Symmetry transitions during gating of the TRPV2 ion channel in lipid membranes

Transient Receptor Potential V (TRPV) channels are part of the larger TRP channel family which plays important roles in numerous physiological processes (Clapham et al., 2001). A subset of TRPV channels, including subtypes TRPV1-TRPV4, possess an intrinsic capability to sense heat and are therefore referred to as thermoTRPV channels (Cao et al., 2013a; Liu and Qin, 2016; Smith et al., 2002; Chung et al., 2003). TRPV1-TRPV4 are non-selective cation channels which play important physiological roles in sensing noxious heat (Bölcskei et al., 2010; Julius, 2013; Marwaha et al., 2016; Mitchell et al., 2014), maintaining cardiac structure (Katanosaka et al., 2014) and maintaining skin (Eytan et al., 2014; Imura et al., 2009; Kim et al., 2016), hair (Asakawa et al., 2006; Imura et al., 2007; Xiao et al., 2008) and bone physiology (Masuyama et al., 2008). A distinctive feature of TRPV1 and TRPV2 is their permeability to large organic cations (Chung et al., 2008), such as the cationic dye YO-PRO-1 and the sodium channel blocker QX-314. This feature has led to proposals to utilize these channels as conduits for delivering small molecules to intracellular targets (Puopolo et al., 2013). The non-conducting structures of TRPV1 and TRPV2 possess two restrictions, one at the selectivity filter (SF) and second one at the intracellular mouth of the pore (termed the common gate) (Liao et al., 2013; Zubcevic et al., 2016; Huynh et al., 2016). Both restrictions must open widely to accommodate the passage of large organic cations. However, the mechanism that enables such opening was long unclear. In order to study the permeation of both metal ions and large organic cations in TRPV2, we recently crystallized the rabbit resiniferatoxin (RTx)-sensitive (Zhang et al., 2016) TRPV2 channel with a truncation in the pore turret in the presence of the agonist RTx (Zubcevic et al., 2018a). This study led to the revelation that the binding of RTx leads to a two-fold symmetric (C2) opening at the selectivity filter that is wide enough to permeate YO-PRO-1. This unexpected result offered the first experimental evidence that the homotetrameric TRPV2 can adopt C2 symmetric conformations during the gating cycle. However, it was unclear if crystal contacts or the crystallization conditions (e.g. high concentration of Ca²⁺) played a role in stabilizing the C2 symmetry. In addition, the minimal TRPV2 construct used in the crystallographic study lacked the pore turret, a region that is not essential for function (Liao et al., 2013; Zubcevic et al., 2016; Zubcevic et al., 2018a; Yao et al., 2010) but had previously been shown to have a modulatory effect on gating in TRPV1 and TRPV2 (Jara-Oseguera et al., 2016; Dosey et al., 2019). It was uncertain if the absence of this region in our crystallographic study affected the symmetry of the channel.

In order to answer these questions and further study the role of two-fold symmetry in TRPV channel gating, we conducted cryo-electronmicroscopy (cryo-EM) studies of the full-length, RTx-sensitive rabbit TRPV2 (Zhang et al., 2016) channel reconstituted into nanodiscs and amphipol. We present four structures of the TRPV2/RTx complex, one obtained in nanodiscs (TRPV2_RTx-ND) and three in amphipol (TRPV2_{RTx-APOL 1-3}) determined to 3.8 Å, 2.9 Å, 3.3 Å and 4.2 Å resolution, respectively (Table 1, Figure 1). Our data shows that binding of RTx induces C2 symmetric conformations in TRPV2, but the extent of C2 symmetry depends on the environment in which the channel is reconstituted. C2 symmetry is particularly pronounced in the dataset collected from nanodisc-reconstituted TRPV2, which better approximates the physiological environment of the channel. Moreover, the data offers further insights into the allosteric coupling between the RTx-binding site and the activation gates in TRPV2, confirms the critical role of the S4-S5 linker π-helix (S4-S5_π-hinge) in ligand-dependent gating of TRPV2, and provides a glimpse of the conformational landscape of TRPV2 gating.

