Cannabidiol sensitizes TRPV2 channels to activation by 2-APB
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eLife assessment
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 solid 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.
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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.
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Author Response
Reviewer #1 (Public Review):
The manuscript by Gochman and colleagues reports the discovery of a very strong sensitization of TRPV2 channels by the herbal compound cannabidiol (CBD) to activation by the synthetic agonist 2aminoethoxydiphenyl borate (2-APB). Using patch-clamp electrophysiology the authors show that the ~100-fold enhancement by micromolar CBD of TRPV2 current responses to low concentrations of 2-APB reflects a robust increase in apparent affinity for the latter agonist. Cryo-EM structures of TRPV2 in lipid nanodiscs in the presence of both drugs report two-channel conformations. One conformation resembles previously solved structures whereas the second conformation reveals two distinct CBD binding sites per subunit, as well as changes in the conformation of the S4-S5 linker. Interestingly, although …
Author Response
Reviewer #1 (Public Review):
The manuscript by Gochman and colleagues reports the discovery of a very strong sensitization of TRPV2 channels by the herbal compound cannabidiol (CBD) to activation by the synthetic agonist 2aminoethoxydiphenyl borate (2-APB). Using patch-clamp electrophysiology the authors show that the ~100-fold enhancement by micromolar CBD of TRPV2 current responses to low concentrations of 2-APB reflects a robust increase in apparent affinity for the latter agonist. Cryo-EM structures of TRPV2 in lipid nanodiscs in the presence of both drugs report two-channel conformations. One conformation resembles previously solved structures whereas the second conformation reveals two distinct CBD binding sites per subunit, as well as changes in the conformation of the S4-S5 linker. Interestingly, although TRPV1 and TRPV3 are highly homologous to TRPV2 and both CBD binding sites are relatively conserved, the CBD-induced sensitization towards 2-APB is observable only for TRPV3 but not for TRPV1. Moreover, the simultaneous substitution of non-conserved residues in the CBD binding sites and the pore region of TRPV1 with the amino acids present in TRPV2 fails to confirm strong CBD-induced sensitization. The authors conclude that CBD-dependent sensitization of TRPV2 channels depends on structural features of the channel that are not restricted to the CBD binding site but involve multiple channel regions.
These are important findings that promote our understanding of the molecular mechanisms of TRPV family channels, and the data provide convincing evidence for the conclusions.
We appreciate the supportive evaluation of the reviewer.
Reviewer #2 (Public Review):
In this manuscript, Gochman et al. studied the molecular mechanism by which cannabidiol (CBD) sensitizes the TRPV2 channel to activation by 2-APB. While CBD itself can activate TRPV2 with low efficacy, it can sensitize TRPV2 current activated by 2-APB by two orders of magnitude. The authors showed, via single-channel recording, that the CBD-dependent sensitization arises from an increase in Po when the channel binds to both CBD and 2-APB. The authors then used cryo-EM to investigate how CBD binds to TRPV2 and identified two CBD binding sites in each subunit, with one site being previously reported and the other being newly discovered.
TRPV1 and TRPV2 are two channels closely related to TRPV2. All three channels can be activated by CBD and 2-APB, but only TRPV2 and 3 are strongly sensitized by CBD. To understand the molecular basis of the different sensitivity to CBD, the authors compared the residues within the CBD binding sites and generated mutants by swapping non-conserved residues between TRPV1 and TRPV2. They then performed patch-clamp recordings on these mutants and found that mutations on non-conserved residues indeed influenced the CBD-dependent sensitization, thereby supporting the observed CBD binding sites.
Unexpectedly, the authors did not identify the binding site of 2-APB, despite its robust effect in electrophysiology recordings, especially when combined with CBD. Although previous structural studies of TRPV2 have reported 2-APB binding sites, the associated densities in these studies were not wellresolved. Therefore, the authors called on the field to re-examine published structural data with regard to the 2-APB binding sites.
Overall, this is an important study with well-designed and well-conducted experiments.
We appreciated the supportive comments of the reviewer.
Reviewer #3 (Public Review):
In this paper, Gochman et al examine TRPV1-3 channel sensitization by CBD, specifically in the context of 2-APB activation. The authors primarily used classic electrophysiological techniques to address their questions about channel behavior but have also used structural biology in the form of cryo-EM to examine drug binding to TRPV2. The authors have carefully observed and quantified sensitization of the rat TRPV2 channel to 2-APB by CBD. While this sensitization has been reported previously (Pumroy et al, Nat Commun 2022), the authors have gone into much more detail here and carefully examined this process from several angles, including a comparison to some other known methods of sensitizing TRPV2. Additionally, the authors have also revealed that CBD sensitizes rat TRPV1 and mouse TRPV3 to 2-APB, which has not been reported previously. Up to this point, the work is well thought through and cohesive.
