Thermoring switch for TRPV2 inactivation

Thermoring switch for TRPV2 inactivation | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Thermoring switch for TRPV2 inactivation Guangyu Wang This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9726674/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The interdomain interaction plays a critical role in regulating protein activity. Although the exogenous nonselective chemical agonist, 2-aminoethoxydiphenyl borate (2-APB), is well-known for activating and inactivating the homotetrameric thermosensitive transient receptor potential vanilloid 2 (TRPV2) channel, the precise structural basis is still missing. In this computational study, the 3D cryo-EM structures of rat TRPV2 in both 2-APB-activated and inactivated states alone or in combination with other exogenous chemical agonists were analyzed and quantified at the tertiary and quaternary levels using a highly sensitive thermoring model. The results indicated that the weakest tertiary bridge between the pre-S1 domain and the ankyrin repeat domain in the pre-open closed state was still present in the activated state but disrupted in the inactivated state along with the highly conserved swapping π bridges broken near the lower gate, regardless of chemical perturbations at different sites. Furthermore, the thermostability difference between the activated and inactivated states may enhance our understanding of the potentiation mechanism by an additional agonist. Finally, three lipid-free non-conductive cryo-EM structures of apo TRPV2 channels in the presence or absence of nanodiscs but without the treatment of methyl-β-cyclodextrin were identified as inactivated or desensitized, rather than closed. Therefore, the thermoring structures of a protein in different functional states can help us pinpoint the precise dynamic allosteric domain-domain communication for the regulation switch of protein activity. Biophysics Computational Biology Chemical Biology Allosteric gating coupling interdomain interaction protein stability thermoring structure Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction The regulation of activity in multidomain thermosensitive transient receptor potential (TRP) vanilloid 1-4 (TRPV1-4) channels by various physical and chemical stimuli is essential for organismal health in various physiological or pathological processes. These polytopic membrane proteins have complex topology and are homotetrameric, with each monomer featuring six ankyrin repeat domain (ARD), six transmembrane helices (S1-S6) and the C-terminal domain (CTD). The first S1-S4 bundle forms the voltage sensor-like domain (VSLD) while the later S5-S6 bundle, together with two pore turrets and the pore helix (PH) between them, fold as the pore domain (PD). Along with the S4-S5 linker at the active gating core, the VSLD interfaces the ARD and the CTD via the pre-S1 domain and the TRP domain, respectively. When each PD from one subunit swaps with the PD’ and the VSLD’ from the adjacent one, the pre-S1 domain of TRPV3 or TRPV2 also forms an inter-protomer interface with the neighboring ARD’. Thus, these global inter-domain allosteric-networks allow the selectivity filter to be an upper gate with a GXGY motif (X=M, L; Y=D, E) on the PH and the LIA motif near the a-to-p transition of S6 to be the lower gate for the activity regulation (1). Given that the TRPV1-4 channels can be activated by heat in the temperature range from 25 °C to 54 °C (2-10), recent studies have shown that the weakest tertiary bridge serves as a unique thermal sensor in TRPV1, TRPV3 or TRPV4. This specific sensor connects allosterically with the highly conserved swapping p interaction between an aromatic residue on the S4-S5 linker and a positively charged residue on S5’ near the lower gate for heat activation. For example, in rat TRPV1 (rTRPV1)/human TRPV1 (hTRPV1) with phosphatidylinositol (PI) at the internal vanilloid site, the weakest Y401-R499/E406-K504 bridge at the pre-S1/VSLD interface is linked with the Y565-R579’ bridge. In reduced hTRPV3 with phosphatidylcholine (PC) at the vanilloid site, the weakest E610-K649 bridge at the external interface between two pore turrets is coupled with the Y575-K589’ bridge. In human TRPV4 (hTRPV4), the weakest P498/Y502-Y567 bridge at the external interface between S1-S2 and S3-S4 linkers is coupled with the Y602-R616’ bridge (11). However, during the initial thermal activation of rTRPV2 with phosphatidylethanolamine (PE) in the VSLD and without PE at the vanilloid site, there is not a strict coupling between the least-stable K300-N354, S486-Y497 or L555-Y590 bridge and the strong swapping H521-R539’-Y525-N639’ bridges near the lower gate, partly because the weakest K300-N354 bridge is not disrupted but enhanced in the cold-activated state, which mirrors the heat activation with high thermosensitivity (12). On the other hand, the initial broken Y575-K589’ bridge in TRPV3 disrupts the critical weakest D586-T680 H-bond in the PD, resulting in channel inactivation and pore dilation, along with a tetramer-to-pentamer transition (13-15). Since the chemical perturbation of the swapping H521-R539’-Y525-N639’ bridges near the lower gate by 2-APB, an exogenous nonselective chemical agonist (16), also inactivates the rTRPV2 channel (17), it is hypothesized that disrupting the weakest K300-N354 bridge is allosterically coupled to the highly conserved swapping bridges near the lower gate for channel inactivation. In this computational study, a highly sensitive thermoring model, which has been recently developed (11-13, 18-29), was used to test this specific hypothesis. By examining the 3D cryogenic electron microscopy (cryo-EM) structures of rTRPV2 in both the 2-APB-activated and inactivated states with or without an additional perturbation of cannabidiol (CBD) or probenecid (PBC) at various sites, it was observed that the weakest K300-N354 bridge remained intact in the activated states but was broken in the inactivated states, along with the highly conserved swapping p bridges broken near the lower gate. In this regard, three undefined non-conductive cryo-EM structures of apo TRPV2 lacking PE in the VSLD and at the vanilloid site were categorized as inactivated. Furthermore, considering the distinct thermostabilities of the activated and inactivated states, a potentiation mechanism for TRPV2 activity emerged in response to a combination of two chemical agonists. Methods Cryo-EM structures used Five full-length 3D cryo-EM structures of apo rTRPV2 channels without PE in the VSLD and at the vanillid site in the 2-APB-activated, 2-APB-inactivated, 2-APB/CBD-activated, 2-APB/CBD-inactivated and 2-APB/PBC-activated states were examined to identify the structural basis for the inactivation of rTRPV2 by 2-APB (PDB ID: 7N0N, model resolution = 4.15 Å; 7N0M, model resolution = 3.5 Å; 7T37, model resolution = 3.7 Å; 7T38, model resolution = 3.8 Å; 9B3V, model resolution = 4.2 Å, respectively). These channels were first purified in the decyl maltose neopentyl glycol (DMNG) detergent and then reconstituted in membrane scaffold protein 2N2 (MSP2N2) nanodiscs at 4°C ( 17 ). In addition, the full-length or truncated 3D cryo-EM structures of other native TRPV2 channels were also used to test the putative inactivation motif. Their PDB IDs included 5AN8, 5HI9, 6BO4, 6U84, 6U86, 6U8A, 6U88, 6WKN, 7XEM, 7XEO, 7XEU, 7XEV, 7YEP, 7ZJD, 7ZJE, 7ZJG, 7ZJI, 7ZJH, 8EKP, 8EKQ, 8EKR, 8EKS, 8FFL, 8FFM, 8SLX, 8SLY and 9B3U ( 30 – 40 ). Detecting and Filtering tertiary noncovalent interactions Along the PE-dependent minimal gating pathway of rTRPV2 from V254 to P726 ( 12 ), the tertiary noncovalent interactions between two amino acid side chains or backbones, or combined were detected and filtered using UCSF Chimera with the same strict and consistent standards as previously confirmed ( 11 – 13 , 18 – 29 ). Briefly, salt bridges were identified by scanning pairs of charged residues, lone pair/CH/cation/π-π interactions were identified by scanning aromatic residues such as Phe, Tyr or Trp and nearby residues, and H-bonds were identified in the tertiary structure using UCSF Chimera. Specific cutoff distances and interaction angles for the different noncovalent interactions can be found in the online Supporting Information (Table S1, S2, S3, S4, and S5). It shoud be noted that momentary fluctuation-induced perturbations in tertiary noncovalent interactions during protein dynamics were not considered in this study. Mapping tertiary thermoring structures using the grid thermodynamic model The study utilized the same protocol as previously described and validated to map the systematic fluidic grid-like tertiary noncovalent interaction mesh network as the thermoring structure (11–13, 18–29). First, one interaction group included the pore domain (PD) and the other covered VSLD, TRP domain, ARD, pre-S1 domain and the CTD based on the identified tertiary noncovalent interactions. Second, along the defined PE-dependent minimal gating pathway of rTRPV2 from V254 to P726 (black line), arrowed network nodes and edges with linked nodes represented the involved amino acid residues and tertiary noncovalent bridges, respectively. Thus, each edge and nearby polypeptide segments with or without other edges could form several rings as topological grids without repeating the same edge. For example, in Fig. 2 A, the T604-Y629 H-bond and the peptide chain segment from I605 to A628 could generate a ring. Alternatively, this H-bond could shape another ring with the nearby T604-Y544-Y629 H-bonds (Fig. 2 A). In this case, Graph theory and the Floyd–Warshall algorithm were necessary to constrain these grids so that a unique grid could be obtained for each specific tertiary noncovalent interaction with the shortest round path length or the minimal number of free residues in it as the grid size ( 41 ). As the melting temperature required to unfold the least-stable tertiary noncovalent interaction in a grid is related to this unique grid size, the constained grid was also defined as a thermoring to control the least-stable interaction and denoted as Grid s . As an example, in Fig. 2 A, the Y544-T604-Y629-Y544 H-bonds were controlled by the same smallest Grid 0 (Fig. 2 A). In this approach, along with all the tertiary noncovalent bridges labeled by uncommon grid sizes that corresponded to the minimum energy required to stabilize the interactions, the biggest thermoring could be identified to govern the weakest tertiary bridge along the PE-dependent minimal gating pathway from V254 to P726. Meanwhile, the total numbers of noncovalent interactions ( N ) and total grid sizes ( S ) along the same gating pathway were calculated and shown in black and cyan circles, respectively, next to the mesh network map for the following calculations. Calculating the systematic thermal instability The systematic thermal instability (T i ) was calculated using the following expression as previously examined ( 11 – 13 , 18 – 29 ): T i = S/N ( 1 ) Calculating the melting temperature threshold for heat unfolding The melting temperature threshold (T m,th ) for the heat unfolding of the least-stable noncovalent interaction within a specific grid at a normal salt concentration of 150 mM NaCl was calculated using the following expression as previously examined ( 11 – 13 , 18 – 29 ): T m,th (°C) = 34 + (n − 2) × 10 + (20 – s) × 2 ( 2 ) where, n denotes the total number of basic H-bonds (~ 1 kcal/mol for each in a hydrophilic environment) that are energetically equivalent to the weakest noncovalent bridge controlled by the given grid, and s is the size of the grid that controls the weakest tertiary bridge. Thus, the heat capacity of the grid will be higher with a smaller grid size or the more equivalent basic H-bonds. Results Similar internal intersubunit interactions at the PBC site in both the activated and inactivated states A recent study has revealed that PBC targets K118, L126, Y162, H165, I170 and K174 from one monomer, as well as W333’ and Y335’ from the adjacent monomer of rTRPV2, preventing the inactivation of rTRPV2 ( 17 , 40 ). Therefore, it is necessary to examine the swapping interactions at the PBC site in the activated and inactivated states in the presence of the agonist 2-APB with or without CBD or PBC treatment. In the 2-APB-activated state (PDB ID: 7N0N), aside from the Y162-F161-F198-F199 π interactions, the inactivation-sensitive H165 also formed π interactions with nearby H169 and F199 from the same subunit, as well as heat-sensitive Y335’ from the adjacent subunit ( 17 , 42 ). In contrast, the additional Y335’-F198/F199 and W333’-Y162 π bridges were found in the 2-APB-inactivated state (PDB ID: 7N0M), and all of these interactions were disrupted in the 2-APB/PBC-activated state. Thus, the tight swapping interface at the PBC site seemed to favor the inactivated state. However, similar swapping interactions were also observed in the 2-APB/CBD-activated and inactivated states (Fig. 1 ). Accordingly, strictly speaking, the PBC site is not directly responsible for channel inactivation. Further identification of a structural motif is necessary for TRPV2 inactivation. The K300-N354 H-bond is present in the 2-APB-activated state but not in the inactivated state Along the PE-dependent minimal gating pathway from N254 to P726 in the 2-APB-activated state without PE in the VSLD and at the vanilloid site (PDB ID: 7N0N), a total of 105 