Decision letter: Cannabidiol sensitizes TRPV2 channels to activation by 2-APB

Abstract

Full text Figures and data Side by side Abstract Editor's evaluation Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract The cation-permeable TRPV2 channel is important for cardiac and immune cell function. Cannabidiol (CBD), a non-psychoactive cannabinoid of clinical relevance, is one of the few molecules known to activate TRPV2. Using the patch-clamp technique, we discover that CBD can sensitize current responses of the rat TRPV2 channel to the synthetic agonist 2-aminoethoxydiphenyl borate (2-APB) by over two orders of magnitude, without sensitizing channels to activation by moderate (40°C) heat. Using cryo-EM, we uncover a new small-molecule binding site in the pore domain of rTRPV2 in addition to a nearby CBD site that had already been reported. The TRPV1 and TRPV3 channels are also activated by 2-APB and CBD and share multiple conserved features with TRPV2, but we find that strong sensitization by CBD is only observed in TRPV3, while sensitization for TRPV1 is much weaker. Mutations at non-conserved positions between rTRPV2 and rTRPV1 in either the pore domain or the CBD sites failed to confer strong sensitization by CBD in mutant rTRPV1 channels. Together, our results indicate that CBD-dependent sensitization of rTRPV2 channels engages multiple channel regions, and that the difference in sensitization strength between rTRPV2 and rTRPV1 channels does not originate from amino acid sequence differences at the CBD binding site or the pore domain. The remarkably robust effect of CBD on TRPV2 and TRPV3 channels offers a promising new tool to both understand and overcome one of the major roadblocks in the study of these channels – their resilience to activation. Editor's evaluation This is an important report on the discovery of a strong sensitizing effect of cannabidiol on the activation of TRPV2 channels by 2-APB. The conclusions are convincingly supported by electrophysiological recordings and cryo-EM structures, but identification of a clear molecular mechanism will require additional structural work. The paper will be of interest to the ion channel research community. https://doi.org/10.7554/eLife.86166.sa0 Decision letter Reviews on Sciety eLife's review process Introduction The non-selective, cation-permeable transient receptor potential vanilloid 2 (TRPV2) channel is a homo-tetrameric protein (Huynh et al., 2016; Zubcevic et al., 2016) expressed in multiple cell types and tissues in animals (Caterina et al., 1999; Tamura et al., 2005). Adult mice in which TRPV2 channel expression in cardiomyocytes was ablated died within 2 wk and exhibited severe structural abnormalities of the heart (Iwata and Matsumura, 2019). In neonatal mice, TRPV2-deficient cardiomyocytes were also highly dysfunctional after growth in culture (Katanosaka et al., 2014), indicating that TRPV2 channel expression in cardiomyocytes is required for normal development and function of the heart. In macrophages, TRPV2 channel expression is essential for phagocytosis (Link et al., 2010; Lévêque et al., 2018), in pancreatic β-cells it influences insulin secretion (Hisanaga et al., 2009), in red blood cells it contributes to their response to osmotic challenges (Belkacemi et al., 2021), and its expression levels are significantly altered in multiple types of cancers (Monet et al., 2010; Yamada et al., 2010; Kudou et al., 2019; Siveen et al., 2020; Guéguinou et al., 2021). Interestingly, the identity of the stimuli that orchestrate TRPV2 channel activity in each of the examples mentioned above remains unknown. Very few stimuli capable of activating the TRPV2 channel have been identified; the extreme temperatures (>55°C) required to activate rodent TRPV2 channels (Caterina et al., 1999; Tamura et al., 2005; Yao et al., 2011; Liu and Qin, 2016) and the lack of thermo-sensitivity of the human orthologue under basal conditions (Neeper et al., 2007; Yao et al., 2011) exclude a likely role for this protein in thermo-detection. Yet, oxidative modification of specific methionine residues in rodent and human TRPV2 channels resulted in robust channel activation at temperatures <50°C (Fricke et al., 2019), suggesting that redox