The major weakness of this paper is that the authors' efforts to track down the structural and molecular basis for CBD sensitization neither give insight into how sensitization occurs nor provide a solid footing for future work on the topic. The structural work presented in this paper lacks proper controls to interpret the observed states and the authors do nothing to follow up on a potentially interesting second binding site for CBD. Overall, the structural work feels detached from the rest of the paper. The mutations chosen to examine sensitization are based on setting up TRPV1 in opposition to TRPV2 and TRPV3, which makes little sense as all three channels show sensitization by CBD, even if to different extents. The authors chose their mutations based on the assumption that response to CBD is the key difference between the channels for sensitization, yet the overall state of each channel or the different modes of activation by 2-APB seem to be more likely candidates. As a result, it is not particularly surprising that none of the mutations the authors make reduce CBD sensitization in TRPV2 or increase CBD sensitization in TRPV1.
A difficulty in examining TRPV1-3 as a group is that while they are highly conserved in sequence and structure, there are key differences in drug responses. While it does seem likely that CBD would bind to the same location in TRPV1-3, there is extensive evidence that 2-APB binds at different sites in each channel, as the authors discuss in the paper. Without more basic information about where 2-APB binds to each channel and confirmation that CBD does indeed bind TRPV1-3 at the same site, it may not be possible to untangle this particular mode of channel sensitization.
We appreciate this reviewer’s perspective and we too were disappointed that our approach did not yield more definitive answers to why some TRPV channels are more sensitive to CBD. We have revised the results and discussion sections to more clearly articulate what we think our results reveal. We have also added a section to the discussion to present the idea that the differential sensitivity of TRPV channels to CBD may have more to do with where 2-APB binds and how it activates the channel than CBD. These challenging points are all excellent and they have helped us to present our message more clearly.
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eLife assessment
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 solid 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.
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Reviewer #1 (Public Review):
The manuscript by Gochman and colleagues reports the discovery of a very strong sensitization of TRPV2 channels by the herbal compound cannabidiol (CBD) to activation by the synthetic agonist 2-aminoethoxydiphenyl borate (2-APB). Using patch-clamp electrophysiology the authors show that the ~100-fold enhancement by micromolar CBD of TRPV2 current responses to low concentrations of 2-APB reflects a robust increase in apparent affinity for the latter agonist. Cryo-EM structures of TRPV2 in lipid nanodiscs in the presence of both drugs report two-channel conformations. One conformation resembles previously solved structures whereas the second conformation reveals two distinct CBD binding sites per subunit, as well as changes in the conformation of the S4-S5 linker. Interestingly, although TRPV1 and TRPV3 are …
Reviewer #1 (Public Review):
The manuscript by Gochman and colleagues reports the discovery of a very strong sensitization of TRPV2 channels by the herbal compound cannabidiol (CBD) to activation by the synthetic agonist 2-aminoethoxydiphenyl borate (2-APB). Using patch-clamp electrophysiology the authors show that the ~100-fold enhancement by micromolar CBD of TRPV2 current responses to low concentrations of 2-APB reflects a robust increase in apparent affinity for the latter agonist. Cryo-EM structures of TRPV2 in lipid nanodiscs in the presence of both drugs report two-channel conformations. One conformation resembles previously solved structures whereas the second conformation reveals two distinct CBD binding sites per subunit, as well as changes in the conformation of the S4-S5 linker. Interestingly, although TRPV1 and TRPV3 are highly homologous to TRPV2 and both CBD binding sites are relatively conserved, the CBD-induced sensitization towards 2-APB is observable only for TRPV3 but not for TRPV1. Moreover, the simultaneous substitution of non-conserved residues in the CBD binding sites and the pore region of TRPV1 with the amino acids present in TRPV2 fails to confirm strong CBD-induced sensitization. The authors conclude that CBD-dependent sensitization of TRPV2 channels depends on structural features of the channel that are not restricted to the CBD binding site but involve multiple channel regions.
These are important findings that promote our understanding of the molecular mechanisms of TRPV family channels, and the data provide convincing evidence for the conclusions.
-
Reviewer #2 (Public Review):
In this manuscript, Gochman et al. studied the molecular mechanism by which cannabidiol (CBD) sensitizes the TRPV2 channel to activation by 2-APB. While CBD itself can activate TRPV2 with low efficacy, it can sensitize TRPV2 current activated by 2-APB by two orders of magnitude. The authors showed, via single-channel recording, that the CBD-dependent sensitization arises from an increase in Po when the channel binds to both CBD and 2-APB. The authors then used cryo-EM to investigate how CBD binds to TRPV2 and identified two CBD binding sites in each subunit, with one site being previously reported and the other being newly discovered.