tertiary noncovalent interactions formed a local grid-like mesh network constrained with a total of 112 grid sizes (Fig. 2 A, Table S1). Thus, the local systematic thermal instability (T i ) was calculated as 1.07 (Table 1 ). Meanwhile, the biggest Grid 17 was found to control the weakest S559-P589 H-bond via a thermoring from P589 to L600, Y629, Y551, S559 and back to P589 (Fig. 2 B-C). Because this weakest bridge was energetically equivalent to 1.2 basic H-bonds (1.2 kcal/mol), the calculated melting temperature threshold (T m,th ) was about 32°C (Table 1 ), which was lower than the body temperature of 37°C. Thus, this activated state may be unstable at the physiological temperature. Notably, with 2-APB perturbing the swapping interace between the S4-S5 linker and S5’, the swapping H521-R539’-Y525-N639’ π bridges near the lower gate were disrupted along with the broken Y412-R560’ and F618’-W496/P499 and F612’-L594 π interactions near the upper gate (Fig. 2 D) ( 12 ). Table 1 Comparison of ligand-induced thermoring structural changes of rTRPV2 along the PE-dependent minimal gating pathway from V254 to P726. The comparative parameters are highlighted in bold. PDB ID 7N0N 7N0M 7T37 7T38 9B3V Lipid in the VSLD Free Free Free Free PE Lipid at the active vanilloid site Free Free Free Free Free Lipid environment MSP2N2 MSP2N2 MSP2N2 MSP2N2 MSP2N2 Sampling temperature, °C 4 4 4 4 4 Ligand 2-APB 2-APB 2-APB + CBD 2-APB + CBD 2-APB + PBC Gating state Activated Inactivated Activated Inactivated Activated # of the biggest Grids Grid 17 Grid 10 Grid 15 Grid 21 Grid 16 grid size (s) 17 10 15 21 16 # of basic H-bonds (n) in stability equivalent to the weakest noncovalent bridge 1.2 1.5 1.5 1.0 1.2 Total non-covalent interactions (N) 105 110 105 98 124 Total grid sizes (S), a.a 112 119 106 130 100 Systemic thermal instability (Ti) 1.07 1.08 1.00 1.33 0.81 Calculated T m,th °C at E = 1 kcal/mol 32 49 39 22 32 Refs for PDB 17 17 17 17 17 In contrast, in the 2-APB-inactivated state without PE in the VSLD and at the vanilloid site (PDB ID: 7N0M), the total numbers of tertiary noncovalent interactions and grid sizes along the same PE-dependent minimal gating pathway from N254 to P726 increased from 105 and 112 to 110 and 119, respectively (Table S2). Thus, the local T i of 1.08 was similar to 1.07 (Table 1 ). Meanwhile, the biggest Grid 10 appeared to govern the least-stable M468-Q530 H-bond via a thermoring from F467 to Y471, F472, Y514, Y515, F393, F394, F519, Q530, M468, and back to F467 (Fig. 3 B-C). Since this weakest bridge at the S4/S5 interface was energetically equivalent to 1.5 basic H-bonds (1.5 kcal/mol), the calculated T m,th was about 49°C, which was higher than 32°C in the 2-APB-activated state (Table 1 ). Therefore, the inactivated state was much more stable than the activated state after the 2-APB perturbation. Although the swapping of H521-R539’-Y525-N639’ π bridges near the lower gate were still broken, the Y412-R560’ and F618’-W496/P499 and F612’-L594 π interactions near the upper gate were reinstated (Fig. 3 D) ( 12 ). Most notably, the weakest K300-N354 bridge at the interface between the pre-S1 domain and the ARD in the pre-open closed state was found to be in the 2-APB-activated state but not in the inactivated state (Figs. 2 A & 3 A) ( 12 ). This finding was consistent with a significant 4.4 Å downwards movement of the ARDs in the 2-APB-inactivated state ( 17 ). Given that this interfacial H-bond was controlled by a smaller Grid 2 through a thermoring from K300 to T297, L296, N292, Q294, K304, C364, F362, R459, E358, S355, N354 and back to K300 and was energetically equivalent to 1.3 basic H-bonds (1.3 kcal/mol) (Fig. 2 A), the calculated T m,th to unfold it was about 63°C. Accordingly, it is necessary to examine if this stable H-bond at the pre-S1/ARD interface was also present in the 2-APB-activated state but not in the inactivated state upon perturbation by external CBD or internal PBC. External CBD cannot prevent the breaking of the K300-N354 bridge in the 2-APB-inactivated state When CBD additionally interacted with both L537 from one PD and Y634’ from the neighboring PD’ ( 17 , 33 ), the K300-N354 bridge in the 2-APB-activated state was still observed along with a total of 105 tertiary noncovalent bridges and 106 grid sizes (Fig. 4 A, Table S3). In this case, the local grid-based systematic thermal instability (T i ) slightly decreased from 1.07 to 1.00 (Table 1 ). However, the new weakest H438-Q487 H-bond at the interface between the S1-S2 and S3-S4 linkers was governed by the biggest Grid 15 via a thermoring involving H438, Y447, W676, W454, Y455, R459, F362, A361, R369, D469, Y471, F472, Y514, F476, Q487 and back to H438 (Fig. 4 B-C). Since the controlled bridge was energetically equivalent to 1.5 basic H-bonds (1.5 kcal/mol), the calculated T m,th was about 39°C, higher than the 32°C for the 2-APB-activated state (Table 1 ). Therefore, CBD binding stabilized the 2-APB-activated state without affecting the K300-N354 bridge. On the other hand, the L537-CBD-Y634’ bridge restored the Y525-R539’ bridge near the lower gate and the W496-F618’-P499 bridges near the upper gate (Fig. 4 D). Of special note, the K300-N354 bridge was also broken in the 2-APB/CBD-inactivated state, along with a decrease in the total tertiary noncovalent bridges from 110 to 98 but an increase in the total grid sizes from 119 to 130 (Figs. 3 A & 5 A, Tables S2 & S4). As a result, the local systematic thermal instability (T i ) increased from 1.08 to 1.33 (Table 1 ). Hence, CBD destabilized the 2-APB-inactivated state. In line with this notion, the weakest R563-Q615 H-bond was found at the external interface between two pore turrets (Fig. 5 A). It was controlled by the biggest Grid 21 via a thermoring from F551 to R563, Q615, T604, F601, Y544, Y629, and back to F551 (Fig. 5 B-C). For the weakest R563-Q615 bridge to be energetically equivalent to 1.0 basic H-bond (1.0 kcal/mol), the calculated T m,th was 22°C, lower than 49°C in the 2-APB-inactivated state (Table 1 ). Internal PBC prohibits any disruption to the K300-N354 bridge Following the treatment of internal PBC ( 17 , 40 ), the K300-N354 bridge was only present in the 2-APB-activated state, along with a total of 124 tertiary noncovalent interactions and 100 grid sizes (Fig. 6 A, Table S5). As a result, the local systematic thermal instability (T i ) significantly decreased from 1.07 to 0.81 (Table 1 ). Meanwhile, the biggest Grid 16 appeared to control the weakest E561-R617 H-bond via a thermoring from F551 to E561, R617, F618, R619, F612, T604, F544, Y629, and back to F551 (Fig. 6 B-C). Since this weakest bridge between two pore turrets was energetically equivalent to 1.2 basic H-bonds (1.2 kcal/mol), the T m,th was calculated as 32°C, the same as the value in the 2-APB-activated state (Table 1 ). Therefore, after removing the 2-APB-inactivated state, PBC could potentiate the channel activity below the physiological temperature. Notably, the swapping Y625-N639’ bridge near the lower gate and the P499-F618’ and L594-F612’ bridges near the upper gate were restored (Fig. 6 D). The K300-N354 bridge was found to be highly conserved in both activated and closed states Further structural scanning uncovered that the K300-N354 bridge was also present with other agonists such as protons, CBD, phytocannabinoid Δ9-tetrahydrocannabiorcol (C16) and estradiol (E2) alone or in combination with PBC. It should be noteworthy that in the absence of any ligand, this bridge still appeared in three closed states with PE bound (PDB ID: 8EKP, 8EKQ and 8EKR), a PE-free closed state (PDB ID: 6U86) and a PE-free open state (PDB ID: 6BO4) (Table 2 ) ( 32 , 33 , 39 ). However, it disappeared in two PE-free apo rTRPV2 channels: one in DMNG (PDB ID: 5HI9) and the other in the DMNG/MSP2N2 nanodiscs (PDB ID: 6U84) (Table 2 ) ( 31 , 33 ). In addition, it was also absent in a truncated rabbit TRPV2 channel without PE in the VSLD and at the vanilloid site (PDB: 5AN8) (Table 2 ) ( 30 ). Therefore, these three nonconducting TRPV2 channels were actually in an inactivated or desensitized state, rather than a closed state. Table 2 Identification of gating states of rTRPV2 with or without a ligand. Species PDB ID Nanodiscs/ MβCD PE/VSLD PE/VBP Ligand Pre-S1/ARD interface Gating state Refs. Rabbit 5AN8 - - - - - Inactivated 30 Rat 5HI9 - - - - - Inactivated 31 Rat 6BO4 - - - - K300-N354 Open 32 Rat 6U84 + - - - - Inactivated 33 Rat 6U86 + - - - K300-N354 Activated 33 Rat 6U8A + - - CBD-1 K300-N354 Activated 33 Rat 6U88 + - - CBD-2 K300-N354 Activated 33 Rat 6WKN + - - PL(antagonist) K300-N354 Inhibited 34 Mouse 7XEM - - CHL - K295-N349 Inhibited 35 Mouse 7XEO + - - - K295-N349 Closed 35 Mouse 7XEU - - - E2 K295-N349 Activated 35 Mouse 7XEV - - 2-APB E2 - Inactivated 35 Mouse 7YEP + - 2-APB - - Inactivated 35 Rat 7ZJD - - - C16-1 K300-N354 Activated 36 Rat 7ZJE - - - C16-2 K300-N354 Activated 36 Rat 7ZJG - - - PBC K300-N354 Activated 36 Rat 7ZJI - - - C16 + PBC-1 K300-N354 Activated 36 Rat 7ZJH - - - C16 + PBC-2 K300-N354 Activated 36 Rat 8SLX + - - 1 CBD K300-N354 Activated 37 Rat 8SLY + - + 2 CBD K300-N354 Activated 37 Rat 8FFL + + - RR K300-N354 Activated 38 Rat 8FFM + + - RR + 2-APB K300-N354 Activated 38 Rat 8EKP + + + - K300-N354 Closed 39 Rat 8EKQ + + + - K300-N354 Closed 39 Rat 8EKR + + - K300-N354 Closed 39 Rat 8EKS + + - H + K300-N354 Activated 39 Rat 9B3U + - - PBC K300-N354 Activated 40 Discussion The highly conserved swapping interactions between a Tyr residue side chain on the S4-S5 linker of one subunit and a Lys/Arg residue side chain on S5’ from the neighboring subunit near the lower gate play a critical role in channel gating of thermosensitive TRPV1-4 channels ( 11 – 13 , 43 ). On one hand, these swapping interactions must be finally broken for heat activation ( 11 ). On the other hand, when they are first disrupted, the channel can become inactivated ( 13 – 15 ). After illuminating the thermoring basis for the inactivation-induced pore dilation of hTRPV3, this study further demonstrated that the weakest K300-N354 bridge in the pre-open closed state was allosterically coupled to the highly conserved swapping interactions near the lower gate for TRPV2 inactivation. This study also identified other inactivated states that have not been clearly defined. In addition, the various thermostabilities of activated and inactivated states upon two agonist perturbations at different sites may help us understand the potentiation or sensitization mechanism of one by the other. Collectively, the unique interfacial bridge advances our understanding of the thermoring switch for allosteric gating regulation of thermosensitve TRPV1-4 channel. The K300-N354 gating bridge is an important feature that distinguishes the inactivated state from the activated or closed state Despite the activation of TRPV2 by multiple agonists such as CBD, 2-APB, E2, PBC and protons alone or in combination, their cryo-EM structures do not always show the pore opening. On the other hand, the TRPV2 channel is open in the absence of these agonists but in the presence of detergents such as lauryl maltose neopentyl glycol (LMNG) at low temperature (Table 2 ). Therefore, it is unclear whether the channel has been activated or inactivated when it adopts a non-conducting conformation at low temperature. In this computational study, detailed thermoring analyses revealed that the unique K300-N354 bridge at the pre-S1/ARD interface was present in the 2-APB-activated state with or without CBD or PBC treatment but absent in the 2-APB-inactivated state with or without CBD perturbation (Figs. 2 – 6 ). Further structural scanning demonstrated that the K300-N354 bridge (K295-N349 bridge in mTRPV2) also appeared in the presence of other agonists such as C16 and E2 or a mild detergent LMNG to activate or open the channel (Table 2 ) ( 32 , 35 , 36 ). Once the channel is activated, the blocker RR fails to affect this bridge (Table 2 ) ( 38 ). On the other hand, the K300-N354 bridge was also found in three closed states with PE bound in the VSLD or at the vanilloid site (Table 2 ) ( 12 , 39 ). Therefore, the presence of the K300-N354 bridge could distinguish between the activated or closed state and the inactivated state. Based on this characteristic coupling, several cryo-EM structures of apo TRPV2 were identified as inactivated. These included two apo rTRPV2 channels (PDB ID: 5HI9 and 6U84) ( 31 , 33 ), one apo rabbit TRPV2 (PDB ID: 5AN8) ( 30 ), and two mTRPV2 channels with 2-APB or plus E2 (Table 2 ) ( 35 ). Since the weakest K300-N354 bridge in the pre-open closed state is also important for thermal activation and 6U84 without this bridge has a higher threshold for heat activation ( 11 – 12 ), it is possible that 6U84 may also function as a heat-induced inactivated state of rTRPV2 ( 44 ). Given that human TRPV2 (hTRPV2) is insensitive to heat and 2-APB below 60°C, and the pre-S1 domain, the ARD and the CTD of rTRPV2 are involved in responses to heat and 2-APB ( 45 – 49 ), the inactivated 6U86 channel could potentially serve as a homological model of hTRPV2 in a resting state without the K300-N354 bridge but with a heat threshold exceeding 60°C ( 11 ). Notably, the presence of the K300-N354 bridge in rTRPV2 with the antagonist piperlongumine (PL) or mTRPV2 with cholesterol (CHL) suggests that channel activation could be inhibited (Table 2 ) ( 34 – 35 ). Jointly, similar to TRPV3, there is a