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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.


Inserting an unstructured peptide between the transmembrane core domain and the intracellular domains does not affect general channel function.Averaged calcium imaging recordings of wild-type (WT) TRPV1 and two insertion mutants of interest, F430_3aa and E693_8aa, activated by extracellularly applied (as indicated by white bars) 10 μM capsaicin, 130 mM Mg2+ and low pH (pH 4.6) respectively. 3 mM ionomycin (as indicated by gray bars) was applied at the end of each imaging experiment to get the maximum fluorescence change. The total number of cells tested is given in each panel. The error bar represents standard error of means. Change in fluorescence intensity, ΔF, was calculated as the difference between the equilibrium level before stimulus application and after application of ionomycin.
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f4: Inserting an unstructured peptide between the transmembrane core domain and the intracellular domains does not affect general channel function.Averaged calcium imaging recordings of wild-type (WT) TRPV1 and two insertion mutants of interest, F430_3aa and E693_8aa, activated by extracellularly applied (as indicated by white bars) 10 μM capsaicin, 130 mM Mg2+ and low pH (pH 4.6) respectively. 3 mM ionomycin (as indicated by gray bars) was applied at the end of each imaging experiment to get the maximum fluorescence change. The total number of cells tested is given in each panel. The error bar represents standard error of means. Change in fluorescence intensity, ΔF, was calculated as the difference between the equilibrium level before stimulus application and after application of ionomycin.

Mentions: To assess the impact of unstructured peptide insertion at intracellular sites to channel function, we used live-cell fluorescence imaging to record intracellular Ca2+ increases upon activation of TRPV1, a non-selective cation channel with high Ca2+-permeability1. Control experiment with cells expressing the wild-type channels showed that, upon adding capsaicin, there was a rapid increase of the intracellular fluorescence intensity that reached peak level in a couple of seconds (Fig. 4). No increase in fluorescence intensity could be observed from untransfected cells, or cells over-expressing any of the non-functional insertion mutant channels.


Exploring functional roles of TRPV1 intracellular domains with unstructured peptide-insertion screening
Inserting an unstructured peptide between the transmembrane core domain and the intracellular domains does not affect general channel function.Averaged calcium imaging recordings of wild-type (WT) TRPV1 and two insertion mutants of interest, F430_3aa and E693_8aa, activated by extracellularly applied (as indicated by white bars) 10 μM capsaicin, 130 mM Mg2+ and low pH (pH 4.6) respectively. 3 mM ionomycin (as indicated by gray bars) was applied at the end of each imaging experiment to get the maximum fluorescence change. The total number of cells tested is given in each panel. The error bar represents standard error of means. Change in fluorescence intensity, ΔF, was calculated as the difference between the equilibrium level before stimulus application and after application of ionomycin.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4: Inserting an unstructured peptide between the transmembrane core domain and the intracellular domains does not affect general channel function.Averaged calcium imaging recordings of wild-type (WT) TRPV1 and two insertion mutants of interest, F430_3aa and E693_8aa, activated by extracellularly applied (as indicated by white bars) 10 μM capsaicin, 130 mM Mg2+ and low pH (pH 4.6) respectively. 3 mM ionomycin (as indicated by gray bars) was applied at the end of each imaging experiment to get the maximum fluorescence change. The total number of cells tested is given in each panel. The error bar represents standard error of means. Change in fluorescence intensity, ΔF, was calculated as the difference between the equilibrium level before stimulus application and after application of ionomycin.
Mentions: To assess the impact of unstructured peptide insertion at intracellular sites to channel function, we used live-cell fluorescence imaging to record intracellular Ca2+ increases upon activation of TRPV1, a non-selective cation channel with high Ca2+-permeability1. Control experiment with cells expressing the wild-type channels showed that, upon adding capsaicin, there was a rapid increase of the intracellular fluorescence intensity that reached peak level in a couple of seconds (Fig. 4). No increase in fluorescence intensity could be observed from untransfected cells, or cells over-expressing any of the non-functional insertion mutant channels.

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.