Transient receptor potential (TRP) cation channels are involved in a diverse array of physiological functions (Li, 2017), with many of them acting as sensory detectors of stimuli such as temperature, natural products and various cell-signaling molecules (Flockerzi, 2007). Structurally, TRP channels are tetramers, with each subunit containing six transmembrane helices (S1-S6); the region encompassing the S5 through S6 helices forms the central ion-conducting pore, and the S1-S4 helices of each subunit form voltage sensor-like domains located peripherally in a domain-swapped arrangement (Madej and Ziegler, 2018).

Consistent with the remarkable diversity of biological roles that TRP channels play, the types of stimuli that activate these channels also largely differ between TRP subtypes, even among closely related members of the same TRP channel subfamily (Flockerzi, 2007; Li, 2017). However, many important structural features are highly conserved within each TRP channel subfamily, and between TRP channels in general (Palovcak et al., 2015; Bae et al., 2018; Kasimova et al., 2018; Madej and Ziegler, 2018; McGoldrick et al., 2018; Zheng et al., 2018a; Zheng et al., 2018b; Zubcevic et al., 2018a; Zubcevic et al., 2018b), raising the possibility that TRP channels that are sensitive to different stimuli share mechanisms of activation downstream of their ligand-interaction sites. This is well exemplified by TRPV1 and its closest homologue, TRPV2 (48.4% of sequence identity). TRPV1, an integrator of pain-producing stimuli in nociceptors (Moore et al., 2018), is the only member of the TRPV (vanilloid) subfamily of TRP channels that can be activated by vanilloid compounds (Caterina et al., 1997; Jordt and Julius, 2002), cysteine-reactive molecules (Salazar et al., 2008), and some agonists that directly interact with the extracellular face of the pore domain, such as protons (Tominaga et al., 1998; Jordt et al., 2000) and the double-knot toxin (DkTx) from tarantula venom (Bohlen et al., 2010; Cao et al., 2013; Bae et al., 2016; Gao et al., 2016). In contrast, TRPV2 is activated by very few known stimuli, including heat (Caterina et al., 1999; Yao et al., 2011), the non-selective channel modulator 2-aminoethoxydiphenyl borate (2-APB) (Hu et al., 2004) and cannabinoid compounds (Qin et al., 2008), all of which also influence the activity of TRPV1 (Hu et al., 2004; Qin et al., 2008). In this context, it is remarkable that only four mutations in TRPV2 (TRPV2-4M) are sufficient to enable robust activation by the TRPV1-specific vanilloid agonist resiniferatoxin (RTx) (Yang et al., 2016; Zhang et al., 2016; Zubcevic et al., 2018b), suggesting that the sites where vanilloids interact with TRPV1 and TRPV2-4M are very similar, and that these channels share mechanisms of activation. Indeed, structural studies have confirmed that RTx binds to the same site between the S1-S4 domains and the pore domain in both TRPV1 (Cao et al., 2013; Gao et al., 2016) and TRPV2-4M (Zubcevic et al., 2018b) (Figure 1—figure supplement 1A).

In the present study, we explored whether vanilloid sensitivity could be engineered into the TRPV3 channel, which shares 42% sequence identity with TRPV1. TRPV3 is expressed primarily in mammalian keratinocytes, where it is required for the formation of the skin barrier (Cheng et al., 2010), and mutations in the channel can lead to skin diseases such as Olmsted Syndrome (Lin et al., 2012; Zhi et al., 2016). TRPV3 is a polymodal detector of warm temperatures (Peier et al., 2002; Smith et al., 2002; Xu et al., 2002), chemicals such as 2-APB (Chung et al., 2004; Hu et al., 2004) and multiple natural products such as camphor, eugenol and thymol (Xu et al., 2006; Vogt-Eisele et al., 2007). To probe for RTx sensitivity in TRPV3, we mutated six residues in mouse TRPV3 (TRPV3-6M) corresponding to the vanilloid-binding pocket of rat TRPV1, and found that although RTx can bind to the mutant channel, activation of TRPV3-6M by RTx requires facilitation of the opening transition, either by introducing mutations in the pore domain, or sensitizing the channel with heat or 2-APB. Our results provide functional evidence for conserved mechanisms of activation within the TRPV family, while also providing important information on TRPV3-specific activation properties.