TRPV1 and TRPV2 are two channels closely related to TRPV2. All three channels can be activated by CBD and 2-APB, but only TRPV2 and 3 are strongly sensitized by CBD. To …
Reviewer #2 (Public Review):
In this manuscript, Gochman et al. studied the molecular mechanism by which cannabidiol (CBD) sensitizes the TRPV2 channel to activation by 2-APB. While CBD itself can activate TRPV2 with low efficacy, it can sensitize TRPV2 current activated by 2-APB by two orders of magnitude. The authors showed, via single-channel recording, that the CBD-dependent sensitization arises from an increase in Po when the channel binds to both CBD and 2-APB. The authors then used cryo-EM to investigate how CBD binds to TRPV2 and identified two CBD binding sites in each subunit, with one site being previously reported and the other being newly discovered.
TRPV1 and TRPV2 are two channels closely related to TRPV2. All three channels can be activated by CBD and 2-APB, but only TRPV2 and 3 are strongly sensitized by CBD. To understand the molecular basis of the different sensitivity to CBD, the authors compared the residues within the CBD binding sites and generated mutants by swapping non-conserved residues between TRPV1 and TRPV2. They then performed patch-clamp recordings on these mutants and found that mutations on non-conserved residues indeed influenced the CBD-dependent sensitization, thereby supporting the observed CBD binding sites.
Unexpectedly, the authors did not identify the binding site of 2-APB, despite its robust effect in electrophysiology recordings, especially when combined with CBD. Although previous structural studies of TRPV2 have reported 2-APB binding sites, the associated densities in these studies were not well-resolved. Therefore, the authors called on the field to re-examine published structural data with regard to the 2-APB binding sites.
Overall, this is an important study with well-designed and well-conducted experiments.
-
Reviewer #3 (Public Review):
In this paper, Gochman et al examine TRPV1-3 channel sensitization by CBD, specifically in the context of 2-APB activation. The authors primarily used classic electrophysiological techniques to address their questions about channel behavior but have also used structural biology in the form of cryo-EM to examine drug binding to TRPV2. The authors have carefully observed and quantified sensitization of the rat TRPV2 channel to 2-APB by CBD. While this sensitization has been reported previously (Pumroy et al, Nat Commun 2022), the authors have gone into much more detail here and carefully examined this process from several angles, including a comparison to some other known methods of sensitizing TRPV2. Additionally, the authors have also revealed that CBD sensitizes rat TRPV1 and mouse TRPV3 to 2-APB, which has …
Reviewer #3 (Public Review):
In this paper, Gochman et al examine TRPV1-3 channel sensitization by CBD, specifically in the context of 2-APB activation. The authors primarily used classic electrophysiological techniques to address their questions about channel behavior but have also used structural biology in the form of cryo-EM to examine drug binding to TRPV2. The authors have carefully observed and quantified sensitization of the rat TRPV2 channel to 2-APB by CBD. While this sensitization has been reported previously (Pumroy et al, Nat Commun 2022), the authors have gone into much more detail here and carefully examined this process from several angles, including a comparison to some other known methods of sensitizing TRPV2. Additionally, the authors have also revealed that CBD sensitizes rat TRPV1 and mouse TRPV3 to 2-APB, which has not been reported previously. Up to this point, the work is well thought through and cohesive.
The major weakness of this paper is that the authors' efforts to track down the structural and molecular basis for CBD sensitization neither give insight into how sensitization occurs nor provide a solid footing for future work on the topic. The structural work presented in this paper lacks proper controls to interpret the observed states and the authors do nothing to follow up on a potentially interesting second binding site for CBD. Overall, the structural work feels detached from the rest of the paper. The mutations chosen to examine sensitization are based on setting up TRPV1 in opposition to TRPV2 and TRPV3, which makes little sense as all three channels show sensitization by CBD, even if to different extents. The authors chose their mutations based on the assumption that response to CBD is the key difference between the channels for sensitization, yet the overall state of each channel or the different modes of activation by 2-APB seem to be more likely candidates. As a result, it is not particularly surprising that none of the mutations the authors make reduce CBD sensitization in TRPV2 or increase CBD sensitization in TRPV1.
A difficulty in examining TRPV1-3 as a group is that while they are highly conserved in sequence and structure, there are key differences in drug responses. While it does seem likely that CBD would bind to the same location in TRPV1-3, there is extensive evidence that 2-APB binds at different sites in each channel, as the authors discuss in the paper. Without more basic information about where 2-APB binds to each channel and confirmation that CBD does indeed bind TRPV1-3 at the same site, it may not be possible to untangle this particular mode of channel sensitization.
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