state-dependent coupling between cytoplasmic and transmembrane domains in rTRPV2 that helps regulate normal channel gating ( 50 ). Tight swapping interface between the S4-S5 linker and S5’ is necessary for initial TRPV2 closure Recent studies have indicated that the swapping bridge near the lower gate, Y565-R579’ in rTRPV1/hTRPV1, Y575-K589’ in reduced hTRPV3, or Y602-R616’ in hTRPV4 is involved in channel activation of TRPV1, TRPV3 or TRPV4 ( 11 , 13 , 43 ). However, in TRPV2, the swapping bridges H521-R539’-Y525-N639’ are present in three closed states with PE bound ( 12 ). This study further demonstrated that these bridges were partially or fully broken in the 2-APB-activated or inactivated states with or without CBD or PBC perturbation. Therefore, at least the H521-R539’-Y525 bridges at the interface between the S4-S5 linker and S5’ are necessary for initial rTRPV2 closure. Nanodiscs or MβCD is necessary for cryo-EM structures of TRPV2 with normal gating In the presence of MSP2N2 nanodiscs or methyl-β-cyclodextrin (MβCD), rTRPV2 was inactivated or desensitized upon 2-APB perturbation (Table 2 ) ( 17 , 35 ). However, in the absence of MSP2N2/MβCD and 2-APB, the channel was also inactivated (PDB ID: 5AN8, 5HI9) (Table 2 ) ( 30 – 31 ). This is reminiscent of the inactivated TRPV3 channel in the absence of MSP2N2 ( 13 – 15 ). Therefore, although the treatment of MβCD does not affect the 2-APB sensitivity of mTRPV2 ( 51 ), after detergent solubilization, the MSP2N2 nanodisc or MβCD treatment favors the cryo-EM structures of initial TRPV2 closure with the H521-R539’-Y525 bridges (H516- R534’-Y521 bridges in mTRPV2) in a near-native lipid environment. Thermostability-based activity potentiation Agonist-induced dynamic intrasubunit allosteric gating pathways may be involved in the global unfolding/folding and posttranslational stability of TRPV2. Along with 2-APB binding at the interface between the S4-S5 linker and S5’, rTRPV2 can be activated and inactivated along with the ARD-up and ARD-down conformations, respectively (Fig. 7 ). Since the melting threshold of the inactivated state was higher than that of the activated one (Table 1 ), a minor activated state (PDB 7N0N) and a dominant inactivated state (PDB 7N0M) resulted in limited activity ( 17 ). However, the L537-CBD-Y634’ bridge significantly enhanced the thermostability of the 2-APB-activated state but weakened the thermostability of the 2-APB-inactivated state (Table 1 ). Therefore, it is reasonable that CBD can sensitize the 2-APB activation rather than the heat activation at 40°C ( 17 , 37 ). Similarly, PBC also decreased the local systematic thermal instability (T i ) of the 2-APB-activated state while inhibiting inactivation (Table 1 ). Thus, a change in the occupancy of the activated and inactivated states may account for the activity potentiation by PBC ( 17 ). Given that the activation of rTRPV2 by PBC involves H165 ( 40 ), and either protonation of H521 at pH 5 or 2-APB perturbation at pH 8 can disrupt the swapping of H521-R539’-Y525-N639’ bridges at the S4-S5 linker/S5’ interface but still maintain the intact K300-N354 bridge for channel activation (Table 2 ) ( 39 – 40 ), protonation of H165 and H521 may stabilize the 2-APB-activated state (Fig. 1 ), allowing acid to enhance the channel activity ( 52 ). In any case, the up and down conformations of the ARD play a pivotal role in switching TRPV2 gating via the unique K300-N354 bridge. The resultant difference in global thermostability between the activated and inactivated states allows for the regulation of channel activity in response to various agonists at different sites. On the other hand, although the tight allosteric coupling between the weakest bridge at the interface between the pre-S1 domain and the VSLD, between two pore turrets or between S1-S2 and S3-S4 linkers in the pre-open closed state and the highly conserved swapping interaction near the lower gate for thermal activation of TRPV1, TRPV3 or TRPV4 allows each to be a highly-sensitive biothermometer for monitoring a change in environment temperature, respectively ( 11 ), the allosteric coupling between the weakest bridge at the pre-S1/ARD interface in the pre-open closed state and the highly conserved swapping interactions near the lower gate for TRPV2 inactivation compromises the channel’s ideal design for detecting acute thermal nociception above 50°C or a relevant target for pain therapy, particularly for hTRPV2. conclusions The cryo-EM structures of ion channels have been extensively used to identify drug binding sites. However, most of these structures, when at low temperatures, adopt a nonconductive conformation, regardless of the presence of an exogenous chemical agonist or antagonist. Therefore, it is still unclear whether the channel is activated, inactivated, inhibited or closed, hindering the understanding of the precise action mechanism of a drug. In this computational study, the tertiary structural changes of TRPV2 from activated to inactivated states were analyzed using a high-sensitivity thermoring model. By comparing five thermoring structures and examining the channel’s thermosensitivity in response to different agonists, the unique weakest interdomain H-bond in the pre-open closed state was found to couple the highly conserved swapping interactions allosterically for channel inactivation, rather than activation. Furthermore, a change in thermostability revealed a unique potentiation mechanism when two agonists were involved. Finally, other gating states under various conditions were further defined based on this coupling hallmark. Therefore, this thermoring model can be used to elucidate the allosteric coupling between the protein stability and channel gating, as well as the precise action mechanism of drugs on ion channels. Abbreviations 2-APB, 2-aminoethoxydiphenyl borate ARD, Ankyrin repeat domain CBD, cannabidiol CHL, cholesterol cryo-EM, cryogenic electron microscopy CTD, C-terminal domain DMNG, decyl maltose neopentyl glycol E2, estradiol LMNG, lauryl maltose neopentyl glycol MSP2N2, membrane scaffold protein 2N2 MβCD, methyl-β-cyclodextrin PE, phosphatidylethanolamine PC, phosphatidylcholine PI, phosphatidylinositol C16, phytocannabinoid Δ9-tetrahydrocannabiorcol PL, piperlongumine PD, pore domain PH, pore helix PBC, probenecid RR, 2-aminoethoxydiphenyl borate T i , systematic thermal instability T m,th , melting temperature threshold TRP, transient receptor potential TRPV, TRP vanilloid TRPVi (i=1, 2, 3, 4) hTRPV1, human TRPV1 hTRPV2, human TRPV2 hTRPV3, human TRPV3 hTRPV4, human TRPV4 rTRPV1, rat TRPV1 rTRPV2, rat TRPV2 VBP, vanilloid binding pocket VSLD, voltage-sensor-like domain Declarations Data availability All data generated or analysed during this study are included in this published article and supplementary material. Supporting information This article contains supplementary material (Tables S1, S2, S3, S4 and S5). Funding Declaration The work was supported by the American Heart Association (AHA) Grant (10SDG4120011 to GW). Author contributions G. W. wrote the main manuscript text and prepared Figures 1, 2, 3, 4, 5, 6, 7 and Tables 1 and 2 and supplementary material (Tables S1, S2, S3, S4 and S5), and reviewed the manuscript. Competing interests The author declares no conflict of interest. References Cao E (2020) Structural mechanisms of transient receptor potential ion channels. J Gen Physiol 152:e201811998 Caterina MJ, Schumacher MA, Tominaga M et al (1997) The capsaicin receptor: A heat-activated ion channel in the pain pathway. Nature 389:816–824 Caterina MJ, Rosen TA, Tominaga M et al (1999) A capsaicin-receptor homologue with a high threshold for noxious heat. Nature 398:436–441 Peier AM, Reeve AJ, Andersson DA et al (2002) A heat-sensitive TRP channel expressed in keratinocytes. 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Mol Pharmacol 107:100060 Gao L et al (2016) Selective potentiation of 2-APB-induced activation of TRPV1-3 channels by acid. Sci Rep 6:20791 Additional Declarations The authors declare no competing interests. Supplementary Files Supplementarymaterial2APBinactivationinTRPV2v1.0.pdf Supplemental Tables S1-S5 Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9726674","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":640969701,"identity":"559edaef-6d25-4805-9f28-d7c5fcbe082a","order_by":0,"name":"Guangyu Wang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA60lEQVRIiWNgGAWjYFADZsbGB0CKh494LezMhw1AWtiI18LPliYBoglqkXc/+0yCsc0uT96Zx6zya46dDBsD88NHN/BoMTyTbgbUklxseJjH7LbstmSgw9iMjXPwaWlIY5Ng3MacuLEZqEVyGzNQCw+bNF4t/c9AWurBWoolt9UT1iIvAbblcOJ8ZrY0xo/bDhPWYiDxjNki8d/xxA3MzIelGbcd52FjJuAX+f40xhsfzlQnzu8/2Pjx57Zqe3725oeP8dpygIFFIgHCYGDmAQkx41EOtqWBgfkDlMHA+IOA6lEwCkbBKBiZAACt70H8iuWZ5QAAAABJRU5ErkJggg==","orcid":"","institution":"University of California Davis","correspondingAuthor":true,"prefix":"","firstName":"Guangyu","middleName":"","lastName":"Wang","suffix":""}],"badges":[],"createdAt":"2026-05-15 16:26:45","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":false,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false},"doi":"10.21203/rs.3.rs-9726674/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9726674/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":109438698,"identity":"552faecc-201a-4769-ad5e-f27ea4c37237","added_by":"auto","created_at":"2026-05-18 06:51:23","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":157609,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eComparison of intersubunit interactions in the activated and inactivated states of rTRPV2 with different agonists at low temperatures. \u003c/strong\u003eThe cryo-EM structures of rTRPV2 in response to various agonists at 4 °C (PDB ID: 7N0N, 7N0M, 7T37, 7T38 and 9B3V) were used for the model.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9726674/v1/c4f3583006799eb6a468c1fb.jpg"},{"id":109759766,"identity":"9ed98c84-e512-48ec-8561-f233f91b43b7","added_by":"auto","created_at":"2026-05-22 07:27:39","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":257956,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThermoring structures of 2-APB-activated rTRPV2 at a low temperature.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e(\u003cstrong\u003eA) \u003c/strong\u003eThe grid-like noncovalently interacting mesh network along the PE-dependent minimal gating pathway of a single subunit of 2-APB-activated rTRPV2 in DMNG/MSP2N2 nanodisc at pH 8 and 4 °C (PDB: 7N0N).\u0026nbsp; The same colored residues and arrows are used for convenient tracking.\u0026nbsp; Salt bridges, p interactions, and H-bonds between paired amino acid side chains along the PE-dependent gating pathway from V254 to P726 are denoted in purple, green, and orange lines, respectively. The specific constrained grid sizes necessary to regulate the least-stable noncovalent interactions in the grids are indicated with black numbers.\u0026nbsp; The identified least-stable S559-P589 H-bond in the biggest Grid\u003csub\u003e17\u003c/sub\u003e is highlighted in orange.\u0026nbsp; The K300-N354 H-bond is also highlighted\u0026nbsp; in yellow.\u0026nbsp; The total grid sizes and the total grid size-controlled noncovalent interactions along the PE-dependent minimal gating pathway are displayed in cyan and black circles, respectively. The inset shows the structure of the PE sites in the VSLD and at the vanilloid site.\u0026nbsp; (\u003cstrong\u003eB) \u003c/strong\u003eThe structure of the biggest Grid\u003csub\u003e17 \u003c/sub\u003ewith a 17-residue size to regulate the weakest S559-P589 H-bond at the external interface of two pore turrets. (\u003cstrong\u003eC)\u003c/strong\u003e The sequence of the biggest Grid\u003csub\u003e17\u003c/sub\u003e to control the weakest S559-P589 bridge highlighted in the blue box.\u0026nbsp; (\u003cstrong\u003eD\u003c/strong\u003e) Swapping interactions at the VSLD/PD’/PD interfaces near the PE site and upper and lower gates in the 2-APB-activated state.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9726674/v1/10dff91bc6916a4c28dc8568.jpg"},{"id":109438699,"identity":"6c969977-a7b9-432b-9075-f0f53af536b1","added_by":"auto","created_at":"2026-05-18 06:51:23","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":279077,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThermoring structures of 2-APB-inactivated rTRPV2 at a low temperature.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e(\u003cstrong\u003eA) \u003c/strong\u003eThe grid-like noncovalently interacting mesh network along the PE-dependent minimal gating pathway of a single subunit of 2-APB-inactivated rTRPV2 in DMNG/MSP2N2 nanodisc at pH 8 and 4 °C (PDB: 7N0M).\u0026nbsp; The same colored residues and arrows are used for convenient tracking.\u0026nbsp; Salt bridges, p interactions, and H-bonds between paired amino acid side chains along the PE-dependent gating pathway from V254 to P726 are denoted in purple, green, and orange lines, respectively. The specific constrained grid sizes necessary to regulate the least-stable noncovalent interactions in the grids are indicated with black numbers.\u0026nbsp; The identified least-stable M468-Q530 H-bond in the biggest Grid\u003csub\u003e10\u003c/sub\u003e is highlighted in orange.\u0026nbsp; The total grid sizes and the total grid size-controlled noncovalent interactions along the PE-dependent minimal gating pathway are displayed in cyan and black circles, respectively. The inset shows the structure of the PE sites in the VSLD and at the vanilloid site.