The goal of the present study was to determine whether sensitivity to vanilloids could be engineered into the TRPV3 channel. Our results demonstrate that like TRPV2, the TRPV3 channel contains a ‘defunct’ vanilloid-binding site that can be readily made functional by mutating a few key positions in the binding pocket. More importantly, our results show that the allosteric machinery required for coupling vanilloid-binding to pore-opening has remained functional through evolution in both TRPV2 and TRPV3 channels, even though these two channels are not naturally sensitive to vanilloid molecules. This points to the presence of conserved mechanisms of gating among structurally related but functionally distinct TRP channels. In contrast to TRPV2, RTx binding to the engineered vanilloid site in TRPV3 was not sufficient to promote channel activation at room temperature, which required further manipulations: either co-stimulation with heat or sensitization with 2-APB, or the introduction of single point mutations at one of 10 identified positions throughout the pore that are conserved in TRPV1 and TRPV2 but differ in TRPV3. This indicates that agonist sensitivity in TRP channels is not only fine-tuned by the structure of the agonist-binding pocket, but also by the intrinsic energetics of activation that are influenced by multiple sites throughout the pore. Out of the five positions in the pore that enabled robust RTx activation in the TRPV3-6M construct, A606 at the extracellular end of the S5 helix had an effect on temperature-activation and moderately increased sensitivity to 2-APB. In contrast, the other four positions had no clear influence on heat-dependent activation and only produced a large (>20 fold) increase in 2-APB sensitivity when introduced cumulatively and together with A606V. These findings point to the presence of non-overlapping allosteric networks in the pore, and likely the rest of the protein, that selectively influence activation by specific stimuli.

It is intriguing that both TRPV2 and TRPV3 can respond to RTx once the vanilloid pocket has been engineered. The presence of densities near the vanilloid pocket in the cryo-EM structures of apo-TRPV1 (Gao et al., 2016), -TRPV2 (Zubcevic et al., 2016), -TRPV3 (Zubcevic et al., 2018a), -TRPV5 (Hughes et al., 2018b) and -TRPV6 (McGoldrick et al., 2018) have led to the hypothesis that the site can also be occupied by lipids in these channels, leading to inhibition of TRPV1 (Gao et al., 2016) or activation of TRPV6 (McGoldrick et al., 2018). Indeed, it has been shown that the endogenous monounsaturated fatty acid oleic acid inhibits TRPV1 by binding to the vanilloid pocket (Morales-Lázaro et al., 2016). This raises the possibility that the functional coupling between the vanilloid site and the pore is maintained in vanilloid-insensitive TRPV channels because it is important for their modulation by lipids or other endogenous modulators.

How similar are the mechanisms for RTx activation in TRPV1, TRPV2-4M and TRPV3-6M? The overall fold of the vanilloid site, located at a key interface between the S1-S4 domain and the pore (Jordt and Julius, 2002; Cao et al., 2013; Gao et al., 2016; Zhang et al., 2016; Singh et al., 2018; Zubcevic et al., 2018b), and the overall orientation of side-chains in the binding site known to establish key interactions for activation by RTx are highly conserved between TRPV1, TRPV2 and TRPV3 channels (Figure 1—figure supplement 1A). Indeed, the crystal structure of the TRPV2-4M channel solved with RTx bound (Zubcevic et al., 2018b) suggests a similar binding pose for the vanilloid when compared to the structure of TRPV1 bound to RTx and the double knot toxin (DkTx) (Figure 1—figure supplement 1A). Interestingly, whereas the cryo-EM structures of closed, apo TRPV2 display four-fold (C4) symmetry relative to the central pore axis (Huynh et al., 2016; Zubcevic et al., 2016), the crystal structures of TRPV2-4M obtained in non-conducting conformations in the absence and presence of RTx (Zubcevic et al., 2018b) have symmetry-breaking features in the S4-S5 linker resulting in C2-symmetry for the tetrameric channel. The break in C4-symmetry was therefore proposed as an intermediate step in the mechanism of activation by RTx in TRPV2-4M (Zubcevic et al., 2018b). A similar symmetry-breaking step was suggested for activation of human TRPV3 by 2-APB (Zubcevic et al., 2018a), where a subpopulation of 2-APB-treated particles adopt non-conducting conformations with central C2-symmetry, whereas structures of a fully open, 2-APB-bound conformation of mouse TRPV3 and of non-conducting, apo states of both human and mouse TRPV3 exhibit C4 symmetry (Singh et al., 2018; Zubcevic et al., 2018a). In contrast to TRPV2 and TRPV3, no intermediate conformations with C2 symmetry have been identified for TRPV1, even if the data-set for the RTx/DkTx/TRPV1 complex is analyzed without imposing a central C4-symmetry axis (Bae et al., 2016; Gao et al., 2016). A comprehensive exploration of the conformational space of these proteins, including structures of RTx-bound TRPV1 in the absence of DkTx, and fully open RTX-bound TRPV2-4M, will be required to understand how changes in symmetry are associated with activation in each of these proteins.