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In order to capture the RTx-induced gating transitions in the rabbit TRPV2 channel, we conducted cryo-EM studies of the TRPV2/RTx complex reconstituted into amphipol (TRPV2_RTx-APOL) and nanodiscs (TRPV2_RTx-ND). Amphipols (Zoonens and Popot, 2014) have been a useful tool in structural studies of membrane proteins, and especially TRP channels (Liao et al., 2013; Zubcevic et al., 2016; Cao et al., 2013b; Paulsen et al., 2015; Yoo et al., 2018; Hirschi et al., 2017; Zubcevic et al., 2018b). Indeed, Amphipol A8-35 enabled the very first structural determination of the TRPV2 channel (Zubcevic et al., 2016). Nanodiscs, on the other hand, represent the closest in vitro approximation to the native lipid membranes used in structural studies (Denisov and Sligar, 2016). The data was processed using RELION (Scheres, 2012) (Materials and methods), with no symmetry imposed during particle classification and 3D reconstruction in order to avoid obscuring any classes with lower symmetry (C1 and C2) that might exist in the sample. Symmetry was only imposed in the last step of the refinement and only if the 3D reconstructions showed clear two-fold (C2) or four-fold (C4) symmetry (Figure 1—figure supplements 1–3). Classification of the TRPV2_RTx-APOL sample revealed the presence of four classes: one low-resolution (~7 Å) class, which was excluded from further analysis, and three higher resolution classes which are representative of three different conformations. These include one C4 symmetric and two distinct C2 symmetric classes refined to 2.9 Å, 3.3 Å and 4.2 Å, respectively (Figure 1, Figure 1—figure supplement 1). By contrast, 3D classification of the TRPV2_RTx-ND converged on a single C2 symmetric conformation resolved to 3.8 Å (Figure 1, Figure 1—figure supplements 2–3). All four maps were of sufficient quality to enable placement of individual structural motifs with confidence (Figure 1—figure supplements 4–7) and the models for all four structures were built to good overall geometry (Table 1).

RTx induces a break in symmetry in TRPV2_RTx-ND

In stark contrast to the amphipol-reconstituted channel, RTx binding induces C2 symmetry in the nanodisc-reconstituted TRPV2 which extends throughout the channel. The symmetry of the TRPV2_RTx-ND map was assessed both visually, and by the Map Symmetry function in Phenix which yielded a CC = 0.9 and score of 1.28 for C2 symmetry. For comparison, C4 symmetry yielded a lower correlation coefficient (CC = 0.8). To further confirm the correctness of the symmetry assignment, we evaluated the fit of the TRPV2_RTx-ND model built into the C2 symmetric map to the non-symmetrized C1 map (Figure 1—figure supplement 3). In addition, we evaluated the fit of the TRPV2_RTx-ND model to the individually refined non-symmetrized classes 1 and 6, which constitute the TRPV2_RTx-ND map (Figure 1—figure supplement 3). All FSC curves indicate that the two-fold symmetric model fits well into the C1 maps and the density of the C1 symmetric TRPV2_RTx-ND map supports the model (Figure 1—figure supplement 3), showing that two-fold symmetry is truly present in the TRPV2_RTx-ND sample.

The pore of TRPV2_RTx-ND adopts a C2 symmetric arrangement (Figure 2A). The pore helices are arranged so that the carbonyl oxygens of the selectivity filter in subunits B and D line the entry to the pore while pore helices of subunits A and C tilt away from the permeation pathway. This arrangement creates a large C2 symmetric opening where the narrowest constriction between SF residues in diametrically opposing subunits A and C and B and D is ~11 Å and ~8 Å, respectively. This results in an SF with ample room to accommodate large organic cations (Figure 2B). A closer look at the pore helices reveals that this arrangement in the SF is achieved through a ~ 10° swivel of the subunit A pore helix, which brings the N-terminal part of the helix closer to S5 while distancing it from S6 (Figure 2C). The position of the pore helices controls the size and the shape of the SF, and appears to exert control over ion permeation in TRPV2. While the SF is widely open, the common gate adopts a putative intermediate conformation where two of the diagonally opposing subunits adopt a closed state, and the remaining two are open. In subunits A and C, the S6 helix adopts a straight α-helical, closed conformation, while the S6 of subunits B and D is bent and the common gate apparently open (Figure 2A). However, the overall functional state of the common gate is likely non-conductive as the gate residues from subunits A and C would presumably hinder ion permeation.

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In order to establish the origin of the C2 symmetry in the TRPV2_RTx-ND structure, we aligned subunits A and B (Cα R.M.S.D = 0.96) (Figure 3—figure supplement 5). Similar to our previous findings, this alignment shows that the two subunits diverge at the S4-S5 linker and the PH and indicates that rotation of subunits around the S4-S5_π-hinge appears to result in the distinct C2 symmetric arrangement observed in TRPV2_RTx-ND (Figure 3—figure supplements 4–5).

