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Exploring functional roles of TRPV1 intracellular domains with unstructured peptide-insertion screening

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ABSTRACT

TRPV1 is a polymodal nociceptor for diverse physical and chemical stimuli that interact with different parts of the channel protein. Recent cryo-EM studies revealed detailed channel structures, opening the door for mapping structural elements mediating activation by each stimulus. Towards this goal, here we have combined unstructured peptide-insertion screening (UPS) with electrophysiological and fluorescence recordings to explore structural and functional roles of the intracellular regions of TRPV1 in mediating various activation stimuli. We found that most of the tightly packed protein regions did not tolerate structural perturbation by UPS when tested, indicating that structural integrity of the intracellular region is critical. In agreement with previous reports, Ca2+-dependent desensitization is strongly dependent on both intracellular N- and C-terminal domains; insertions of an unstructured peptide between these domains and the transmembrane core domain nearly eliminated Ca2+-dependent desensitization. In contrast, channel activations by capsaicin, low pH, divalent cations, and even heat are mostly intact in mutant channels containing the same insertions. These observations suggest that the transmembrane core domain of TRPV1, but not the intracellular domains, is responsible for sensing these stimuli.

No MeSH data available.


Peptide insertion before the S2-S3 linker does not disrupt capsaicin activation.(A) Structural models of the S2-S3 linker (orange) superimposed on the electron density map of TRPV1 in the apo state (EMD-5778). The position of L504 is highlighted in green; the side-chain of Y512 and S513 are shown in ball-and-stick format (purple). (B,C) Representative current trace of WT and L504_8aa mutant channel in response to increasing concentrations of capsaicin, respectively. (D) The concentration-response relationship of L504_8aa is fitted to a Hill function with EC50 of 0.19 ± 0.08 μM and slope factor of 0.6 ± 0.05 (n = 3). The wild-type TRPV1 curve (black dashed curve) is shown as comparison.
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f5: Peptide insertion before the S2-S3 linker does not disrupt capsaicin activation.(A) Structural models of the S2-S3 linker (orange) superimposed on the electron density map of TRPV1 in the apo state (EMD-5778). The position of L504 is highlighted in green; the side-chain of Y512 and S513 are shown in ball-and-stick format (purple). (B,C) Representative current trace of WT and L504_8aa mutant channel in response to increasing concentrations of capsaicin, respectively. (D) The concentration-response relationship of L504_8aa is fitted to a Hill function with EC50 of 0.19 ± 0.08 μM and slope factor of 0.6 ± 0.05 (n = 3). The wild-type TRPV1 curve (black dashed curve) is shown as comparison.

Mentions: Interestingly, cells expressing each of the functional insertion mutants exhibited a fluorescence increase very similar in time course to that of the wild-type channels (Fig. 4). These observations indicated that unstructured peptide insertions in these mutant channels—all at the interface between the transmembrane domain and the intracellular domains—had a minor effect on capsaicin activation. This is particularly interesting for L504_8aa, which contains an eight-amino-acid peptide insertion at the S2-S3 linker just a few amino acids upstream from Y512 and S513, two key residues whose mutations are known to substantially affect capsaicin activation1416. Because the structure of the S2-S3 linker was not determined in the original cryo-EM study22, we used Rosetta-based structural modeling to predict its likely structure. Our result suggested that this region forms a short helix, which is consistent with the recently available TRPV1 cryo-EM structure at a higher resolution40, with Y512 and S513 residing at its junction with S3 (Fig. 5A). In this structural model, the peptide-insertion site is also a flexible junction between two helical segments, S2 and the S2-S3 linker. Patch-clamp recordings revealed that L504_8aa responded to capsaicin with an EC50 value similar to that of the wild-type channel (WT: 0.15 ± 0.02 μM. L504_8aa: 0.19 ± 0.08 μM. P = 0.57) (Fig. 5B to D). Taken together, results from our functional and computational analyses are consistent with the hypothesis that the S2-S3 linker is likely a mobile structure. The anticipated mobility would facilitate the swinging motion of Y512 upon capsaicin binding towards the nearby ligand-binding pocket141623.