Another difference between structurally-inferred activation mechanisms in TRPV1 and other TRPVs involves an α- to π-helical conversion of a segment of the S6 helix, which results in a disruption in the helix-stabilizing backbone hydrogen bonds in that segment and is proposed to function as a hinge through which the lower S6 tilts to open the internal entrance to the pore (Palovcak et al., 2015; Zubcevic et al., 2016; Kasimova et al., 2018; McGoldrick et al., 2018) (Figure 2—figure supplement 1B,D). The S6 helices in the non-conducting, apo structures of TRPV2 (Huynh et al., 2016; Zubcevic et al., 2016; Zubcevic et al., 2018b), TRPV3 (Singh et al., 2018; Zubcevic et al., 2018a) and TRPV6 (McGoldrick et al., 2018) are entirely α-helical, whereas the S6 helices in the open-state structures of TRPV3 and TRPV6 contain a π-helical bulge from which their inner portion tilts, opening the activation gate and exposing a different face of the helix to the central aqueous pore (Figure 2—figure supplement 1B and D) (McGoldrick et al., 2018; Singh et al., 2018; Zubcevic et al., 2018a). In the case of TRPV1, all structures in either open or non-conducting conformations contain a π-bulge in the S6 (Figure 2—figure supplement 1C) (Cao et al., 2013; Liao et al., 2013; Gao et al., 2016). More studies are therefore required to determine whether the S6 of TRPV1 also undergoes an α- to π-helix transition during activation, or whether TRPV1 contains a persistent π-helical bulge that shifts positions along the helix to enable gating (Palovcak et al., 2015).

It is intriguing to consider whether the other members of the TRPV family could be engineered to be activated by RTx. On the one hand, the structure of the zebrafish TRPV4 channel exhibits significant differences relative to all other TRPVs, particularly in the orientation of the S1-S4 domains relative to the pore (Deng et al., 2018). This results in considerable changes in the region of TRPV4 corresponding to the vanilloid pocket of TRPV1 (Figure 1—figure supplement 1B), making it unlikely that RTx could bind in the state represented by the structures. On the other hand, the site corresponding to the vanilloid pocket in the structures of TRPV5 (Hughes et al., 2018a) and TRPV6 (McGoldrick et al., 2018) is very similar to that of RTx-sensitive TRPV channels (Figure 1—figure supplement 1B). Interestingly, non-protein densities in the vanilloid pocket observed in cryo-EM structures of TRPV5 (Hughes et al., 2018a) and TRPV6 (McGoldrick et al., 2018) have been proposed to represent either the bound inhibitor econazole or a channel-activating lipid, respectively, raising the possibility that the pocket may be functionally coupled to the pore in these channels.

It is interesting that in contrast to TRPV1 and TRPV2-4M, binding of RTx to TRPV3-6M does not readily elicit activation at room temperature. Before any previous stimulation, activation of TRPV3 seems to involve a transition with a large energy barrier, as either higher concentrations of 2-APB or temperatures > 50°C are initially required for robust activation when compared to subsequent stimulations, which elicit maximal activation at lower temperatures (<40°C) or 2-APB concentrations (Chung et al., 2004; Liu et al., 2011; Liu and Qin, 2017). This ‘sensitization’ is irreversible over the explored time-scale of several minutes, indicating that the return kinetic barrier towards the initial closed state is also high, and making it possible to shift the net apparent gating equilibrium towards the open state by either surpassing or reducing this kinetic barrier (Liu et al., 2011). Activation of TRPV1 and TRPV2 by heat involves significant hysteresis as well, as both the apparent threshold for activation and the steepness of the current-temperature relations are reduced after channels have been initially activated by heat (Liu and Qin, 2016; Sánchez-Moreno et al., 2018). However, in contrast to TRPV3, TRPV1 channel opening by the vanilloid capsaicin does not involve hysteresis (Liu and Qin, 2016), suggesting that TRPV1 activation by vanilloids does not necessarily require overcoming a large initial energy barrier. Furthermore, initial activation of TRPV1 and TRPV2 with 2-APB sensitizes both channels to subsequent 2-APB applications (Liu and Qin, 2016), but to a lesser degree than in TRPV3 (Chung et al., 2004; Liu et al., 2011) and more importantly, without influencing the apparent threshold or sensitivity to activation by heat (Liu and Qin, 2016). Together, these observations raise the possibility that a large energy barrier is an obligatory component of activation for TRPV3, whereas activation of TRPV1 and TRPV2 by some stimuli follows a path that does not involve a large kinetic barrier. The structural origin of this kinetic barrier is not known, but it is interesting to note that the open and closed TRPV3 structures exhibit far more drastic differences than those of open and closed TRPV1 (Cao et al., 2013; Gao et al., 2016) and TRPV6 (McGoldrick et al., 2018) structures, including significant changes in the length of the S6 and TRP helices, and a 20° rotation of the rest of the channel relative to the pore domain (Singh et al., 2018). Furthermore, a structure of human TRPV3 was recently obtained under conditions that favor sensitization, and it also exhibits significant conformational changes in the S6, the TRP box and the S4-S5-linker helices as compared to the apo closed state (Zubcevic et al., 2018a). Based in these structures it was suggested that the α- to π-transition in the S6 helix observed between the human apo and the 2-APB-sensitized structures may be part of the sensitization mechanism in TRPV3 (Zubcevic et al., 2018a), as no such transition has been structurally observed for TRPV1 or TRPV2 (see discussion above). It is therefore possible that RTx binding to TRPV3-6M channels is not sufficient to overcome this large energy barrier, either because the vanilloid site in TRPV3-6M may lack the allosteric connections to influence this slow transition, or because of energetically weaker coupling of the RTx site to the gating machinery. It is likely that the binding of RTx to TRPV3-6M and TRPV2-4M and/or its coupling to the pore might not function as efficiently as in TRPV1. Indeed, RTx-sensitive TRPV2 and TRPV3 constructs cannot be activated by capsaicin (Yang et al., 2016; Zhang et al., 2016) (Figure 5—figure supplement 1A and data not shown), a vanilloid exhibiting much lower apparent affinity when compared to RTx.