When compared to the TRPV2_APO, the TM domains of the TRPV2_RTx-ND structure appear to contract in an asymmetric manner (Figure 3A), while the ARD assembly expands by ~10 Å and rotates by 3° (Figure 3B). The TM domains and the ARDs appear to move as a single rigid body, which is evident when individual subunits from TRPV2_APO and TRPV2_RTx-ND are superposed (Cα R.M.S.D = 1.9 Å) to reveal that only the S4-S5 linker and the pore helix deviate significantly in the two structures (Figure 3C). This coupled movement of the TM and ARD indicates that RTx-binding to TRPV2 in lipid membranes induces a rigid-body rotation of the entire subunit that originates at the S4-S5_π-hinge (Figure 3D–E).

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Interestingly, the TRPV2_RTx-ND structure exhibits different degrees of reduced symmetry from the previously determined crystal structure of TRPV2 in complex with RTx (TRPV2_RTx-XTAL) (Zubcevic et al., 2018a). Compared to the TRPV2_RTx-XTAL, the TM domains of TRPV2_RTx-ND contract in an two-fold symmetric manner (Figure 4A). This conformational change, which stems from rotation of individual TRPV2_RTx-ND subunits around the S4-S5_π-hinge (Figure 4—figure supplement 1), results in an overall fold that is closer to C4 symmetry than that of the TRPV2_RTx-XTAL (Figure 4B). However, while the TRPV2_RTx-ND helices S1-S6 adopt a more C4 symmetric arrangement, the pore helices and the SF remain distinctly C2 symmetric (Figure 4C). Remarkably, the SF of TRPV2_RTx-ND is wider than that of TRPV2_RTx-XTAL, and the two structures display different C2 symmetric openings at the SF (Figure 4C). The two different conformations result from both the different arrangements of subunits and changes in the position and tilt angle of the pore helices (Figure 4D–E). In the TRPV2_RTx-XTAL structure, the pore helices of subunits B and D, which assume a widened conformation, are free of interactions with the pore domain, while a network of interactions (presumably hydrogen bonds) between Y542-T602-Y627 in subunits A and C tethers the pore helices to S5 and S6. Our previous work showed that disruption of these interactions is detrimental to the permeation of large organic cations but has no effect on permeation of metal ions (Zubcevic et al., 2018a). Interestingly, the putative hydrogen bond triad is disrupted in all four subunits of the TRPV2_RTx-ND structure (Figure 4—figure supplement 2). Nevertheless, the SF assumes a fully open state that can potentially accommodate passage of a large cation. This suggests that the putative hydrogen bond triad, while not a feature of the fully open SF, is an essential part of the transition between closed and open states of the channel.

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It is interesting to point out that extensive rearrangements around the SF and the PH during gating have thus far only been observed in structural studies of TRPV1 (Cao et al., 2013b) and TRPV2 (Zubcevic et al., 2018a) channels. In the non-conductive state, the SFs of the remaining members of the TRPV subfamily (TRPV3-TRPV6 [Zubcevic et al., 2018b; Singh et al., 2018; Deng et al., 2018; Hughes et al., 2018; McGoldrick et al., 2018]) adopt a conformation that is wide enough to accommodate a semi-hydrated cation, and do not move appreciably during channel activation. This may indicate that TRPV1 and TRPV2 are the only members of the TRPV subfamily that possess a gate at the SF, and that the coupling of structural elements necessary for activation of these channels differs from that of TRPV3-TRPV6 (Zhang et al., 2019).

As observed in our previous study (Zubcevic et al., 2018a), RTx assumes different binding poses in subunits of the C2-symmetric structures, both in the amphipol and the nanodisc samples (Figure 4—figure supplement 3) which may lead to the distinct conformations observed in these channels.

Despite the use of a full-length rabbit TRPV2 construct in this study, we were not able to confidently resolve the entire loop connecting S5 to the pore helix known as the ‘pore turret’. Interestingly, a recent structure of rat TRPV2 with the pore turret resolved showed that this region, which contains a large number of charged and polar residues, occupies the space within the membrane plane between S5 and the Voltage Sensor-Like Domain (VSLD) (Dosey et al., 2019). While the density in our cryo-EM maps was not of sufficient quality to build the entire pore turret with confidence, we do observe density following the S5 helix and preceding the pore helix. However, the direction of this density is perpendicular to the membrane and does not agree with the structure reported for rat TRPV2 (Figure 4—figure supplement 4). Indeed, the pore turret is the least conserved region amongst the TRPV2 orthologs, and the variations in its sequence might indicate that the turret adopts different conformations in TRPV2 channels from different species. Nevertheless, our study clearly shows that the omission of this region from the construct used in the crystallographic study of the TRPV2/RTx complex is not the cause of the C2 symmetry.