\u0026nbsp; (\u003cstrong\u003eB) \u003c/strong\u003eThe structure of the biggest Grid\u003csub\u003e10 \u003c/sub\u003ewith a 10-residue size to regulate the weakest M468-Q530 H-bond at the internal S3-S5 interface. (\u003cstrong\u003eC)\u003c/strong\u003e The sequence of the biggest Grid\u003csub\u003e10\u003c/sub\u003e to control the weakest M468-Q530 bridge highlighted in the blue box.\u0026nbsp; (\u003cstrong\u003eD\u003c/strong\u003e) Swapping interactions at the VSLD/PD’/PD interfaces near the PE site and upper and lower gates in the 2-APB-inactivated state.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9726674/v1/d1728b356d9dea76b9e6f31b.jpg"},{"id":109438701,"identity":"17f223d3-752f-4675-a3f5-378a1ac946e0","added_by":"auto","created_at":"2026-05-18 06:51:23","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":275847,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThermoring structures of 2-APB/CBD-activated rTRPV2 at a low temperature.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e(\u003cstrong\u003eA) \u003c/strong\u003eThe grid-like noncovalently interacting mesh network along the PE-dependent minimal gating pathway of a single subunit of 2-APB/CBD-activated rTRPV2 in DMNG/MSP2N2 nanodisc at pH 8 and 4 °C (PDB: 7T37).\u0026nbsp; The same colored residues and arrows are used for convenient tracking.\u0026nbsp; Salt bridges, p interactions, and H-bonds between paired amino acid side chains along the PE-dependent gating pathway from V254 to P726 are denoted in purple, green, and orange lines, respectively. The specific constrained grid sizes necessary to regulate the least-stable noncovalent interactions in the grids are indicated with black numbers.\u0026nbsp; The identified least-stable H438-Q487 H-bond in the biggest Grid\u003csub\u003e15\u003c/sub\u003e is highlighted in orange.\u0026nbsp; The K300-N354 H-bond is also highlighted in yellow. The total grid sizes and the total grid size-controlled noncovalent interactions along the PE-dependent minimal gating pathway are displayed in cyan and black circles, respectively. The inset shows the structure of the PE sites in the VSLD and at the vanilloid site.\u0026nbsp; (\u003cstrong\u003eB) \u003c/strong\u003eThe structure of the biggest Grid\u003csub\u003e15 \u003c/sub\u003ewith a 15-residue size to regulate the weakest H438-Q487 H-bond at the external interface between S1-S2 and S3-S4 linkers. (\u003cstrong\u003eC)\u003c/strong\u003e The sequence of the biggest Grid\u003csub\u003e15\u003c/sub\u003e to control the weakest H438-Q487 bridge highlighted in the blue box.\u0026nbsp; (\u003cstrong\u003eD\u003c/strong\u003e) Swapping interactions at the VSLD/PD’/PD interfaces near the PE site and upper and lower gates in the 2-APB/CBD-activated state.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9726674/v1/71761d374c64921f5a2e9582.jpg"},{"id":109759745,"identity":"d054cf8a-5261-44b3-951f-97beaa71d36a","added_by":"auto","created_at":"2026-05-22 07:27:37","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":256980,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThermoring structures of 2-APB/CBD-inactivated rTRPV2 at a low temperature.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e(\u003cstrong\u003eA) \u003c/strong\u003eThe grid-like noncovalently interacting mesh network along the PE-dependent minimal gating pathway of a single subunit of 2-APB/CBD-inactivated rTRPV2 in DMNG/MSP2N2 nanodisc at pH 8 and 4 °C (PDB: 7T38).\u0026nbsp; The same colored residues and arrows are used for easy tracking.\u0026nbsp; Salt bridges, p interactions, and H-bonds between paired amino acid side chains along the PE-dependent gating pathway from V254 to P726 are denoted in purple, green, and orange lines, respectively. The specific constrained grid sizes necessary to regulate the least-stable noncovalent interactions in the grids are indicated with black numbers.\u0026nbsp; The identified least-stable R563-Q615 H-bond in the biggest Grid\u003csub\u003e21\u003c/sub\u003e is highlighted in orange.\u0026nbsp; The total grid sizes and the total grid size-controlled noncovalent interactions along the PE-dependent minimal gating pathway are displayed in cyan and black circles, respectively. The inset shows the structure of the PE sites in the VSLD and at the vanilloid site.\u0026nbsp; (\u003cstrong\u003eB) \u003c/strong\u003eThe structure of the biggest Grid\u003csub\u003e21 \u003c/sub\u003ewith a 21-residue size to regulate the weakest R563-Q615 H-bond at the external interface of two pore turrets. (\u003cstrong\u003eC)\u003c/strong\u003e The sequence of the biggest Grid\u003csub\u003e21\u003c/sub\u003e to control the weakest R563-Q615 bridge highlighted in the blue box.\u0026nbsp; (\u003cstrong\u003eD\u003c/strong\u003e) Swapping interactions at the VSLD/PD’/PD interfaces near the PE site and upper and lower gates in the 2-APB-inactivated state.\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9726674/v1/04af446671020d0dd4ccd247.jpg"},{"id":109438704,"identity":"5e41f4fc-3fab-4777-91f2-cd53c6959aa6","added_by":"auto","created_at":"2026-05-18 06:51:23","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":271318,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThermoring structures of 2-APB/PBC-activated rTRPV2 at a low temperature.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e(\u003cstrong\u003eA) \u003c/strong\u003eThe grid-like noncovalently interacting mesh network along the PE-dependent minimal gating pathway of a single subunit of 2-APB/PBC-activated rTRPV2 in DMNG/MSP2N2 nanodisc at pH 8 and 4 °C (PDB: 9B3V).\u0026nbsp; The same colored residues and arrows are used for convenient tracking.\u0026nbsp; Salt bridges, p interactions, and H-bonds between paired amino acid side chains along the PE-dependent gating pathway from V254 to P726 are denoted in purple, green, and orange lines, respectively. The specific constrained grid sizes necessary to regulate the least-stable noncovalent interactions in the grids are indicated with black numbers.\u0026nbsp; The identified least-stable E561-R617 H-bond in the biggest Grid\u003csub\u003e16\u003c/sub\u003e is highlighted in orange.\u0026nbsp; The K300-N354 H-bond is also highlighted\u0026nbsp; in yellow.\u0026nbsp; The total grid sizes and the total grid size-controlled noncovalent interactions along the PE-dependent minimal gating pathway are displayed in cyan and black circles, respectively. The inset shows the structure of the PE sites in the VSLD and at the vanilloid site.\u0026nbsp; (\u003cstrong\u003eB) \u003c/strong\u003eThe structure of the biggest Grid\u003csub\u003e16 \u003c/sub\u003ewith a 16-residue size to regulate the weakest E561-R617 H-bond at the external interface of two pore turrets.\u0026nbsp; (\u003cstrong\u003eC)\u003c/strong\u003e The sequence of the biggest Grid\u003csub\u003e16\u003c/sub\u003e to control the weakest E561-R617 bridge highlighted in the blue box.\u0026nbsp; (\u003cstrong\u003eD\u003c/strong\u003e) Swapping interactions at the VSLD/PD’/PD interfaces near the PE site and upper and lower gates in the 2-APB/PBC-activated state.\u003c/p\u003e","description":"","filename":"Figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9726674/v1/0cea5582108d9bb8a957bc7a.jpg"},{"id":109760883,"identity":"ee515c37-9ecc-44b5-9b06-d792458cbe0b","added_by":"auto","created_at":"2026-05-22 07:29:16","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":275490,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eWorkingmodel of the activation and inactivation of rTRPV2 by 2-APB. \u003c/strong\u003eThe homo-tetrameric cryo-EM structures of rTRPV2 in the PE-bound closed state 3, the PE-free activated and inactivated states upon 2-APB perturbations at 4 °C (PDB ID, 8EKR, 7N0N and 7N0M, respectively) are utilized for the model. In the closed state with the ARD-up, the weakest intrasubunit K300-N354 bridges (blue) at the pre-S1/ARD interfaces are present along with swapping H521-R539’-Y525-N539’ bridges near the lower gate. When the channel is activated by 2-APB, these swapping bridges (red) are broken. Simultaneously, disrupting the weakest K300-N354 bridges inactivates the channel, along with the ARD-down.\u003c/p\u003e","description":"","filename":"Figure7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9726674/v1/cec15498c0ab38e860ee4717.jpg"},{"id":109907171,"identity":"f7d6f693-dbfe-428a-8252-58859ec03ffe","added_by":"auto","created_at":"2026-05-25 06:41:46","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2187090,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9726674/v1/4e67fa89-6380-4a36-9c3f-a61c2ec62cd8.pdf"},{"id":109759714,"identity":"5c2df23e-d809-4b7c-9c91-33d0dfece9f8","added_by":"auto","created_at":"2026-05-22 07:27:35","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":329730,"visible":true,"origin":"","legend":"\u003cp\u003eSupplemental Tables S1-S5\u003c/p\u003e","description":"","filename":"Supplementarymaterial2APBinactivationinTRPV2v1.0.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9726674/v1/919ee5bc0c56c25095805e41.pdf"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003e\u003cstrong\u003eThermoring switch for TRPV2 inactivation\u003c/strong\u003e\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe regulation of activity in multidomain thermosensitive transient receptor potential (TRP) vanilloid 1-4 (TRPV1-4) channels by various physical and chemical stimuli is essential for organismal health\u0026nbsp;in various physiological or pathological processes. \u0026nbsp;These polytopic membrane proteins have complex topology and are homotetrameric, with each monomer featuring six ankyrin repeat domain (ARD), six transmembrane helices (S1-S6) and the C-terminal domain (CTD). \u0026nbsp;The first S1-S4 bundle forms the voltage sensor-like domain (VSLD) while the later S5-S6 bundle, together with two pore turrets and the pore helix (PH) between them, fold as the pore domain (PD). \u0026nbsp;Along with the S4-S5 linker at the active gating core, the VSLD interfaces the ARD and the CTD via the pre-S1 domain and the TRP domain, respectively. \u0026nbsp;When each PD from one subunit swaps with the PD\u0026rsquo; and the VSLD\u0026rsquo; from the adjacent one, the pre-S1 domain of TRPV3 or TRPV2 also forms an inter-protomer interface with the neighboring ARD\u0026rsquo;. \u0026nbsp;Thus, these global inter-domain allosteric-networks allow the selectivity filter to be an upper gate with a GXGY motif (X=M, L; Y=D, E) on the PH and the LIA motif near the\u0026nbsp;a-to-p\u0026nbsp;transition of S6 to be the lower gate for the activity regulation (1).\u003c/p\u003e\n\u003cp\u003eGiven that the TRPV1-4 channels can be activated by heat in the temperature range from 25 \u0026deg;C to 54 \u0026deg;C (2-10), recent studies have shown that the weakest tertiary bridge serves as a unique thermal sensor in TRPV1, TRPV3 or TRPV4. \u0026nbsp;This specific sensor connects allosterically with the highly conserved swapping\u0026nbsp;p\u0026nbsp;interaction between an aromatic residue on the S4-S5 linker and a positively charged residue on S5\u0026rsquo; near the lower gate for heat activation. \u0026nbsp;For example, in rat TRPV1 (rTRPV1)/human TRPV1 (hTRPV1) with phosphatidylinositol (PI) at the internal vanilloid site, the weakest Y401-R499/E406-K504 bridge at the pre-S1/VSLD interface is linked with the Y565-R579\u0026rsquo; bridge. In reduced hTRPV3 with phosphatidylcholine (PC) at the vanilloid site, the weakest E610-K649 bridge at the external interface between two pore turrets is coupled with the Y575-K589\u0026rsquo; bridge. \u0026nbsp;In human TRPV4 (hTRPV4), the weakest P498/Y502-Y567 bridge at the external interface between S1-S2 and S3-S4 linkers is coupled with the Y602-R616\u0026rsquo; bridge (11). \u0026nbsp; However, during the initial thermal activation of rTRPV2 with phosphatidylethanolamine (PE) in the VSLD and without PE at the vanilloid site, there is not a strict coupling between the least-stable K300-N354, S486-Y497 or L555-Y590 bridge and the strong swapping H521-R539\u0026rsquo;-Y525-N639\u0026rsquo; bridges near the lower gate, partly because the weakest K300-N354 bridge is not disrupted but enhanced in the cold-activated state, which mirrors the heat activation with high thermosensitivity (12).\u003c/p\u003e\n\u003cp\u003eOn the other hand, the initial broken Y575-K589\u0026rsquo; bridge in TRPV3 disrupts the critical weakest D586-T680 H-bond in the PD, resulting in channel inactivation and pore dilation, along with a tetramer-to-pentamer transition (13-15). \u0026nbsp;Since the chemical perturbation of the swapping H521-R539\u0026rsquo;-Y525-N639\u0026rsquo; bridges near the lower gate by 2-APB, an exogenous nonselective chemical agonist (16), also inactivates the rTRPV2 channel (17), it is hypothesized that disrupting the weakest K300-N354 bridge is allosterically coupled to the highly conserved swapping bridges near the lower gate for channel inactivation.