally opens TRPV1 in response to noxious heat still operates in the absence of Na⁺, yet without external Na⁺ the channel would be spontaneously open at physiological temperatures, many channels would be inactivated, and responses to noxious heat would therefore be diminished. Our results also show that the Na⁺ sites will play physiologically important roles in activation of the channel by acid, as protonation of acidic residues involved in binding Na⁺ leads to diminished Na⁺ occupancy and activation of the channel. This connection between Na⁺ and pH regulation provides a mechanistic explanation for previous observations where mildly acidic solutions potentiate activation of TRPV1 by heat (Jordt et al., 2000). Thus, this Na⁺ regulatory mechanism plays a fundamental role in tuning the properties of TRPV1 to serve as a detector of noxious heat and acid, and possibly other stimuli that have yet to be examined.

Second, our results reveal the existence of multiple temperature-sensitive transitions that are coupled to the opening transition (see model in Figure 7A ). Although we see only hints of the presence of distinct temperature-dependent transitions in the presence of external Na⁺ (Figure 4C), they become unambiguous when Na⁺ is removed (Figure 5D)and when studied in the presence of capsaicin with the turret deletion construct (Figure 9C). Our results suggest that the role of the first temperature-sensitive transition is to maintain the channel in a closed state at physiological temperatures, whereas the second transition functions to activate the channel in response to noxious heat. It is interesting that both Na⁺ and capsaicin do not seem to have a substantial impact on the second transition, at least to the extent that we could study this component in the presence of inactivation. In contrast, external Na⁺ exerts strong control over the first transition. Capsaicin also modifies the shape of macroscopic P_o-T relations (Figure 8A), but we were able to detect temperature-dependent transitions at low temperatures even in the presence of near-saturating capsaicin concentrations (e.g. 100 nM) that increase the P_o at room temperature to values higher than those measured in the absence of external Na⁺ or in the presence of DkTx (Figure 3C). In contrast, we observed marginal to no temperature-dependence in the P_o-T relations at low temperatures in the absence of external Na⁺ or in the presence of DkTx (Figure 5D and Figure 10). These observations suggest that the vanilloid has a larger influence on the open/closed equilibrium than on the temperature-sensing machinery. The existence of multiple temperature-sensitive transitions also provides a possible explanation for why P_o-T relations at negative voltages are at least two-fold steeper than at positive voltages (Figure 4B) (Voets et al., 2004; Yao et al., 2010). If hyperpolarization shifts the temperature-sensitive transition operating at lower temperatures so as to coincide with the second temperature-sensitive transition, the △H^o of the two transitions would add to make the P_o-T steeper. Overall, the present results on the influence of Na⁺ and capsaicin on temperature-dependent gating in TRPV1 provide a set of essential constraints for understanding the gating mechanism of the channel using an allosteric framework.

Third, our results provide clues about where these important Na⁺ binding sites are located. Because Na⁺ inhibition has only very weak voltage-dependence, these Na⁺ binding sites are likely to be located outside the ion permeation pathway within each subunit. Indeed, the large outward Na⁺ currents we observe on removing external Na⁺ at positive voltages indicate that the permeation pathway must have a high Na⁺ occupancy even in the absence of external Na⁺. Mutating acidic residues forming the external Na⁺ site would weaken Na⁺ binding affinity, possibly leading to lower Na⁺ occupancy in the presence of 130 mM external Na⁺. However, such an effect would not necessarily cause spontaneous TRPV1 activity because the lower Na⁺ occupancy would be expected to promote the form of inactivation that we observed on removing external Na⁺. Additionally, removing external Na⁺ would be expected to cause less activation of the channel compared to the WT protein because ion occupancy would be diminished by the mutant. E600 in the extracellular loop just after the S5 helix (Figure 3—figure supplement 1A) has been proposed to be responsible for proton-mediated potentiation of heat- and capsaicin-dependent activation of the channel (Jordt et al., 2000), making it an excellent candidate for forming the Na⁺ binding site. The E600Q mutant displayed modest spontaneous activity when recording at room temperature (Ohta et al., 2008), and only weak activation when removing external Na⁺ or applying DkTx when compared with the WT channel, consistent with a role in forming the Na⁺ binding site. Interestingly, E600 is located near the pore turret (Liao et al., 2013) (Figure 3—figure supplement 1A), which when deleted from TRPV1 causes a dramatic increase in the apparent affinity of Na⁺ (Figure 3F), and this residue is also located very close to where DkTx binds (Cao et al., 2013) (Figure 3—figure supplement 1A and Figure 10—figure supplement 1A).

Finally, our results have important implications for interpreting the recent cryo-EM structures of TRPV1 and in localizing the region of the protein involved in temperature sensing. In the cryo-EM studies on the turret deletion construct of TRPV1, capsaicin appears to produce a partial opening of the internal pore, and the combined influence of RTx and DkTx to produce further expansion of the internal pore together with a conformational change within the selectivity filter (Cao et al., 2013) (Figure 10—figure supplement 1A). These differences between the two structures might indicate that there are two gates that can be differentially regulated by activating stimuli (Cao et al., 2013) (Figure 1A). However, we discovered that capsaicin is a partial agonist of the turret deletion construct, suggesting that the partially open features of the capsaicin-bound channel may not represent a unique conformation of the protein, but an average of closed and open states. The cryo-EM structure of the apo state of TRPV1 (Liao et al., 2013), however, reveals that the internal pore must expand for the pore to support ion permeation, consistent with accessibility studies (Salazar et al., 2009). The observation of temperature-independent plateaus in P_o-T relations under diverse conditions that produce submaximal open probabilities suggests that the opening of the pore does not require the two temperature-dependent transitions to occur, and that the open/closed equilibrium itself is not the source of the temperature-sensitivity of TRPV1. If opening of the internal pore is indeed temperature-insensitive, what is the significance of the structural changes in the external pore observed in the structure of the RTx/DkTx-bound channel? The present findings with external Na⁺ and DkTx suggest that these structural changes in the external pore are tightly associated with the temperature-sensing machinery, either by participating in allosteric coupling or in the actual mechanism of temperature-sensing.

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Author details

1. Andres Jara-Oseguera

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

   Contribution

   AJO, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Competing interests

   No competing interests declared.

2. Chanhyung Bae

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

   Contribution

   CB, Produced recombinant DkTx, Conception and design, Acquisition of data, Drafting or revising the article, Contributed unpublished essential data or reagents

   Competing interests

   No competing interests declared.

3. Kenton J Swartz

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

   Contribution

   KJS, Conception and design, Analysis and interpretation of data, Drafting or revising the article

   For correspondence

   swartzk@ninds.nih.gov

   Competing interests

   KJS: Reviewing editor, eLife.

   "This ORCID iD identifies the author of this article:" 0000-0003-3419-0765

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