processes, temperature, and membrane depolarization together could promote channel activity in native tissues under physiological or pathological conditions. The synthetic agonist 2-aminoethoxydiphenyl borate (2-APB) robustly activates rodent but not human TRPV2 channels (Hu et al., 2004; Juvin et al., 2007), and even rodent TRPV2 channels have very low apparent affinity for 2-APB, with an EC50 > 1 mM (Gao et al., 2016a; Liu and Qin, 2016) that is close to the solubility limit of the compound. Importantly, 2-APB also targets other channels, including TRP channels (Hu et al., 2004; Xu et al., 2005; Togashi et al., 2008; Kovacs et al., 2012), STIM-Orai channels (Bootman et al., 2002), and gap-junctions (Bai et al., 2006). Cannabidiol (CBD), a non-psychotropic compound from the cannabis plant that has received much attention recently for its potential use in treating a variety of disorders (Pauli et al., 2020), has also been found to activate rodent TRPV2 channels with much higher apparent affinity than 2-APB (Qin et al., 2008). Other cannabis-derived compounds, including Δ9-tetrahydrocannabinol (THC), are also reported to activate TRPV2 channels (Neeper et al., 2007; Qin et al., 2008; Zhang et al., 2022). In stark contrast with the limited number of stimuli that activate the TRPV2 channel, the TRPV1 channel expressed in nociceptive neurons can be activated by the stimuli that activate the TRPV2 channel, but also by a remarkably diverse set of inflammatory mediators (Zygmunt et al., 1999; Hwang et al., 2000; Shin et al., 2002; Nieto-Posadas et al., 2011; Joseph et al., 2019), animal toxins (Bohlen et al., 2010; Yang et al., 2015), and natural products (Caterina et al., 1997; Salazar et al., 2008). Paradoxically, both channels are >40% identical in amino acid sequence, have an identical structural fold (Gao et al., 2016b; Huynh et al., 2016; Zubcevic et al., 2016), and utilize similar mechanisms to gate the passage of cations through their ion conduction pathway in response to stimulation with 2-APB (Jara-Oseguera et al., 2019). Notably, high sensitivity to a TRPV1-specific agonist, resiniferatoxin (RTx), could be engineered into the rat TRPV2 channel simply by substituting four non-conserved residues in a pocket that is otherwise equivalent to where RTx binds in TRPV1 (Yang et al., 2016; Zhang et al., 2016; Zhang et al., 2019). Cryo-electron microscopy (cryo-EM) structures of rat TRPV2 channels obtained in the presence of CBD (Pumroy et al., 2019) and CBD together with 2-APB (Pumroy et al., 2022) suggest that the cannabinoid binds close to the regions in the channel that directly gate the passage of cations through the pore in response to stimulation. Here we set out to use the patch-clamp technique and cryo-EM structural determination to characterize the actions of CBD on rat TRPV2 channels, and learn more about how the mechanisms of TRPV2 channel activation contrast with those of its closest homologue, the TRPV1 channel that can be activated by so many different types of stimuli. We discovered that CBD potently sensitizes the rTRPV2 channel to activation by 2-APB, without an effect on channel sensitivity to moderate heat. We obtained structures of rTRPV2 in the presence of CBD and 2-APB representing two non-conducting conformations; one has CBD bound to a previously identified site (Pumroy et al., 2019; Pumroy et al., 2022) providing confirmatory evidence for the site. The other conformation exhibits an additional non-protein density at a site located at an allosteric nexus between the pore domain, the S4-S5 linker, and a bound lipid molecule. We assign this density to CBD, but it is possible that other molecules, including endogenous ligands, could occupy that site to modulate channel function physiologically. When comparing the effect of CBD on the TRPV1 and TRPV3 channels, which are also activated by CBD and 2-APB (Hu et al., 2004), we find that only TRPV3 channels are potently sensitized by CBD. Further, mutations at rTRPV1 sites that differ between TRPV1 and TRPV2 and that are located in the pore domain or the CBD binding region failed to confer strong sensitization by CBD. These findings establish that the robust sensitization by CBD observed in TRPV2 channels involves a