\u003c/p\u003e\n\u003cp\u003eIn this computational study, a highly sensitive thermoring model, which has been recently developed (11-13, 18-29), was used to test this specific hypothesis. \u0026nbsp;By examining the 3D cryogenic electron microscopy (cryo-EM) structures of rTRPV2 in both the 2-APB-activated and inactivated states with or without an additional perturbation of cannabidiol (CBD) or probenecid (PBC) at various sites, it was observed that the weakest K300-N354 bridge remained intact in the activated states but was broken in the inactivated states, along with the highly conserved swapping p bridges broken near the lower gate. \u0026nbsp;In this regard, three undefined non-conductive cryo-EM structures of apo TRPV2 lacking PE in the VSLD and at the vanilloid site were categorized as inactivated. \u0026nbsp;Furthermore, considering the distinct thermostabilities of the activated and inactivated states, a potentiation mechanism for TRPV2 activity emerged in response to a combination of two chemical agonists.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec2\" class=\"Section2\"\u003e \u003ch2\u003eCryo-EM structures used\u003c/h2\u003e \u003cp\u003eFive full-length 3D cryo-EM structures of apo rTRPV2 channels without PE in the VSLD and at the vanillid site in the 2-APB-activated, 2-APB-inactivated, 2-APB/CBD-activated, 2-APB/CBD-inactivated and 2-APB/PBC-activated states were examined to identify the structural basis for the inactivation of rTRPV2 by 2-APB (PDB ID: 7N0N, model resolution\u0026thinsp;=\u0026thinsp;4.15 \u0026Aring;; 7N0M, model resolution\u0026thinsp;=\u0026thinsp;3.5 \u0026Aring;; 7T37, model resolution\u0026thinsp;=\u0026thinsp;3.7 \u0026Aring;; 7T38, model resolution\u0026thinsp;=\u0026thinsp;3.8 \u0026Aring;; 9B3V, model resolution\u0026thinsp;=\u0026thinsp;4.2 \u0026Aring;, respectively). These channels were first purified in the decyl maltose neopentyl glycol (DMNG) detergent and then reconstituted in membrane scaffold protein 2N2 (MSP2N2) nanodiscs at 4\u0026deg;C (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). In addition, the full-length or truncated 3D cryo-EM structures of other native TRPV2 channels were also used to test the putative inactivation motif. Their PDB IDs included 5AN8, 5HI9, 6BO4, 6U84, 6U86, 6U8A, 6U88, 6WKN, 7XEM, 7XEO, 7XEU, 7XEV, 7YEP, 7ZJD, 7ZJE, 7ZJG, 7ZJI, 7ZJH, 8EKP, 8EKQ, 8EKR, 8EKS, 8FFL, 8FFM, 8SLX, 8SLY and 9B3U (\u003cspan additionalcitationids=\"CR31 CR32 CR33 CR34 CR35 CR36 CR37 CR38 CR39\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eDetecting and Filtering tertiary noncovalent interactions\u003c/h2\u003e \u003cp\u003eAlong the PE-dependent minimal gating pathway of rTRPV2 from V254 to P726 (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e), the tertiary noncovalent interactions between two amino acid side chains or backbones, or combined were detected and filtered using UCSF Chimera with the same strict and consistent standards as previously confirmed (\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan additionalcitationids=\"CR19 CR20 CR21 CR22 CR23 CR24 CR25 CR26 CR27 CR28\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). Briefly, salt bridges were identified by scanning pairs of charged residues, lone pair/CH/cation/π-π interactions were identified by scanning aromatic residues such as Phe, Tyr or Trp and nearby residues, and H-bonds were identified in the tertiary structure using UCSF Chimera. Specific cutoff distances and interaction angles for the different noncovalent interactions can be found in the online Supporting Information (Table S1, S2, S3, S4, and S5). It shoud be noted that momentary fluctuation-induced perturbations in tertiary noncovalent interactions during protein dynamics were not considered in this study.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMapping tertiary thermoring structures using the grid thermodynamic model\u003c/h3\u003e\n\u003cp\u003eThe study utilized the same protocol as previously described and validated to map the systematic fluidic grid-like tertiary noncovalent interaction mesh network as the thermoring structure (11\u0026ndash;13, 18\u0026ndash;29). First, one interaction group included the pore domain (PD) and the other covered VSLD, TRP domain, ARD, pre-S1 domain and the CTD based on the identified tertiary noncovalent interactions. Second, along the defined PE-dependent minimal gating pathway of rTRPV2 from V254 to P726 (black line), arrowed network nodes and edges with linked nodes represented the involved amino acid residues and tertiary noncovalent bridges, respectively. Thus, each edge and nearby polypeptide segments with or without other edges could form several rings as topological grids without repeating the same edge. For example, in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, the T604-Y629 H-bond and the peptide chain segment from I605 to A628 could generate a ring. Alternatively, this H-bond could shape another ring with the nearby T604-Y544-Y629 H-bonds (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). In this case, Graph theory and the Floyd\u0026ndash;Warshall algorithm were necessary to constrain these grids so that a unique grid could be obtained for each specific tertiary noncovalent interaction with the shortest round path length or the minimal number of free residues in it as the grid size (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e). As the melting temperature required to unfold the least-stable tertiary noncovalent interaction in a grid is related to this unique grid size, the constained grid was also defined as a thermoring to control the least-stable interaction and denoted as Grid\u003csub\u003es\u003c/sub\u003e. As an example, in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, the Y544-T604-Y629-Y544 H-bonds were controlled by the same smallest Grid\u003csub\u003e0\u003c/sub\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). In this approach, along with all the tertiary noncovalent bridges labeled by uncommon grid sizes that corresponded to the minimum energy required to stabilize the interactions, the biggest thermoring could be identified to govern the weakest tertiary bridge along the PE-dependent minimal gating pathway from V254 to P726. Meanwhile, the total numbers of noncovalent interactions (\u003cem\u003eN\u003c/em\u003e) and total grid sizes (\u003cem\u003eS\u003c/em\u003e) along the same gating pathway were calculated and shown in black and cyan circles, respectively, next to the mesh network map for the following calculations.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eCalculating the systematic thermal instability\u003c/h3\u003e\n\u003cp\u003eThe systematic thermal instability (T\u003csub\u003ei\u003c/sub\u003e) was calculated using the following expression as previously examined (\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan additionalcitationids=\"CR19 CR20 CR21 CR22 CR23 CR24 CR25 CR26 CR27 CR28\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e):\u003c/p\u003e \u003cp\u003eT\u003csub\u003ei\u003c/sub\u003e = \u003cem\u003eS/N\u003c/em\u003e (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e)\u003c/p\u003e\n\u003ch3\u003eCalculating the melting temperature threshold for heat unfolding\u003c/h3\u003e\n\u003cp\u003eThe melting temperature threshold (T\u003csub\u003em,th\u003c/sub\u003e) for the heat unfolding of the least-stable noncovalent interaction within a specific grid at a normal salt concentration of 150 mM NaCl was calculated using the following expression as previously examined (\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan additionalcitationids=\"CR19 CR20 CR21 CR22 CR23 CR24 CR25 CR26 CR27 CR28\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e):\u003c/p\u003e \u003cp\u003eT\u003csub\u003em,th\u003c/sub\u003e (\u0026deg;C)\u0026thinsp;=\u0026thinsp;34 + (n\u0026thinsp;\u0026minus;\u0026thinsp;2) \u0026times; 10 + (20 \u0026ndash; s) \u0026times; 2 (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e)\u003c/p\u003e \u003cp\u003ewhere, n denotes the total number of basic H-bonds (~\u0026thinsp;1 kcal/mol for each in a hydrophilic environment) that are energetically equivalent to the weakest noncovalent bridge controlled by the given grid, and s is the size of the grid that controls the weakest tertiary bridge. Thus, the heat capacity of the grid will be higher with a smaller grid size or the more equivalent basic H-bonds.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eSimilar internal intersubunit interactions at the PBC site in both the activated and inactivated states\u003c/h2\u003e \u003cp\u003eA recent study has revealed that PBC targets K118, L126, Y162, H165, I170 and K174 from one monomer, as well as W333\u0026rsquo; and Y335\u0026rsquo; from the adjacent monomer of rTRPV2, preventing the inactivation of rTRPV2 (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e). Therefore, it is necessary to examine the swapping interactions at the PBC site in the activated and inactivated states in the presence of the agonist 2-APB with or without CBD or PBC treatment.\u003c/p\u003e \u003cp\u003eIn the 2-APB-activated state (PDB ID: 7N0N), aside from the Y162-F161-F198-F199 π interactions, the inactivation-sensitive H165 also formed π interactions with nearby H169 and F199 from the same subunit, as well as heat-sensitive Y335\u0026rsquo; from the adjacent subunit (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e). In contrast, the additional Y335\u0026rsquo;-F198/F199 and W333\u0026rsquo;-Y162 π bridges were found in the 2-APB-inactivated state (PDB ID: 7N0M), and all of these interactions were disrupted in the 2-APB/PBC-activated state. Thus, the tight swapping interface at the PBC site seemed to favor the inactivated state. However, similar swapping interactions were also observed in the 2-APB/CBD-activated and inactivated states (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Accordingly, strictly speaking, the PBC site is not directly responsible for channel inactivation. Further identification of a structural motif is necessary for TRPV2 inactivation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eThe K300-N354 H-bond is present in the 2-APB-activated state but not in the inactivated state\u003c/h3\u003e\n\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eAlong the PE-dependent minimal gating pathway from N254 to P726 in the 2-APB-activated state without PE in the VSLD and at the vanilloid site (PDB ID: 7N0N), a total of 105 tertiary noncovalent interactions formed a local grid-like mesh network constrained with a total of 112 grid sizes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, Table S1). Thus, the local systematic thermal instability (T\u003csub\u003ei\u003c/sub\u003e) was calculated as 1.07 (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Meanwhile, the biggest Grid\u003csub\u003e17\u003c/sub\u003e was found to control the weakest S559-P589 H-bond via a thermoring from P589 to L600, Y629, Y551, S559 and back to P589 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003eB-C). Because this weakest bridge was energetically equivalent to 1.2 basic H-bonds (1.2 kcal/mol), the calculated melting temperature threshold (T\u003csub\u003em,th\u003c/sub\u003e) was about 32\u0026deg;C (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), which was lower than the body temperature of 37\u0026deg;C. Thus, this activated state may be unstable at the physiological temperature. Notably, with 2-APB perturbing the swapping interace between the S4-S5 linker and S5\u0026rsquo;, the swapping H521-R539\u0026rsquo;-Y525-N639\u0026rsquo; π bridges near the lower gate were disrupted along with the broken Y412-R560\u0026rsquo; and F618\u0026rsquo;-W496/P499 and F612\u0026rsquo;-L594 π interactions near the upper gate (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003eD) (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eComparison of ligand-induced thermoring structural changes of rTRPV2 along the PE-dependent minimal gating pathway from V254 to P726. The comparative parameters are highlighted in bold.