If RTx-binding to TRPV3-6M does not lead to channel activation because it is not capable of overcoming the initial energy barrier, it is expected that stimuli capable of directly activating TRPV3 channels, such as heat or 2-APB, could enable RTx activation, as we show in the present manuscript. How is RTx-sensitivity enabled in TRPV3-6M by each of the identified mutations in the pore? The A606V mutation facilitates activation by all tested stimuli, suggesting that it has a global effect on gating, possibly by favoring activation of the heat-sensing machinery, enhancing the efficacy of coupling between the stimulus-sensing domains and the pore, directly reducing the large kinetic barrier in the activation pathway and/or directly stabilizing the open state. A global effect of the mutation on gating is consistent with the lower Q₁₀ values for all heat-sensitive TRPV3 constructs containing the A606V mutation (Figure 3—figure supplement 2) relative to the Q₁₀ for TRPV1, as expected for ‘sensitized’ TRPV3 channels (Liu et al., 2011). This ‘global’ effect on gating of A606V could also explain the non-additivity in the increase in apparent affinity for 2-APB observed in TRPV3-5PM (~20 fold) relative to TRPV3-4PM (2-fold), which lacks the A606V mutation, and TRPV3-A606V (3.5-fold) (Figure 4F). Structurally, residue A606 at the top of the S5 helix is tightly packed against the pore helix of the same subunit (Figure 2—figure supplement 1A,B), so that the A606V mutation might influence the relative positioning of these two helices, resulting in a global effect on gating. It will be interesting to investigate how A606 and RTx sensitivity relate to position S404 in the membrane-proximal N-terminal domain that has been associated with temperature-dependent sensitization in TRPV3 (Liu and Qin, 2017).

The mechanism for the other four pore mutations that also enable robust RTx activation is more puzzling, as they all had negligible effects on activation by heat and 2-APB, suggesting that they specifically influence activation by RTx. The side-chain of V587L could potentially contribute to RTx-binding, as the residues at equivalent positions in the RTx/DkTx-bound TRPV1 (Figure 2—figure supplement 1C) (Cao et al., 2013; Gao et al., 2016) and RTx-bound TRPV2-4M (Zubcevic et al., 2018b) structures are ~4 Å from the vanilloid. However, this position has not been previously identified as an important determinant for vanilloid binding in TRPV1 (Yang et al., 2015; Elokely et al., 2016; Zhang et al., 2016). Importantly, V587 is located at a critical junction between the S4-S5 linker- and S5-helices that undergoes an α to π transition between the apo-TRPV2 and RTx-bound TRPV2-4M structures (Zubcevic et al., 2018b). This transition appears to be fundamental for coupling RTx-binding to gating in this channel, which is consistent with a key role of the S4-S5 linker in transducing the binding of capsaicin or RTx to the opening of the pore proposed for TRPV1 based on structural (Cao et al., 2013; Gao et al., 2016) and functional data (Yang et al., 2015; Yang et al., 2018). It is therefore possible that V587L specifically enhances the coupling between RTx binding and gating by influencing the conformational dynamics of the S4-S5 linker helix.