CBD-specific allosteric mechanism that engages channel regions distant from the CBD binding site and the pore. Results CBD strongly sensitizes rTRPV2 channels to activation by 2-APB We expressed rat TRPV2 (rTRPV2) channels in HEK293 cells and began by measuring the magnitude of the currents elicited by a low concentration of 2-APB (0.5 mM) or a near-saturating concentration (10 µM) of CBD (Qin et al., 2008) in the whole-cell configuration of the patch clamp at a holding potential of –80 mV. Even at this low concentration, 2-APB elicited currents that were much larger than those by CBD (Figure 1A). When 2-APB and CBD were applied together, we observed large currents that were comparable to those measured in response to a concentration of 2-APB (6 mM) that maximally activates rTRPV2 channels (Figure 1A). Currents in the presence of 0.5 mM 2-APB were over two orders of magnitude larger in the presence of CBD than in its absence (Figure 1H). Importantly, 2-APB and CBD applied together elicited no increase in whole-cell currents from un-transfected cells recorded in response to voltage pulses from –100 to +100 mV (Figure 1B). In contrast, the same voltage-stimulation protocol elicited robust currents in rTRPV2-transfected cells when exposed to 0.5 mM 2-APB or 10 µM CBD applied separately (Figure 1C and D). The sensitization caused by CBD is so strong that it becomes challenging to quantitate; at 0.5 mM 2-APB, channel activity is barely detectable in the whole-cell configuration, and yet with CBD added currents reach maximal activation levels (Figure 1A and H). The magnitude of sensitization we measured (Figure 1H) likely represents a lower bound for the sensitizing effect of CBD on rTRPV2 channels. We therefore analyzed data in a semi-quantitative manner without attempting to quantify the energetics associated with sensitization. Figure 1 with 3 supplements see all Download asset Open asset Cannabidiol (CBD) strongly sensitizes rTRPV2 channels to activation by 2-aminoethoxydiphenyl borate (2-APB) in whole-cell and in excised patch recordings. (A) Representative whole-cell gap-free current recording at –80 mV from a cell expressing rTRPV2 channels. The colored horizontal lines denote the duration of exposure to test compounds, and the red dotted line denotes the zero-current level. The inset shows a magnified view of a segment of the recording. (B) Mean current–voltage relations recorded in control solution and in the presence of 0.5 mM 2-APB + 10 µM obtained from un-transfected cells in the whole-cell configuration (n = 8). (C) Representative current families elicited by voltage steps from –100 to +100 mV obtained from an rTRPV2-expressing cell exposed to control solution, 0.5 mM 2-APB or 10 µM CBD. The dotted lines indicate the zero-current level. (D) Mean current–voltage relations obtained from data as in (C) and normalized to the mean value at +100 mV in the presence of 0.5 mM 2-APB. Data are shown as mean ± SEM (n = 5). (E) Dose–response relation for rTRPV2 channel activation by CBD measured at –80 mV in the whole-cell configuration (mean ± SEM; n = 4). The continuous curve is a fit to the Hill equation with parameters: EC50 = 4.3 ± 1.4 µM and Hill coefficient (nH) = 1.7 ± 0.1. (F) Concentration–response relations for rTRPV2 channel activation by 2-APB at –80 mV in the whole-cell configuration measured in the absence (blue symbols) or presence (black symbols) of 10 µM CBD (mean ± SEM; n = 7). Hill equation parameters: no CBD, EC50 = 1.5 ± 0.05 mM, Hill coefficient (nH) = 3.3 ± 0.2; 10 µM CBD, EC50 = 159.9 ± 7.7 µM, Hill coefficient (nH) = 2.9 ± 0.3. (G) Representative whole-cell gap-free recording at –80 mV in an rTRPV2-expressing cell. The bottom-left inset shows group data for the mean current amplitude at each of the three stimulations with 6 mM 2-APB, leak-subtracted and normalized to the amplitude during the first 6 mM 2-APB stimulation (solid squares – mean ± SEM, n = 5; empty circles – data from individual cells). The bottom-right inset displays steady-state data measured over each of the five intervals (① - ⑤) identified by the circled numbers on the current time course, normalized to the response to 2-APB in ① denoted by the horizontal blue line. Black squares are mean ± SEM, and circles are data from individual cells. (H) Leak-subtracted and normalized group data for: experiments in the whole-cell configuration as in (A) (n = 5) or post-sensitization by 6 mM 2-APB as in (G) (n = 5); experiments from outside-out patches as in Figure 1—figure supplement 3A (n = 6); experiments from inside-out patches as in Figure 1—figure supplement 3B (n = 9). Data was normalized to the first stimulation with 0.5 mM 2-APB (denoted by the horizontal blue lines) and shown as mean ± SEM (black squares) or values from individual cells (circles). Figure 1—source data 1 Excel file with group data from electrophysiological recordings shown in Figure 1. https://cdn.elifesciences.org/articles/86166/elife-86166-fig1-data1-v1.xlsx Download elife-86166-fig1-data1-v1.xlsx We were surprised at the minimal efficacy with which CBD activates rTRPV2 channels (Figure 1A, C and D), so we measured the magnitude of currents elicited by increasing concentrations of CBD in rTRPV2-expressing cells. The resulting concentration–response relations (Figure 1E) confirm that CBD activates rTRPV2 channels with much higher affinity (EC50 ~ 4 µM) than 2-APB but also with much lower efficacy, and that rTRPV2 channels can be assumed to be fully bound by CBD at a concentration of 10 µM CBD. We next tested whether rTRPV2 channels display an increased apparent affinity for 2-APB when bound to CBD by measuring rTRPV2 channel activation at increasing concentrations of 2-APB in the presence and absence of 10 µM CBD, and found that the EC50 for 2-APB activation decreased approximately tenfold in the presence of CBD (Figure 1F). The TRPV2 channel is reported to undergo sensitization upon stimulation with 2-APB that is irreversible over the duration of a patch-clamp experiment, leading to hysteresis in the 2-APB dose–response relations obtained pre- and post-sensitization: in most but not all patches, repeated short exposures to low concentrations of 2-APB were found to elicit progressively larger currents until reaching a plateau ~15-fold larger than the initial response (Liu and Qin, 2016). Sensitization also reached saturation after a single short stimulation with a concentration of 2-APB that maximally activates channels (Liu and Qin, 2016). To investigate whether 2-APB and CBD sensitize rTRPV2 channels to a similar extent, we performed experiments where we first briefly exposed cells to 0.5 mM 2-APB, then we stimulated cells with a maximally activating concentration of 2-APB (6 mM), which we repeated three times to ensure all channels had become sensitized (I1, I2, and I3, Figure 1G), and finally exposed cells to 0.5 mM 2-APB and 10 µM CBD first applied separately and then together. We found that currents activated by 0.5 mM 2-APB increased nearly tenfold after repeated stimulation with 6 mM 2-APB in some but not all cells (Figure 1G, ① vs. ②), whereas co-application with CBD increased currents by >100-fold in all cells (Figure 1G and H). The observed magnitude and cell-to-cell variability of 2-APB-dependent sensitization is consistent with previously reported measurements (Liu and Qin, 2016), and markedly smaller than sensitization by CBD. These results indicate that the predominant states adopted by rTPRV2 channels when bound to CBD must be energetically different than those adopted by 2-APB-sensitized channels in the absence of ligands. Recording solutions containing 2-APB and CBD also included dimethyl sulfoxide (DMSO) that we used to make stock solutions of both compounds, so the amount of DMSO applied to cells was larger in solutions that contained both agonists. To rule out an influence of DMSO on our results, in the same experiments described above we exposed cells to solutions with either 2-APB or CBD with added DMSO so that its concentration was the same as in solutions that contained both agonists together. We found that the additional DMSO did not change the magnitude of the currents in 0.5 mM 2-APB (Figure 1G, ② vs. ③) or 10 µM CBD (Figure 1G, ④ vs. ⑤). To rule out a direct chemical reaction between 2-APB and CBD, yielding a product with enhanced potency to activate the channel, we ran samples containing our recording solution (blank), 2-APB, CBD, and 2-APB and CBD mixed together, through