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePDB ID\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7N0N\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7N0M\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7T37\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e7T38\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e9B3V\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLipid in the VSLD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFree\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFree\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eFree\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eFree\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003ePE\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLipid at the active vanilloid site\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFree\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFree\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eFree\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eFree\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eFree\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLipid environment\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMSP2N2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMSP2N2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMSP2N2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMSP2N2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eMSP2N2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSampling temperature, \u0026deg;C\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLigand\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2-APB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2-APB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2-APB\u0026thinsp;+\u0026thinsp;CBD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2-APB\u0026thinsp;+\u0026thinsp;CBD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2-APB\u0026thinsp;+\u0026thinsp;PBC\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGating state\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eActivated\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eInactivated\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eActivated\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eInactivated\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eActivated\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e# of the biggest Grids\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGrid\u003csub\u003e17\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGrid\u003csub\u003e10\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eGrid\u003csub\u003e15\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eGrid\u003csub\u003e21\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eGrid\u003csub\u003e16\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003egrid size (s)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e# of basic H-bonds (n) in stability equivalent to the weakest noncovalent bridge\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal non-covalent interactions (N)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e105\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e110\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e105\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e124\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal grid sizes (S), a.a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e112\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e119\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e106\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e130\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSystemic thermal instability (Ti)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e1.07\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e1.08\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e1.00\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e1.33\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e0.81\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCalculated T\u003csub\u003em,th\u003c/sub\u003e \u0026deg;C at E\u0026thinsp;=\u0026thinsp;1 kcal/mol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e32\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e49\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e39\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e22\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e32\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRefs for PDB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e17\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e17\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e17\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e17\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e17\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eIn contrast, in the 2-APB-inactivated state without PE in the VSLD and at the vanilloid site (PDB ID: 7N0M), the total numbers of tertiary noncovalent interactions and grid sizes along the same PE-dependent minimal gating pathway from N254 to P726 increased from 105 and 112 to 110 and 119, respectively (Table S2). Thus, the local T\u003csub\u003ei\u003c/sub\u003e of 1.08 was similar to 1.07 (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Meanwhile, the biggest Grid\u003csub\u003e10\u003c/sub\u003e appeared to govern the least-stable M468-Q530 H-bond via a thermoring from F467 to Y471, F472, Y514, Y515, F393, F394, F519, Q530, M468, and back to F467 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB-C). Since this weakest bridge at the S4/S5 interface was energetically equivalent to 1.5 basic H-bonds (1.5 kcal/mol), the calculated T\u003csub\u003em,th\u003c/sub\u003e was about 49\u0026deg;C, which was higher than 32\u0026deg;C in the 2-APB-activated state (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Therefore, the inactivated state was much more stable than the activated state after the 2-APB perturbation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAlthough the swapping of H521-R539\u0026rsquo;-Y525-N639\u0026rsquo; π bridges near the lower gate were still broken, the Y412-R560\u0026rsquo; and F618\u0026rsquo;-W496/P499 and F612\u0026rsquo;-L594 π interactions near the upper gate were reinstated (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD) (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). Most notably, the weakest K300-N354 bridge at the interface between the pre-S1 domain and the ARD in the pre-open closed state was found to be in the 2-APB-activated state but not in the inactivated state (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003eA \u0026amp; \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA) (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). This finding was consistent with a significant 4.4 \u0026Aring; downwards movement of the ARDs in the 2-APB-inactivated state (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eGiven that this interfacial H-bond was controlled by a smaller Grid\u003csub\u003e2\u003c/sub\u003e through a thermoring from K300 to T297, L296, N292, Q294, K304, C364, F362, R459, E358, S355, N354 and back to K300 and was energetically equivalent to 1.3 basic H-bonds (1.3 kcal/mol) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003eA), the calculated T\u003csub\u003em,th\u003c/sub\u003e to unfold it was about 63\u0026deg;C. Accordingly, it is necessary to examine if this stable H-bond at the pre-S1/ARD interface was also present in the 2-APB-activated state but not in the inactivated state upon perturbation by external CBD or internal PBC.\u003c/p\u003e\n\u003ch3\u003eExternal CBD cannot prevent the breaking of the K300-N354 bridge in the 2-APB-inactivated state\u003c/h3\u003e\n\u003cp\u003eWhen CBD additionally interacted with both L537 from one PD and Y634\u0026rsquo; from the neighboring PD\u0026rsquo; (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e), the K300-N354 bridge in the 2-APB-activated state was still observed along with a total of 105 tertiary noncovalent bridges and 106 grid sizes (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, Table S3). In this case, the local grid-based systematic thermal instability (T\u003csub\u003ei\u003c/sub\u003e) slightly decreased from 1.07 to 1.00 (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). However, the new weakest H438-Q487 H-bond at the interface between the S1-S2 and S3-S4 linkers was governed by the biggest Grid\u003csub\u003e15\u003c/sub\u003e via a thermoring involving H438, Y447, W676, W454, Y455, R459, F362, A361, R369, D469, Y471, F472, Y514, F476, Q487 and back to H438 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB-C). Since the controlled bridge was energetically equivalent to 1.5 basic H-bonds (1.5 kcal/mol), the calculated T\u003csub\u003em,th\u003c/sub\u003e was about 39\u0026deg;C, higher than the 32\u0026deg;C for the 2-APB-activated state (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Therefore, CBD binding stabilized the 2-APB-activated state without affecting the K300-N354 bridge. On the other hand, the L537-CBD-Y634\u0026rsquo; bridge restored the Y525-R539\u0026rsquo; bridge near the lower gate and the W496-F618\u0026rsquo;-P499 bridges near the upper gate (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eOf special note, the K300-N354 bridge was also broken in the 2-APB/CBD-inactivated state, along with a decrease in the total tertiary noncovalent bridges from 110 to 98 but an increase in the total grid sizes from 119 to 130 (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA \u0026amp; \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, Tables S2 \u0026amp; S4). As a result, the local systematic thermal instability (T\u003csub\u003ei\u003c/sub\u003e) increased from 1.08 to 1.33 (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Hence, CBD destabilized the 2-APB-inactivated state. In line with this notion, the weakest R563-Q615 H-bond was found at the external interface between two pore turrets (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). It was controlled by the biggest Grid\u003csub\u003e21\u003c/sub\u003e via a thermoring from F551 to R563, Q615, T604, F601, Y544, Y629, and back to F551 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB-C). For the weakest R563-Q615 bridge to be energetically equivalent to 1.0 basic H-bond (1.0 kcal/mol), the calculated T\u003csub\u003em,th\u003c/sub\u003e was 22\u0026deg;C, lower than 49\u0026deg;C in the 2-APB-inactivated state (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eInternal PBC prohibits any disruption to the K300-N354 bridge\u003c/h2\u003e \u003cp\u003eFollowing the treatment of internal PBC (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e), the K300-N354 bridge was only present in the 2-APB-activated state, along with a total of 124 tertiary noncovalent interactions and 100 grid sizes (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, Table S5). As a result, the local systematic thermal instability (T\u003csub\u003ei\u003c/sub\u003e) significantly decreased from 1.07 to 0.81 (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Meanwhile, the biggest Grid\u003csub\u003e16\u003c/sub\u003e appeared to control the weakest E561-R617 H-bond via a thermoring from F551 to E561, R617, F618, R619, F612, T604, F544, Y629, and back to F551 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB-C). Since this weakest bridge between two pore turrets was energetically equivalent to 1.2 basic H-bonds (1.2 kcal/mol), the T\u003csub\u003em,th\u003c/sub\u003e was calculated as 32\u0026deg;C, the same as the value in the 2-APB-activated state (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Therefore, after removing the 2-APB-inactivated state, PBC could potentiate the channel activity below the physiological temperature. Notably, the swapping Y625-N639\u0026rsquo; bridge near the lower gate and the P499-F618\u0026rsquo; and L594-F612\u0026rsquo; bridges near the upper gate were restored (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eThe K300-N354 bridge was found to be highly conserved in both activated and closed states\u003c/h2\u003e \u003cp\u003eFurther structural scanning uncovered that the K300-N354 bridge was also present with other agonists such as protons, CBD, phytocannabinoid Δ9-tetrahydrocannabiorcol (C16) and estradiol (E2) alone or in combination with PBC. It should be noteworthy that in the absence of any ligand, this bridge still appeared in three closed states with PE bound (PDB ID: 8EKP, 8EKQ and 8EKR), a PE-free closed state (PDB ID: 6U86) and a PE-free open state (PDB ID: 6BO4) (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e). However, it disappeared in two PE-free apo rTRPV2 channels: one in DMNG (PDB ID: 5HI9) and the other in the DMNG/MSP2N2 nanodiscs (PDB ID: 6U84) (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e). In addition, it was also absent in a truncated rabbit TRPV2 channel without PE in the VSLD and at the vanilloid site (PDB: 5AN8) (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). Therefore, these three nonconducting TRPV2 channels were actually in an inactivated or desensitized state, rather than a closed state.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eIdentification of gating states of rTRPV2 with or without a ligand.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"9\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSpecies\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePDB ID\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNanodiscs/ MβCD\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePE/VSLD\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePE/VBP\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eLigand\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003ePre-S1/ARD interface\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eGating state\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eRefs.