Together with V587L, mutations F625L (pore helix) and F656I (S6 helix), which promote strong responses to RTx, and I595L (S5 helix) and Y650F (S6 helix), which promote weaker responses, are all located at a protein/membrane interface that includes the vanilloid-binding pocket (Figure 2—figure supplement 1A,B). Multiple non-protein densities observed at that interface in the apo- or RTx/DkTx-bound structures of TRPV1 in nanodiscs have been proposed to correspond to bound lipids (Gao et al., 2016), which take different positions in the two structures and suggest gating-associated lipid rearrangements (Figure 2—figure supplement 1C). For TRPV3 it was suggested that activation by 2-APB involves the expulsion of a lipid bound near side chain Y650, based on a non-protein density close to Y650 in the apo structure of mouse TRPV3 that is absent in the 2-APB-bound open structure (Singh et al., 2018). Channel-lipid interactions at that interface could play a role in channel activation by RTx, raising the possibility that mutations in that interface that enable RTx activation of TRPV3-6M do so by altering protein-lipid interactions.

The lack of a global effect on gating by mutation F666Y, which enables strong responses to RTx in TRPV3-6M and is located at the π-helical portion of S6 (Figure 2—figure supplement 1B,D), suggests that the mutation does not largely influence the α- to π-helix transition. The equivalent residue Y671 in TRPV1 has been proposed to be responsible for coupling capsaicin-induced conformational changes in S6 to the selectivity filter and pore-helices (Palovcak et al., 2015; Kasimova et al., 2018). It is therefore possible that F666Y has a similar role in activation of TRPV3-6M by RTx, while activation by 2-APB and temperature could engage other mechanisms to influence the conformation of the filter and pore-helices. L635F and L639M also enable weak responses to RTx, and are in the pore-helix or selectivity filter, respectively, consistent with the hypothesis that conformational changes in those regions are important in the late stages of the mechanism of TRPV1 activation by capsaicin (Kasimova et al., 2018; Yang et al., 2018). However, contrary to the stimulating effect of the L635F mutation in TRPV3-6M, Phe to Leu substitutions at equivalent positions in TRPV1 (F640L) or TRPV2 (F603L) also result in potentiated responses to heat or 2-APB, respectively, and higher basal channel activity (Myers et al., 2008). It will be interesting to investigate the apparently opposite effects on gating that Leu or Phe side-chains have at that position in TRPV1 and TRPV2 vs TRPV3 channels. Finally, the five identified pore mutations added cumulatively to TRPV3 did not have an effect on activation by other TRPV1-specific stimuli, such as extracellular protons (Jordt et al., 2000) or double knot toxin (DkTx) (Bohlen et al., 2010; Cao et al., 2013; Gao et al., 2016) (Figure 5—figure supplement 1B,C), consistent with the absence of binding sites for these two modulators in TRPV3.

The fact that conservative side-chain modifications, such as V587L or I595L, can have such a dramatic effect on the response to RTx in TRPV3-6M raises the possibility that other relatively subtle structural alterations, such as those caused by the binding of another ligand, a post-translational modification, or an alteration in the properties of the membrane, could enable responses to channel modulators that would otherwise seem inactive. The picture of TRPV channel gating that emerges from this study is one where fine structural differences dictate the response of a channel to an agonist or combination of agonists. Understanding these differences might be key to unraveling the mechanisms of allosteric signal integration in TRP channels.

All data generated and analyzed for this manuscript can be visualized in the figures.

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Author details

1. Feng Zhang

   1. Molecular Physiology and Biophysics Section, Porter Neuroscience Research Center, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, United States
   2. Department of Biochemistry, University of Utah, Salt Lake City, United States

   Contribution

   Conceptualization, Data curation, Formal analysis, Investigation, Writing—original draft, Performed experiments

   For correspondence

   feng.zhang2@nih.gov

   Competing interests

   No competing interests declared

2. Kenton Jon 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

   Conceptualization, Resources, Supervision, Funding acquisition, Validation, Project administration, Writing—review and editing

   Competing interests

   Reviewing editor, eLife

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

3. 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

   Conceptualization, Data curation, Software, Formal analysis, Supervision, Funding acquisition, Validation, Visualization, Methodology, Writing—original draft, Writing—review and editing

   For correspondence

   andres.jara-oseguera@nih.gov

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-5921-9320

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