a high-performance liquid chromatography (HPLC) system. We found that the elution time and height of the two peaks present in a sample with both agonists perfectly matched each single peak observed in samples with CBD or 2-APB alone, suggesting the two do not interact chemically (Figure 1—figure supplement 1). In addition to being weakly sensitized by 2-APB, rTRPV2 channels are strongly and irreversibly sensitized by heat, in which channel activation with extreme heat resulted in a tenfold decrease in the EC50 for 2-APB (Liu and Qin, 2016). In addition, heat-sensitized rTRPV2 channels no longer require extreme temperatures (>55°C) to activate, showing current responses at temperatures at or below 40°C (Liu and Qin, 2016). We therefore tested whether the mechanisms of rTRPV2 channel sensitization by CBD and heat are related. We reasoned that if this was the case, then the presence of CBD should facilitate channel activation by heat and enable current responses at around 40°C. Using rTRPV1 channel currents as a positive control for their high sensitivity to temperature changes in the 20–40°C range, we successfully detected steeply temperature-dependent currents as previously described (Figure 1—figure supplement 2A and E; Caterina et al., 1997; Yao et al., 2010; Jara-Oseguera et al., 2016). When we performed experiments using cells expressing rTRPV2 channels in the absence of CBD, we were unable to detect any temperature-dependent changes in current that were noticeably different from those measured under identical conditions in un-transfected cells (Figure 1—figure supplement 2B, C, E) as expected because the threshold for heat activation for rTRPV2 channels is >55°C (Yao et al., 2011; Liu and Qin, 2016). Interestingly, the responses in rTRPV2-expressing cells were the same in the absence and presence of CBD (Figure 1—figure supplement 2D and E), indicating that CBD does not strongly sensitize rTRPV2 channels to activation by heat. We also tested whether CBD sensitizes rTRPV2 channels to activation by the synthetic agonist probenecid (Bang et al., 2007), but we failed to detect any probenecid-elicited currents in the absence or presence of CBD (data not shown). Finally, we tested whether the sensitizing effect of CBD on activation of rTRPV2 channels by 2-APB also occurs in excised membrane patches devoid of many cellular components. Exposure of outside-out or inside-out patches expressing rTRPV2 channels to 0.5 mM 2-APB or 10 µM CBD alone elicited negligible currents (Figure 1—figure supplement 3), and similarly to our results in the whole-cell configuration, application of both agonists together elicited very large currents of the same magnitude as those elicited in response to 6 mM 2-APB that maximally activates channels (Figure 1H, Figure 1—figure supplement 3). These results indicate that patch excision does not disrupt sensitization of 2-APB responses by CBD, and that both agonists are capable of reaching their sites in the channel regardless of the side of the membrane to which they are applied. rTRPV2 channel sensitization by CBD increases channel open probability To determine whether CBD sensitizes rTRPV2 channels to activation by 2-APB by increasing channel open probability (Po), we undertook single-channel recordings. Although we were unable to obtain patches containing a single channel because the rTRPV2 channel expresses extremely well in HEK293 cells, we obtained recordings from inside-out patches containing multiple channels under conditions where the Po is very low and gating transitions of individual channels can be readily distinguished. For each patch, we recorded channel activity in the absence of agonists (i.e. control), and in the presence of 2-APB and CBD applied separately or together. All recorded data are displayed in Figure 2A as data blocks organized into two 3 × 4 arrays: each row of data blocks in the array contains data from a different inside-out patch (n = 6), and columns separate data obtained under different experimental conditions. For each individual data block in the array, current sweeps are stacked along the vertical axis (50 sweeps per block) and the horizontal axis within each block corresponds to the recoding duration of 500 ms per