While both TRPV2_RTx-ND and TRPV2_RTx-XTAL structures adopt C2 symmetry, the distinct arrangement of subunits within the two channels suggests that the structures represent different functional states. We propose that TRPV2_RTx-XTAL precedes TRPV2_RTx-ND in the conformational activation trajectory based on two observations. Firstly, the common gate is fully closed in the TRPV2_RTx-XTAL while it adopts an apparently partially open conformation in TRPV2_RTx-ND (Figure 4—figure supplement 5). Secondly, our previous studies have shown that the putative hydrogen bond network between S5 and S6 and the pore helix is essential for the channel’s ability to transition to a fully open SF that can accommodate large organic cations (Zubcevic et al., 2018a). Nevertheless, in TRPV2_RTx-ND the pore helices do not interact with S5 and S6 and the SF is fully open. We therefore propose that the conformational step that requires the presence of the putative hydrogen bond triad precedes the open SF conformation seen in TRPV2_RTx-ND.

Here, we have conducted a study that reveals symmetry transitions associated with gating of the TRPV2 channel by RTx. Interestingly, our data shows that RTx induces C2 symmetric conformations of TRPV2 in both amphipol and nanodiscs, and it thereby negates the hypothetical role of crystallization artefacts and crystal packing bias in stabilising two-fold symmetry. Similarly, C2 symmetry in TRPV2 is independent of the presence or absence of the pore turret region, suggesting that this region does not play an essential role in the regulation of the SF in rabbit TRPV2. Our study, similar to a previously published study of the magnesium channel CorA (Matthies et al., 2016), also emphasizes the notion that careful inspection of the intermediate maps and conservative application of symmetry during refinement of cryo-EM data can result in valuable insights into gating transitions and intermediate states. In addition, we have also investigated how amphipols and nanodiscs affect the conformational space that can be accessed during ligand gating of TRPV2.

While both TRPV2_RTx-APOL and TRPV2_RTx-ND are C2 symmetric, the two-fold symmetry in TRPV2_RTx-APOL is confined to regions that are not bound by the amphipol polymer. This is evident in the fact that the TM domains, which are in contact with the amphipol, largely retain four-fold symmetry and the common gate and the SF remain firmly closed, while the ARD exhibit symmetry breaking, rotation and lateral expansion. These data, while adding valuable data points to the conformational landscape of TRPV2, also illustrate the potential caveats of using amphipols in studies of conformational changes in the transmembrane domains of proteins, as they appear to constrict the TM domains and stabilize low-energy pre-open states (Figure 4—figure supplement 6). However, at this time we caution against any generalized conclusions about the effect of amphipols and look forward to more systematic studies that will address these issues in the future. The TRPV2_RTx-ND dataset yielded a single, two-fold symmetric structure thus giving strong evidence that RTx stabilizes two-fold symmetric conformational states in the TRPV2 channel in lipid membranes. The ARDs in the TRPV2_RTx-ND structure echo the conformational changes observed in TRPV2_RTx-APOL. However, in nanodiscs TRPV2 is captured with its SF fully open and its common gate in an intermediate conformation where the gate is apparently open in one set of diagonally opposing subunits and closed in the other. In this structure, the opening of the SF occurs according to a mechanism previously observed in the crystallographic study of the TRPV2/RTx complex where RTx binding in the vanilloid pocket, above the S4-S5_π-hinge, induces a rigid body rotation of the entire subunit. In turn, the rotation causes a break in the interaction network between the pore helix and helices S5 and S6, allowing the pore helices to reposition and the SF to open (Zubcevic et al., 2018a).

Interestingly, however, the TRPV2_RTx-ND structure differs from the previously obtained TRPV2_RTx-XTAL. While both structures assume C2 symmetric conformations, the TRPV2_RTx-ND channel appears to make a return toward C4 symmetry. Because the SF in TRPV2_RTx-ND is fully open, and the common gate adopts an apparently partially open conformation, we reason that TRPV2_RTx-ND follows the TRPV2_RTx-XTAL structure in the conformational trajectory of the channel. Therefore, it is possible that TRPV2, as it travels toward the final open state where both the SF and the common gate are fully open, would adopt further conformations that increasingly approximate C4 symmetry (Figure 5). However, it is interesting to note that while the overall fold of TRPV2_RTx-ND indeed is more C4 symmetric than that of TRPV2_RTx-XTAL, the extent of C2 symmetry is not diminished in its SF. Because the symmetry of the SF does not appear to be dictated by the symmetry of the overall channel, we cannot exclude the possibility that the final open state might indeed possess a C2 symmetric SF while otherwise adopting a nearly C4 symmetric conformation. Our previous functional studies have suggested that C2 symmetric states play a role in the permeation of large organic cations and consequently in the full opening of the SF (Zubcevic et al., 2018a). Hence, the channel might be utilizing C2 symmetric states as means to achieve full opening in a step-wise manner. Similar C2 symmetric states elicited by ligand binding have been observed in TRPV3 (Zubcevic et al., 2018b) and TRPM2 (Yin et al., 2018) channels, which opens up the possibility that C2 symmetry might be widely associated with gating in members of the TRP channel superfamily. Intriguingly, a recent cryo-EM study of the human BK channel reconstituted in liposomes showed that this channel also enters C2 symmetric states (Tonggu and Wang, 2018), suggesting that two-fold symmetry might also play a role in the molecular mechanisms of other tetrameric ion channels.