Exploring functional roles of TRPV1 intracellular domains with unstructured peptide-insertion screening
Peptide insertion before the S2-S3 linker does not disrupt capsaicin activation.(A) Structural models of the S2-S3 linker (orange) superimposed on the electron density map of TRPV1 in the apo state (EMD-5778). The position of L504 is highlighted in green; the side-chain of Y512 and S513 are shown in ball-and-stick format (purple). (B,C) Representative current trace of WT and L504_8aa mutant channel in response to increasing concentrations of capsaicin, respectively. (D) The concentration-response relationship of L504_8aa is fitted to a Hill function with EC50 of 0.19 ± 0.08 μM and slope factor of 0.6 ± 0.05 (n = 3). The wild-type TRPV1 curve (black dashed curve) is shown as comparison.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5035920&req=5

f5: Peptide insertion before the S2-S3 linker does not disrupt capsaicin activation.(A) Structural models of the S2-S3 linker (orange) superimposed on the electron density map of TRPV1 in the apo state (EMD-5778). The position of L504 is highlighted in green; the side-chain of Y512 and S513 are shown in ball-and-stick format (purple). (B,C) Representative current trace of WT and L504_8aa mutant channel in response to increasing concentrations of capsaicin, respectively. (D) The concentration-response relationship of L504_8aa is fitted to a Hill function with EC50 of 0.19 ± 0.08 μM and slope factor of 0.6 ± 0.05 (n = 3). The wild-type TRPV1 curve (black dashed curve) is shown as comparison.
Mentions: Interestingly, cells expressing each of the functional insertion mutants exhibited a fluorescence increase very similar in time course to that of the wild-type channels (Fig. 4). These observations indicated that unstructured peptide insertions in these mutant channels—all at the interface between the transmembrane domain and the intracellular domains—had a minor effect on capsaicin activation. This is particularly interesting for L504_8aa, which contains an eight-amino-acid peptide insertion at the S2-S3 linker just a few amino acids upstream from Y512 and S513, two key residues whose mutations are known to substantially affect capsaicin activation1416. Because the structure of the S2-S3 linker was not determined in the original cryo-EM study22, we used Rosetta-based structural modeling to predict its likely structure. Our result suggested that this region forms a short helix, which is consistent with the recently available TRPV1 cryo-EM structure at a higher resolution40, with Y512 and S513 residing at its junction with S3 (Fig. 5A). In this structural model, the peptide-insertion site is also a flexible junction between two helical segments, S2 and the S2-S3 linker. Patch-clamp recordings revealed that L504_8aa responded to capsaicin with an EC50 value similar to that of the wild-type channel (WT: 0.15 ± 0.02 μM. L504_8aa: 0.19 ± 0.08 μM. P = 0.57) (Fig. 5B to D). Taken together, results from our functional and computational analyses are consistent with the hypothesis that the S2-S3 linker is likely a mobile structure. The anticipated mobility would facilitate the swinging motion of Y512 upon capsaicin binding towards the nearby ligand-binding pocket141623.

View Article: PubMed Central - PubMed

ABSTRACT

TRPV1 is a polymodal nociceptor for diverse physical and chemical stimuli that interact with different parts of the channel protein. Recent cryo-EM studies revealed detailed channel structures, opening the door for mapping structural elements mediating activation by each stimulus. Towards this goal, here we have combined unstructured peptide-insertion screening (UPS) with electrophysiological and fluorescence recordings to explore structural and functional roles of the intracellular regions of TRPV1 in mediating various activation stimuli. We found that most of the tightly packed protein regions did not tolerate structural perturbation by UPS when tested, indicating that structural integrity of the intracellular region is critical. In agreement with previous reports, Ca2+-dependent desensitization is strongly dependent on both intracellular N- and C-terminal domains; insertions of an unstructured peptide between these domains and the transmembrane core domain nearly eliminated Ca2+-dependent desensitization. In contrast, channel activations by capsaicin, low pH, divalent cations, and even heat are mostly intact in mutant channels containing the same insertions. These observations suggest that the transmembrane core domain of TRPV1, but not the intracellular domains, is responsible for sensing these stimuli.

No MeSH data available.