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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.

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Heat activation of insertion mutants.(A) Representative calcium imaging of WT, F430_3aa and E693_8aa with corresponding fluorescence signal intensity traces. Increased calcium influx was observed upon channel activation by temperature changes. Due to inactivation, intracellular calcium level dropped after the peak even at persistent high temperature. (B) Box-and-whisker plot of heat activation threshold temperature of WT and insertion mutants. The threshold temperature in imaging studies was defined as the temperature at the starting point of the rapid raising phase of the fluorescence signal. The whisker top, box top, line inside the box, box bottom, and whisker bottom represent the maximum, 75th percentile, median, 25th percentile, and minimum value of each pool of measurements, respectively. (C) Representative heat activation of WT and insertion mutants by patch-clamp recording at +80 mV. Two linear functions (blue dotted lines) were fitted to determine the heat activation threshold temperature. (D,E) Comparison of the threshold temperature and the R value of WT and insertions mutants. n.s., not statistically significant by t-test; n = 4–8.
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f7: Heat activation of insertion mutants.(A) Representative calcium imaging of WT, F430_3aa and E693_8aa with corresponding fluorescence signal intensity traces. Increased calcium influx was observed upon channel activation by temperature changes. Due to inactivation, intracellular calcium level dropped after the peak even at persistent high temperature. (B) Box-and-whisker plot of heat activation threshold temperature of WT and insertion mutants. The threshold temperature in imaging studies was defined as the temperature at the starting point of the rapid raising phase of the fluorescence signal. The whisker top, box top, line inside the box, box bottom, and whisker bottom represent the maximum, 75th percentile, median, 25th percentile, and minimum value of each pool of measurements, respectively. (C) Representative heat activation of WT and insertion mutants by patch-clamp recording at +80 mV. Two linear functions (blue dotted lines) were fitted to determine the heat activation threshold temperature. (D,E) Comparison of the threshold temperature and the R value of WT and insertions mutants. n.s., not statistically significant by t-test; n = 4–8.

Mentions: TRPV1 is a prototypical heat-sensing ion channel1; however, how heat is sensed and used to promote activation conformational change remains unclear. Both N- and C-terminal regions, as well as extracellular regions, have been previously suggested to mediate heat activation10. If the structural element(s) responsible for high-sensitivity heat activation resides in the N- or C-terminal domain, F430_3aa and E693_8aa would offer an opportunity to test the coupling of heat-sensing and pore opening events. Indeed, for the TREK-1 channel a triple-glycine insertion that decoupled C terminus was found to largely reduce temperature sensitivity28. We first measured heat response of F430_3aa and E693_8aa with live-cell calcium imaging (Fig. 7A). Cells expressing the wild-type channel, F430_3aa, or E693_8aa exhibited clear increase in fluorescence intensity when temperature rose above similar threshold values (Fig. 7B. wild-type: 38.1 ± 0.3 °C, n = 58; F430_3aa: 39.2 ± 0.2 °C, n = 36; E693_8aa: 39.7 ± 0.2 °C, n = 28). Upon continuous heating, the fluorescence signal exhibited characteristic transient nature for all three channel-types.


Exploring functional roles of TRPV1 intracellular domains with unstructured peptide-insertion screening
Heat activation of insertion mutants.(A) Representative calcium imaging of WT, F430_3aa and E693_8aa with corresponding fluorescence signal intensity traces. Increased calcium influx was observed upon channel activation by temperature changes. Due to inactivation, intracellular calcium level dropped after the peak even at persistent high temperature. (B) Box-and-whisker plot of heat activation threshold temperature of WT and insertion mutants. The threshold temperature in imaging studies was defined as the temperature at the starting point of the rapid raising phase of the fluorescence signal. The whisker top, box top, line inside the box, box bottom, and whisker bottom represent the maximum, 75th percentile, median, 25th percentile, and minimum value of each pool of measurements, respectively. (C) Representative heat activation of WT and insertion mutants by patch-clamp recording at +80 mV. Two linear functions (blue dotted lines) were fitted to determine the heat activation threshold temperature. (D,E) Comparison of the threshold temperature and the R value of WT and insertions mutants. n.s., not statistically significant by t-test; n = 4–8.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5035920&req=5

f7: Heat activation of insertion mutants.(A) Representative calcium imaging of WT, F430_3aa and E693_8aa with corresponding fluorescence signal intensity traces. Increased calcium influx was observed upon channel activation by temperature changes. Due to inactivation, intracellular calcium level dropped after the peak even at persistent high temperature. (B) Box-and-whisker plot of heat activation threshold temperature of WT and insertion mutants. The threshold temperature in imaging studies was defined as the temperature at the starting point of the rapid raising phase of the fluorescence signal. The whisker top, box top, line inside the box, box bottom, and whisker bottom represent the maximum, 75th percentile, median, 25th percentile, and minimum value of each pool of measurements, respectively. (C) Representative heat activation of WT and insertion mutants by patch-clamp recording at +80 mV. Two linear functions (blue dotted lines) were fitted to determine the heat activation threshold temperature. (D,E) Comparison of the threshold temperature and the R value of WT and insertions mutants. n.s., not statistically significant by t-test; n = 4–8.
Mentions: TRPV1 is a prototypical heat-sensing ion channel1; however, how heat is sensed and used to promote activation conformational change remains unclear. Both N- and C-terminal regions, as well as extracellular regions, have been previously suggested to mediate heat activation10. If the structural element(s) responsible for high-sensitivity heat activation resides in the N- or C-terminal domain, F430_3aa and E693_8aa would offer an opportunity to test the coupling of heat-sensing and pore opening events. Indeed, for the TREK-1 channel a triple-glycine insertion that decoupled C terminus was found to largely reduce temperature sensitivity28. We first measured heat response of F430_3aa and E693_8aa with live-cell calcium imaging (Fig. 7A). Cells expressing the wild-type channel, F430_3aa, or E693_8aa exhibited clear increase in fluorescence intensity when temperature rose above similar threshold values (Fig. 7B. wild-type: 38.1 ± 0.3 °C, n = 58; F430_3aa: 39.2 ± 0.2 °C, n = 36; E693_8aa: 39.7 ± 0.2 °C, n = 28). Upon continuous heating, the fluorescence signal exhibited characteristic transient nature for all three channel-types.

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