Figure 5

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Two-fold symmetry is a well-stablished feature of mammalian Na⁺ selective Two Pore Channels (TPCs) and Voltage Gated Sodium channels (Na_V) (She et al., 2018; Shen et al., 2017; Pan et al., 2018; Shen et al., 2018). Interestingly, the arrangement of pore helices in TRPV2_RTx-ND resembles that observed in TPC and Na_V (Figure 6) and the selectivity filters in all three channels form a 'coin-slot' (Hille, 1971) opening. However, while the selectivity filters of TPC and Na_V remain static during channel gating in order to maintain the structure necessary for Na⁺ selectivity, the SF of TRPV2 displays a large degree of plasticity. Moreover, the two-fold symmetry observed in TRPV2 is unique in that it arises in response to conformational changes in the TM domains induced by ligand binding. By contrast, the two-fold symmetry in TPC and Na_V stems from the arrangement of their respective homologous tandem repeats.

Figure 6

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The EM maps and atomic models have been deposited with the Electron Microscopy Data Bank (accession numbers EMD-20143, EMD-20145, EMD-20146, and EMD-20148) and the Protein Data Back (entry codes 6OO3, 6OO4, 6OO5, and 6OO7), respectively.

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   (2019) Electron Microscopy Data Bank

   ID EMD-20143. Cryo-EM structure of the C4-symmetric TRPV2/RTx complex in amphipol resolved to 2.9 A.

   http://www.ebi.ac.uk/pdbe/entry/emdb/EMD-20143
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   ID EMD-20145. Cryo-EM structure of the C2-symmetric TRPV2/RTx complex in amphipol resolved to 3.3 A.

   http://www.ebi.ac.uk/pdbe/entry/emdb/EMD-20145
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   http://www.ebi.ac.uk/pdbe/entry/emdb/EMD-20146
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   http://www.ebi.ac.uk/pdbe/entry/emdb/EMD-20148
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   ID 6OO3. Cryo-EM structure of the C4-symmetric TRPV2/RTx complex in amphipol resolved to 2.9 A.

   http://www.rcsb.org/structure/6OO3
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   (2019) Protein Data Bank

   ID 6OO4. Cryo-EM structure of the C2-symmetric TRPV2/RTx complex in amphipol resolved to 3.3 A.

   http://www.rcsb.org/structure/6OO4
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   ID 6OO5. Cryo-EM structure of the C2-symmetric TRPV2/RTx complex in amphipol resolved to 4.2 A.

   http://www.rcsb.org/structure/6OO5
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   ID 6OO7. Cryo-EM structure of the C2-symmetric TRPV2/RTx complex in nanodiscs.

   http://www.rcsb.org/structure/6OO7

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Author details

1. Lejla Zubcevic

   Department of Biochemistry, Duke University School of Medicine, Durham, United States

   Contribution

   Conceptualization, Data curation, Validation, Writing—original draft, Writing—review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-1884-9235

2. Allen L Hsu

   Genome Integrity and Structural Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Department of Health and Human Services, Research Triangle Park, United States

   Contribution

   Data curation, Validation, Writing—review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-2065-3802

3. Mario J Borgnia

   1. Department of Biochemistry, Duke University School of Medicine, Durham, United States
   2. Genome Integrity and Structural Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Department of Health and Human Services, Research Triangle Park, United States

   Contribution

   Supervision, Funding acquisition, Validation, Writing—review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-9159-1413

4. Seok-Yong Lee

   Department of Biochemistry, Duke University School of Medicine, Durham, United States

   Contribution

   Conceptualization, Supervision, Funding acquisition, Validation, Writing—original draft, Writing—review and editing

   For correspondence

   seok-yong.lee@duke.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-0662-9921

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