\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRabbit\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5AN8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eInactivated\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRat\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5HI9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eInactivated\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e31\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRat\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6BO4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eK300-N354\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eOpen\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRat\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6U84\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eInactivated\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e33\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRat\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6U86\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eK300-N354\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eActivated\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e33\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRat\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6U8A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eCBD-1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eK300-N354\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eActivated\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e33\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRat\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6U88\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eCBD-2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eK300-N354\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eActivated\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e33\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRat\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6WKN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003ePL(antagonist)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eK300-N354\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eInhibited\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e34\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMouse\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7XEM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eCHL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eK295-N349\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eInhibited\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e35\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMouse\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7XEO\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eK295-N349\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eClosed\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e35\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMouse\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7XEU\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eE2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eK295-N349\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eActivated\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e35\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMouse\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7XEV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2-APB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eE2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eInactivated\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e35\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMouse\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7YEP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2-APB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eInactivated\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e35\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRat\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7ZJD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eC16-1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eK300-N354\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eActivated\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e36\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRat\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7ZJE\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eC16-2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eK300-N354\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eActivated\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e36\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRat\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7ZJG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003ePBC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eK300-N354\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eActivated\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e36\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRat\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7ZJI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eC16\u0026thinsp;+\u0026thinsp;PBC-1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eK300-N354\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eActivated\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e36\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRat\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7ZJH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eC16\u0026thinsp;+\u0026thinsp;PBC-2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eK300-N354\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eActivated\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e36\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRat\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8SLX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1 CBD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eK300-N354\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eActivated\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e37\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRat\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8SLY\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2 CBD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eK300-N354\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eActivated\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e37\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRat\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8FFL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eRR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eK300-N354\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eActivated\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e38\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRat\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8FFM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eRR\u0026thinsp;+\u0026thinsp;2-APB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eK300-N354\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eActivated\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e38\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRat\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8EKP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eK300-N354\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eClosed\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e39\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRat\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8EKQ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eK300-N354\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eClosed\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e39\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRat\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8EKR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eK300-N354\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eClosed\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e39\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRat\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8EKS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eH\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eK300-N354\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eActivated\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e39\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRat\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e9B3U\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003ePBC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eK300-N354\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eActivated\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e40\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe highly conserved swapping interactions between a Tyr residue side chain on the S4-S5 linker of one subunit and a Lys/Arg residue side chain on S5\u0026rsquo; from the neighboring subunit near the lower gate play a critical role in channel gating of thermosensitive TRPV1-4 channels (\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e). On one hand, these swapping interactions must be finally broken for heat activation (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). On the other hand, when they are first disrupted, the channel can become inactivated (\u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). After illuminating the thermoring basis for the inactivation-induced pore dilation of hTRPV3, this study further demonstrated that the weakest K300-N354 bridge in the pre-open closed state was allosterically coupled to the highly conserved swapping interactions near the lower gate for TRPV2 inactivation. This study also identified other inactivated states that have not been clearly defined. In addition, the various thermostabilities of activated and inactivated states upon two agonist perturbations at different sites may help us understand the potentiation or sensitization mechanism of one by the other. Collectively, the unique interfacial bridge advances our understanding of the thermoring switch for allosteric gating regulation of thermosensitve TRPV1-4 channel.\u003c/p\u003e \u003cp\u003e \u003cb\u003eThe K300-N354 gating bridge is an important feature that distinguishes the inactivated state from the activated or closed state\u003c/b\u003e \u003c/p\u003e \u003cp\u003eDespite the activation of TRPV2 by multiple agonists such as CBD, 2-APB, E2, PBC and protons alone or in combination, their cryo-EM structures do not always show the pore opening. On the other hand, the TRPV2 channel is open in the absence of these agonists but in the presence of detergents such as lauryl maltose neopentyl glycol (LMNG) at low temperature (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Therefore, it is unclear whether the channel has been activated or inactivated when it adopts a non-conducting conformation at low temperature.\u003c/p\u003e \u003cp\u003eIn this computational study, detailed thermoring analyses revealed that the unique K300-N354 bridge at the pre-S1/ARD interface was present in the 2-APB-activated state with or without CBD or PBC treatment but absent in the 2-APB-inactivated state with or without CBD perturbation (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Further structural scanning demonstrated that the K300-N354 bridge (K295-N349 bridge in mTRPV2) also appeared in the presence of other agonists such as C16 and E2 or a mild detergent LMNG to activate or open the channel (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e). Once the channel is activated, the blocker RR fails to affect this bridge (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eOn the other hand, the K300-N354 bridge was also found in three closed states with PE bound in the VSLD or at the vanilloid site (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e). Therefore, the presence of the K300-N354 bridge could distinguish between the activated or closed state and the inactivated state. Based on this characteristic coupling, several cryo-EM structures of apo TRPV2 were identified as inactivated. These included two apo rTRPV2 channels (PDB ID: 5HI9 and 6U84) (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e), one apo rabbit TRPV2 (PDB ID: 5AN8) (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e), and two mTRPV2 channels with 2-APB or plus E2 (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSince the weakest K300-N354 bridge in the pre-open closed state is also important for thermal activation and 6U84 without this bridge has a higher threshold for heat activation (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e), it is possible that 6U84 may also function as a heat-induced inactivated state of rTRPV2 (\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e). Given that human TRPV2 (hTRPV2) is insensitive to heat and 2-APB below 60\u0026deg;C, and the pre-S1 domain, the ARD and the CTD of rTRPV2 are involved in responses to heat and 2-APB (\u003cspan additionalcitationids=\"CR46 CR47 CR48\" citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e), the inactivated 6U86 channel could potentially serve as a homological model of hTRPV2 in a resting state without the K300-N354 bridge but with a heat threshold exceeding 60\u0026deg;C (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). Notably, the presence of the K300-N354 bridge in rTRPV2 with the antagonist piperlongumine (PL) or mTRPV2 with cholesterol (CHL) suggests that channel activation could be inhibited (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e). Jointly, similar to TRPV3, there is a state-dependent coupling between cytoplasmic and transmembrane domains in rTRPV2 that helps regulate normal channel gating (\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e).\u003c/p\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eTight swapping interface between the S4-S5 linker and S5\u0026rsquo; is necessary for initial TRPV2 closure\u003c/h2\u003e \u003cp\u003eRecent studies have indicated that the swapping bridge near the lower gate, Y565-R579\u0026rsquo; in rTRPV1/hTRPV1, Y575-K589\u0026rsquo; in reduced hTRPV3, or Y602-R616\u0026rsquo; in hTRPV4 is involved in channel activation of TRPV1, TRPV3 or TRPV4 (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e). However, in TRPV2, the swapping bridges H521-R539\u0026rsquo;-Y525-N639\u0026rsquo; are present in three closed states with PE bound (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). This study further demonstrated that these bridges were partially or fully broken in the 2-APB-activated or inactivated states with or without CBD or PBC perturbation. Therefore, at least the H521-R539\u0026rsquo;-Y525 bridges at the interface between the S4-S5 linker and S5\u0026rsquo; are necessary for initial rTRPV2 closure.