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

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.


Related in: MedlinePlus

Short peptide insertions significantly impaired the coupling between the intracellular domains and the transmembrane core domain.(A,B) Representative traces of F430_3aa and E693_8aa activated by capsaicin. (C) Capsaicin concentration-response curves of WT (EC50: 0.15 ± 0.02 μM; slope factor: 1.82 ± 0.22), F430_3aa (EC50: 0.07 ± 0.03 μM; slope factor: 1.39 ± 0.38) and E693_8aa (EC50: 0.26 ± 0.06 μM; slope factor: 1.10 ± 0.23). (n = 3–4). (D) Representative whole-cell patch recordings (with 2 mM Ca2+ in solutions) of WT TRPV1 (black), F430_3aa (red) and E693_8aa (green) activated by 1 μM capsaicin and desensitized through Ca2+-dependent desensitization mechanisms. WT TRPV1 exhibited little desensitization in the absence of Ca2+ in the solution (blue). Current decay over time from the capsaicin-activated peak current was superimposed with an exponential fit (smooth curve). (E to G) Raw current traces of each channel at time point 1 (colored) and 2 (grey). The X- and Y-axis scales are 40 ms and 1 nA respectively. (H) Comparison of current decay time constants. *, p < 0.05. (I) Averaged fraction of desensitization, calculated as (Ipeak − Idesensitized)/Ipeak *100, where Ipeak is the peak current amplitude potentiated by 1 μM capsaicin and Idesensitized is the current level when current decay caused by desensitization has reached steady-state. **, p < 0.01; ***, p < 0.001.
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f6: Short peptide insertions significantly impaired the coupling between the intracellular domains and the transmembrane core domain.(A,B) Representative traces of F430_3aa and E693_8aa activated by capsaicin. (C) Capsaicin concentration-response curves of WT (EC50: 0.15 ± 0.02 μM; slope factor: 1.82 ± 0.22), F430_3aa (EC50: 0.07 ± 0.03 μM; slope factor: 1.39 ± 0.38) and E693_8aa (EC50: 0.26 ± 0.06 μM; slope factor: 1.10 ± 0.23). (n = 3–4). (D) Representative whole-cell patch recordings (with 2 mM Ca2+ in solutions) of WT TRPV1 (black), F430_3aa (red) and E693_8aa (green) activated by 1 μM capsaicin and desensitized through Ca2+-dependent desensitization mechanisms. WT TRPV1 exhibited little desensitization in the absence of Ca2+ in the solution (blue). Current decay over time from the capsaicin-activated peak current was superimposed with an exponential fit (smooth curve). (E to G) Raw current traces of each channel at time point 1 (colored) and 2 (grey). The X- and Y-axis scales are 40 ms and 1 nA respectively. (H) Comparison of current decay time constants. *, p < 0.05. (I) Averaged fraction of desensitization, calculated as (Ipeak − Idesensitized)/Ipeak *100, where Ipeak is the peak current amplitude potentiated by 1 μM capsaicin and Idesensitized is the current level when current decay caused by desensitization has reached steady-state. **, p < 0.01; ***, p < 0.001.

Mentions: A possible explanation for the lack of difference in fluorescence signal between wild-type and mutant channels might be insensitivity of the non-quantitative optical method to subtle changes. Therefore, we examined the functional mutant channels with patch-clamp recordings, which confirmed observations from fluorescence recordings. We found that F430_3aa produced rapid capsaicin-induced current responses just like the wild-type channel (Fig. 6A,C). Extended recordings, however, revealed that there was a substantial difference in the current decline phase. The current decline represents Ca2+-dependent acute desensitization, a process thought to be mediated by multiple mechanisms, including binding of Ca2+-calmodulin to the ankyrin-like repeat domain34354142, phosphorylation43, and binding of PIP2 to the C-terminus4445464748, that allosterically modulate the stability of the open pore conformation. For the wild-type channel, as expected little desensitization was observed in the absence of Ca2+. With 2 mM Ca2+ present in the solution, desensitization proceeded with a time constant of 31.4 ± 6.2 s (n = 6). In comparison, the current decline of F430_3aa channels proceeded slowly, with a time constant of 162.4 ± 53.5 s (n = 5; p < 0.05). As a result, most of the current remained at the end of the 3 min recording (Fig. 6D to I). Therefore, insertion of an unstructured peptide at the junction between the N-terminus and the transmembrane domain weakened functional coupling between these domains, substantially limited the ability of intracellular Ca2+-mediated events to modulate gating of the channel pore.