sweep. Individual data points are colored by current amplitude – see the color bar and the representative sweeps in Figure 2C for reference. Figure 2 Download asset Open asset Cannabidiol (CBD) sensitization of rTRPV2 channels involves an increase in open probability. (A) Data obtained from six rTRPV2-expressing inside-out patches at +80 mV under conditions of low open probability. Each row of data blocks shows data from a different patch, and columns are for each of the different experimental conditions. Each data block contains 50 vertically stacked current sweeps of 500 ms duration. Data points at each sweep are colored by their current amplitude as indicated by the color bar at the top. (B) Data obtained from nine inside-out patches at +80 mV obtained from un-transfected cells, displayed as in (A). (C) Representative current traces from the same rTRPV2-expressing patch in the presence of 50 µM 2-aminoethoxydiphenyl borate (2-APB) and 10 µM CBD showing two distinct open current amplitudes (S1 and O1) and simultaneous opening of two channels (O2). Data points in each trace are colored as in (A). The red dotted line indicates the zero-current amplitude measured when all channels are closed. (D) All-points histograms for data in (A). Each vertical lane is a histogram, with the single-channel current amplitude bins along the y-axis and the number of points per bin shown as a log-scale color heatmap (see scale bar on the left) and normalized to the peak centered at 0 pA (black dotted line). Histograms from each patch are displayed in the same order as in (A). Asterisks at peak values of 8.5 or 10.5 pA indicate the patches where these single-channel amplitudes were observed. With the exception of a high-Po burst at 250 µM 2-APB observed in one patch, channel activity in the absence of agonists or in the presence of either 2-APB or CBD applied separately was negligible, with no clearly identifiable opening events (Figure 2A). In contrast, exposure of patches to 2-APB and CBD together resulted in robust channel activity in all six patches, with multiple simultaneous channel opening events observed in many of the patches (Figure 2A and C). Exposure to 2-APB and CBD did not elicit changes in channel activity in patches from un-transfected cells (Figure 2B), strongly suggesting that the increases in channel activity observed in rTRPV2-expressing cells reflect a dramatic increase in Po in rTRPV2 channels when CBD and 2-APB are simultaneously present. To compare between data from different patches at each experimental condition, we generated all-points current amplitude histograms from each of the 24 data blocks shown in Figure 2A; each vertical lane in Figure 2D is a histogram, with current–amplitude bins on the vertical axis and a color scale to denote the logarithm of the normalized number of points per bin. For almost all patches, the histograms in control, 2-APB, or CBD have a single peak centered at 0 pA, the mean current amplitude when no channels are open. Consistent with a much greater Po in the presence of 2-APB and CBD together, the corresponding histograms all have robust peaks at larger amplitudes representing the opening of one or more channels (Figure 2D, right panel). We could not accurately determine single-channel current amplitudes in 2-APB or CBD because of the short duration and sparsity of openings when the agonists were applied separately. In the presence of 2-APB and CBD together, the single-channel current amplitude was centered at around 4.3 pA in five out of the six patches, with one patch exhibiting a much larger open amplitude of 8.2 pA (Figure 2D). In one of the patches in the presence of the two agonists, the open-channel current amplitude was initially ~4 pA but we also began observing openings with a larger current amplitude of 10.5 pA that became predominant for the rest of the experiment (Figure 2C and D). We are fairly confident that both amplitudes correspond to open rTPRV2 channels because we were able to observe multiple transitions between the two single-channel current amplitude levels when only one channel was open (Figure 2C). Together, these findings imply that rTRPV2 channels can undergo transitions between open states with different cation-conducting