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eNanodiscs or MβCD is necessary for cryo-EM structures of TRPV2 with normal gating\u003c/h2\u003e \u003cp\u003eIn the presence of MSP2N2 nanodiscs or methyl-β-cyclodextrin (MβCD), rTRPV2 was inactivated or desensitized upon 2-APB perturbation (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e). However, in the absence of MSP2N2/MβCD and 2-APB, the channel was also inactivated (PDB ID: 5AN8, 5HI9) (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). This is reminiscent of the inactivated TRPV3 channel in the absence of MSP2N2 (\u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). Therefore, although the treatment of MβCD does not affect the 2-APB sensitivity of mTRPV2 (\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e), after detergent solubilization, the MSP2N2 nanodisc or MβCD treatment favors the cryo-EM structures of initial TRPV2 closure with the H521-R539\u0026rsquo;-Y525 bridges (H516- R534\u0026rsquo;-Y521 bridges in mTRPV2) in a near-native lipid environment.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eThermostability-based activity potentiation\u003c/h2\u003e \u003cp\u003eAgonist-induced dynamic intrasubunit allosteric gating pathways may be involved in the global unfolding/folding and posttranslational stability of TRPV2. Along with 2-APB binding at the interface between the S4-S5 linker and S5\u0026rsquo;, rTRPV2 can be activated and inactivated along with the ARD-up and ARD-down conformations, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). Since the melting threshold of the inactivated state was higher than that of the activated one (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), a minor activated state (PDB 7N0N) and a dominant inactivated state (PDB 7N0M) resulted in limited activity (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). However, the L537-CBD-Y634\u0026rsquo; bridge significantly enhanced the thermostability of the 2-APB-activated state but weakened the thermostability of the 2-APB-inactivated state (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Therefore, it is reasonable that CBD can sensitize the 2-APB activation rather than the heat activation at 40\u0026deg;C (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e). Similarly, PBC also decreased the local systematic thermal instability (T\u003csub\u003ei\u003c/sub\u003e) of the 2-APB-activated state while inhibiting inactivation (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Thus, a change in the occupancy of the activated and inactivated states may account for the activity potentiation by PBC (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). Given that the activation of rTRPV2 by PBC involves H165 (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e), and either protonation of H521 at pH 5 or 2-APB perturbation at pH 8 can disrupt the swapping of H521-R539\u0026rsquo;-Y525-N639\u0026rsquo; bridges at the S4-S5 linker/S5\u0026rsquo; interface but still maintain the intact K300-N354 bridge for channel activation (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e), protonation of H165 and H521 may stabilize the 2-APB-activated state (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003e), allowing acid to enhance the channel activity (\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e). In any case, the up and down conformations of the ARD play a pivotal role in switching TRPV2 gating via the unique K300-N354 bridge. The resultant difference in global thermostability between the activated and inactivated states allows for the regulation of channel activity in response to various agonists at different sites.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eOn the other hand, although the tight allosteric coupling between the weakest bridge at the interface between the pre-S1 domain and the VSLD, between two pore turrets or between S1-S2 and S3-S4 linkers in the pre-open closed state and the highly conserved swapping interaction near the lower gate for thermal activation of TRPV1, TRPV3 or TRPV4 allows each to be a highly-sensitive biothermometer for monitoring a change in environment temperature, respectively (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e), the allosteric coupling between the weakest bridge at the pre-S1/ARD interface in the pre-open closed state and the highly conserved swapping interactions near the lower gate for TRPV2 inactivation compromises the channel\u0026rsquo;s ideal design for detecting acute thermal nociception above 50\u0026deg;C or a relevant target for pain therapy, particularly for hTRPV2.\u003c/p\u003e \u003c/div\u003e"},{"header":"conclusions","content":"\u003cp\u003eThe cryo-EM structures of ion channels have been extensively used to identify drug binding sites. However, most of these structures, when at low temperatures, adopt a nonconductive conformation, regardless of the presence of an exogenous chemical agonist or antagonist. Therefore, it is still unclear whether the channel is activated, inactivated, inhibited or closed, hindering the understanding of the precise action mechanism of a drug. In this computational study, the tertiary structural changes of TRPV2 from activated to inactivated states were analyzed using a high-sensitivity thermoring model. By comparing five thermoring structures and examining the channel\u0026rsquo;s thermosensitivity in response to different agonists, the unique weakest interdomain H-bond in the pre-open closed state was found to couple the highly conserved swapping interactions allosterically for channel inactivation, rather than activation. Furthermore, a change in thermostability revealed a unique potentiation mechanism when two agonists were involved. Finally, other gating states under various conditions were further defined based on this coupling hallmark. Therefore, this thermoring model can be used to elucidate the allosteric coupling between the protein stability and channel gating, as well as the precise action mechanism of drugs on ion channels.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003e2-APB, 2-aminoethoxydiphenyl borate\u003c/p\u003e\n\u003cp\u003eARD, Ankyrin repeat domain\u003c/p\u003e\n\u003cp\u003eCBD, cannabidiol\u003c/p\u003e\n\u003cp\u003eCHL, cholesterol\u003c/p\u003e\n\u003cp\u003ecryo-EM,\u0026nbsp;cryogenic electron microscopy\u003c/p\u003e\n\u003cp\u003eCTD, C-terminal domain\u003c/p\u003e\n\u003cp\u003eDMNG, decyl maltose neopentyl glycol\u003c/p\u003e\n\u003cp\u003eE2, estradiol\u003c/p\u003e\n\u003cp\u003eLMNG, lauryl maltose neopentyl glycol\u003c/p\u003e\n\u003cp\u003eMSP2N2, membrane scaffold protein 2N2\u003c/p\u003e\n\u003cp\u003eM\u0026beta;CD, methyl-\u0026beta;-cyclodextrin\u003c/p\u003e\n\u003cp\u003ePE, phosphatidylethanolamine\u003c/p\u003e\n\u003cp\u003ePC, phosphatidylcholine\u003c/p\u003e\n\u003cp\u003ePI, phosphatidylinositol\u003c/p\u003e\n\u003cp\u003eC16, phytocannabinoid \u0026Delta;9-tetrahydrocannabiorcol\u003c/p\u003e\n\u003cp\u003ePL, piperlongumine\u003c/p\u003e\n\u003cp\u003ePD, pore domain\u003c/p\u003e\n\u003cp\u003ePH, pore helix\u003c/p\u003e\n\u003cp\u003ePBC, probenecid\u003c/p\u003e\n\u003cp\u003eRR, 2-aminoethoxydiphenyl borate\u003c/p\u003e\n\u003cp\u003eT\u003csub\u003ei\u003c/sub\u003e, systematic thermal instability\u003c/p\u003e\n\u003cp\u003eT\u003csub\u003em,th\u003c/sub\u003e, melting temperature threshold\u003c/p\u003e\n\u003cp\u003eTRP, transient receptor potential\u003c/p\u003e\n\u003cp\u003eTRPV, TRP vanilloid\u003c/p\u003e\n\u003cp\u003eTRPVi (i=1, 2, 3, 4)\u003c/p\u003e\n\u003cp\u003ehTRPV1, human TRPV1\u003c/p\u003e\n\u003cp\u003ehTRPV2, human TRPV2\u003c/p\u003e\n\u003cp\u003ehTRPV3, human TRPV3\u003c/p\u003e\n\u003cp\u003ehTRPV4, human TRPV4\u003c/p\u003e\n\u003cp\u003erTRPV1, rat TRPV1\u003c/p\u003e\n\u003cp\u003erTRPV2, rat TRPV2\u003c/p\u003e\n\u003cp\u003eVBP, vanilloid binding pocket\u003c/p\u003e\n\u003cp\u003eVSLD, voltage-sensor-like domain\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eData availability\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analysed during this study are included in this published article and supplementary material.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eSupporting information\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis article contains supplementary material (Tables S1, S2, S3, S4 and S5).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eFunding Declaration\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe work was supported by the American Heart Association (AHA) Grant (10SDG4120011 to GW).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAuthor contributions\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eG. W. wrote the main manuscript text and prepared Figures 1, 2, 3, 4, 5, 6, 7 and Tables 1 and 2 and supplementary material (Tables S1, S2, S3, S4 and S5), and reviewed the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eCompeting interests\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe author declares no conflict of interest.\u003c/p\u003e\n"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eCao E (2020) Structural mechanisms of transient receptor potential ion channels. J Gen Physiol 152:e201811998\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCaterina MJ, Schumacher MA, Tominaga M et al (1997) The capsaicin receptor: A heat-activated ion channel in the pain pathway. Nature 389:816\u0026ndash;824\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCaterina MJ, Rosen TA, Tominaga M et al (1999) A capsaicin-receptor homologue with a high threshold for noxious heat. 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J Physiol 599:4831\u0026ndash;4844\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZubcevic L, Borschel WF, Hsu AL, Borgnia MJ, Lee S (2019) Y. Regulatory switch at the cytoplasmic interface controls TRPV channel gating. Elife 8:e47746\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFricke TC et al (2025) Molecular determinants of 2-aminoethoxydiphenyl borate sensitivity of transient receptor potential vanilloid 2-unexpected differences between 2 rodent orthologs. Mol Pharmacol 107:100060\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGao L et al (2016) Selective potentiation of 2-APB-induced activation of TRPV1-3 channels by acid. Sci Rep 6:20791\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"University of California, Davis","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Allosteric gating coupling, interdomain interaction, protein stability, thermoring structure","lastPublishedDoi":"10.21203/rs.3.rs-9726674/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9726674/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe interdomain interaction plays a critical role in regulating protein activity. Although the exogenous nonselective chemical agonist, 2-aminoethoxydiphenyl borate (2-APB), is well-known for activating and inactivating the homotetrameric thermosensitive transient receptor potential vanilloid 2 (TRPV2) channel, the precise structural basis is still missing. In this computational study, the 3D cryo-EM structures of rat TRPV2 in both 2-APB-activated and inactivated states alone or in combination with other exogenous chemical agonists were analyzed and quantified at the tertiary and quaternary levels using a highly sensitive thermoring model. The results indicated that the weakest tertiary bridge between the pre-S1 domain and the ankyrin repeat domain in the pre-open closed state was still present in the activated state but disrupted in the inactivated state along with the highly conserved swapping π bridges broken near the lower gate, regardless of chemical perturbations at different sites. Furthermore, the thermostability difference between the activated and inactivated states may enhance our understanding of the potentiation mechanism by an additional agonist. Finally, three lipid-free non-conductive cryo-EM structures of apo TRPV2 channels in the presence or absence of nanodiscs but without the treatment of methyl-β-cyclodextrin were identified as inactivated or desensitized, rather than closed. Therefore, the thermoring structures of a protein in different functional states can help us pinpoint the precise dynamic allosteric domain-domain communication for the regulation switch of protein activity.\u003c/p\u003e","manuscriptTitle":"Thermoring switch for TRPV2 inactivation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-05-18 06:51:16","doi":"10.21203/rs.3.rs-9726674/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"43ee4efc-79ce-40f4-ace3-56b2fcffba09","owner":[],"postedDate":"May 18th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":68196136,"name":"Biophysics"},{"id":68196137,"name":"Computational Biology"},{"id":68196138,"name":"Chemical Biology"}],"tags":[],"updatedAt":"2026-05-18T06:51:16+00:00","versionOfRecord":[],"versionCreatedAt":"2026-05-18 06:51:16","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9726674","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9726674","identity":"rs-9726674","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}