Exploring functional roles of TRPV1 intracellular domains with unstructured peptide-insertion screening
Short peptide insertions significantly impaired the coupling between the intracellular domains and the transmembrane core domain.(A,B) Representative traces of F430_3aa and E693_8aa activated by capsaicin. (C) Capsaicin concentration-response curves of WT (EC50: 0.15 ± 0.02 μM; slope factor: 1.82 ± 0.22), F430_3aa (EC50: 0.07 ± 0.03 μM; slope factor: 1.39 ± 0.38) and E693_8aa (EC50: 0.26 ± 0.06 μM; slope factor: 1.10 ± 0.23). (n = 3–4). (D) Representative whole-cell patch recordings (with 2 mM Ca2+ in solutions) of WT TRPV1 (black), F430_3aa (red) and E693_8aa (green) activated by 1 μM capsaicin and desensitized through Ca2+-dependent desensitization mechanisms. WT TRPV1 exhibited little desensitization in the absence of Ca2+ in the solution (blue). Current decay over time from the capsaicin-activated peak current was superimposed with an exponential fit (smooth curve). (E to G) Raw current traces of each channel at time point 1 (colored) and 2 (grey). The X- and Y-axis scales are 40 ms and 1 nA respectively. (H) Comparison of current decay time constants. *, p < 0.05. (I) Averaged fraction of desensitization, calculated as (Ipeak − Idesensitized)/Ipeak *100, where Ipeak is the peak current amplitude potentiated by 1 μM capsaicin and Idesensitized is the current level when current decay caused by desensitization has reached steady-state. **, p < 0.01; ***, p < 0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

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f6: Short peptide insertions significantly impaired the coupling between the intracellular domains and the transmembrane core domain.(A,B) Representative traces of F430_3aa and E693_8aa activated by capsaicin. (C) Capsaicin concentration-response curves of WT (EC50: 0.15 ± 0.02 μM; slope factor: 1.82 ± 0.22), F430_3aa (EC50: 0.07 ± 0.03 μM; slope factor: 1.39 ± 0.38) and E693_8aa (EC50: 0.26 ± 0.06 μM; slope factor: 1.10 ± 0.23). (n = 3–4). (D) Representative whole-cell patch recordings (with 2 mM Ca2+ in solutions) of WT TRPV1 (black), F430_3aa (red) and E693_8aa (green) activated by 1 μM capsaicin and desensitized through Ca2+-dependent desensitization mechanisms. WT TRPV1 exhibited little desensitization in the absence of Ca2+ in the solution (blue). Current decay over time from the capsaicin-activated peak current was superimposed with an exponential fit (smooth curve). (E to G) Raw current traces of each channel at time point 1 (colored) and 2 (grey). The X- and Y-axis scales are 40 ms and 1 nA respectively. (H) Comparison of current decay time constants. *, p < 0.05. (I) Averaged fraction of desensitization, calculated as (Ipeak − Idesensitized)/Ipeak *100, where Ipeak is the peak current amplitude potentiated by 1 μM capsaicin and Idesensitized is the current level when current decay caused by desensitization has reached steady-state. **, p < 0.01; ***, p < 0.001.
Mentions: A possible explanation for the lack of difference in fluorescence signal between wild-type and mutant channels might be insensitivity of the non-quantitative optical method to subtle changes. Therefore, we examined the functional mutant channels with patch-clamp recordings, which confirmed observations from fluorescence recordings. We found that F430_3aa produced rapid capsaicin-induced current responses just like the wild-type channel (Fig. 6A,C). Extended recordings, however, revealed that there was a substantial difference in the current decline phase. The current decline represents Ca2+-dependent acute desensitization, a process thought to be mediated by multiple mechanisms, including binding of Ca2+-calmodulin to the ankyrin-like repeat domain34354142, phosphorylation43, and binding of PIP2 to the C-terminus4445464748, that allosterically modulate the stability of the open pore conformation. For the wild-type channel, as expected little desensitization was observed in the absence of Ca2+. With 2 mM Ca2+ present in the solution, desensitization proceeded with a time constant of 31.4 ± 6.2 s (n = 6). In comparison, the current decline of F430_3aa channels proceeded slowly, with a time constant of 162.4 ± 53.5 s (n = 5; p < 0.05). As a result, most of the current remained at the end of the 3 min recording (Fig. 6D to I). Therefore, insertion of an unstructured peptide at the junction between the N-terminus and the transmembrane domain weakened functional coupling between these domains, substantially limited the ability of intracellular Ca2+-mediated events to modulate gating of the channel pore.

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.


Related in: MedlinePlus