properties, as described for the closely related rTRPV1 channel (Canul-Sánchez et al., 2018; Geron et al., 2018) and the RTx-sensitive TRPV2-QM variant (Zhang et al., 2016), and establish that the CBD-dependent sensitization of rTRPV2 channels arises from increased open probability of channels bound to CBD and 2-APB. Interaction sites for CBD in the rTRPV2 channel To further explore the mechanism by which CBD sensitizes rTRPV2 to activation by 2-APB, we set out to confirm where CBD binds (Pumroy et al., 2019; Pumroy et al., 2022) and to solve structures of rTRPV2 with both ligands. We expressed an mVenus-tagged construct of full-length rTRPV2 in mammalian cells, purified and reconstituted the protein into lipid nanodiscs using MSP1E3D1 (Matthies et al., 2018), and solved its structure using cryo-EM in the presence of both CBD and 2-APB (Figure 3—figure supplements 1–4, Table 1). In our initial classification and refinement, we determined the structure of rTRPV2 spanning residues F75 to S728, including the transmembrane (TM) regions along with portions of the N- and C-termini and observed reasonably well-defined density for CBD between the S5 and S6 helices (Figure 3A), which we termed conformation A. The overall structure of rTRPV2 in conformation A and the location where CBD binds are remarkably similar to a previously published structure of rTPRV2 with CBD bound (Pumroy et al., 2019), as well as a more recent structure obtained in the presence of CBD and 2-APB (Pumroy et al., 2022) – in each of these instances, the internal pore remains closed and the structures are likely to represent a desensitized state. Although the overall resolution of conformation A was 3.23 Å, we could not see any density corresponding to 2-APB, including regions where 2-APB has been reported to bind to either TRPV2 or TRPV3 (Figure 3—figure supplement 5A; Singh et al., 2018b; Singh et al., 2018a; Zubcevic et al., 2019; Pumroy et al., 2022; Su et al., 2023). Figure 3 with 6 supplements see all Download asset Open asset Identifying cannabidiol (CBD) binding sites in TRPV2. (A) Overall structure of the rTRPV2 channel in lipid nanodiscs in conformation A with one CBD molecule bound to each monomer (PDB: 8SLX). Magnified view of the single CBD binding site is shown in the right panel. Both CBD and interacting residues are presented in stick with the cryo-EM density (EMD-40582) corresponding to CBD shown as a white surface. (B) Overall structure of the rTRPV2 channel in lipid nanodiscs in conformation B with two CBD molecules bound to each monomer (PDB: 8SLY). Magnified view of the two CBD binding sites are shown in the right panel. CBD with interacting residues and lipid were presented in stick configuration and cryo-EM density (EMD-40583) corresponding to CBD and lipid are shown as a white surface. (C) Cryo-EM density for the two CBD binding sites in conformation B modeled using CBD and shown in two orientations. The top density corresponds to that closer to the extracellular side of the channel and the bottom towards the intracellular side. (D) Same cryo-EM densities shown in (C), but in this case modeling the more intracellular density using 2-aminoethoxydiphenyl borate (2-APB). (E) Cryo-EM density for the lipid near the more intracellular CBD binding site fitted with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). (F) Same cryo-EM density shown in (E), but fitted with phosphatidylinositol. Table 1 Cryo-EM data collection, refinement, and validation statistics. Conformation AConformation BMagnification105,000105,000Voltage (kV)300300Electron exposure (e-/Å2)5252Defocus range (µm)–0.5 to –1.5–0.5 to –1.5Pixel size (Å)0.4150.415Symmetry imposedC4C4Initial particle images (no.)1,665,2711,665,271Final particle images (no.)321,71743,071Map resolution (Å)3.233.32 FSC threshold0.1430.143Refinement Initial model used (PDB code)6U846U84Model resolution (Å)3.43.5 FSC threshold0.50.5Map sharpening B-factor (Å2)–50–50Model composition Non-hydrogen atoms17,90118,313Protein residues23802376Ligands917R.m.s deviations Bond lengths (Å)0.0020.004Bond angles (°)0.4550.566B factor(Å2) Protein93.4978.75Ligand94.5841.01Validation MolProbity score1.291.34 Clashscore5.456.18 Poor rotamers (%)00Ramachandran Plot