Recovery from tachyphylaxis of TRPV1 coincides with recycling to the surface membrane

Abstract

The transient receptor potential vanilloid-1 (TRPV1) ion channel is essential for sensation of thermal and chemical pain. TRPV1 activation is accompanied by Ca²⁺-dependent desensitization; acute desensitization reflects rapid reduction in channel activity during stimulation, whereas tachyphylaxis denotes the diminution in TRPV1 responses to repetitive stimulation. Acute desensitization has been attributed to conformational changes of the TRPV1 channel; however, the mechanisms underlying the establishment of tachyphylaxis remain to be defined. Here, we report that the degree of whole-cell TRPV1 tachyphylaxis is regulated by the strength of inducing stimulation. Using light-sheet microscopy and pH-sensitive sensor pHluorin to follow TRPV1 endocytosis and exocytosis trafficking, we provide real-time information that tachyphylaxis of different degrees concurs with TRPV1 recycling to the plasma membrane in a proportional manner. This process controls TRPV1 surface expression level thereby the whole-cell nociceptive response. We further show that activity-gated TRPV1 trafficking associates with intracellular Ca²⁺ signals of distinct kinetics, and recruits recycling routes mediated by synaptotagmin 1 and 7, respectively. These results suggest that activity-dependent TRPV1 recycling contributes to the establishment of tachyphylaxis.

Keywords: TRP channel; calcium; desensitization; pain; synaptotagmin.

Fig. 1.

Tachyphylaxis is regulated by the strength of preimposed stimulation. (A–D) Representative traces showing repetitive activation of TRPV1 channels after desensitization induced by 0.3 µM, 1 µM, 3 µM, and 10 µM capsaicin in the presence of Ca²⁺ (1.8 mM), as indicated by the color bars. Responses shown under gray bars were recorded in the absence of external Ca²⁺ to prevent further desensitization and gain whole-cell responses. A small current appeared upon the washing out of Ca²⁺, reflecting its inhibitory effect on TRPV1 activity as reported (42). The pipette solution contained no adenosine triphosphate (ATP). (E) Dose–response curves for whole-cell TRPV1 currents obtained before and after tachyphylaxis induction. Data are shown as relative values to the reference current evoked by 1 μM capsaicin at the beginning of each recording. Solid lines are fits to Hill equation (n = 7–18 cells for each condition). (F) Dose–response curves normalized to their intrinsic maximum. Recordings were from transiently transfected HEK 293 cells held at −60 mV. Cap, capsaicin. Error bars, SEM.

Fig. 2.

TRPV1 channels undergo activity-dependent endocytosis and exocytosis recycling. (A) HEK cells expressing TRPV1-pHluorin and stimulated by 1 µM capsaicin. Each single trace was derived from an individual cell, with a ring-shape region of interest (ROI) traced along the cell surface while excluding nucleus hole. Fluorescence change is expressed as dF/F₀ with F₀ as the mean intensity of prestimulation baseline. (B) HEK cells expressing TRPV1-pHluorin and stimulated by 10 µM capsaicin. (C) The peak loss in TRPV1-pHluorin fluorescence for 1 µM (n = 10 cells) and 10 µM capsaicin (13 cells) stimulation. (D) Recovery in TRPV1-pHluorin level quantified as the fraction to the prestimulation control level. The timing when the values were measured is indicated by blue arrowheads in A and B. (E) TRPV1-pHluorin recycling triggered by varied doses of capsaicin (0.3–10 µM, n = 7–13 cells for each condition). (F) Imaging of TRPV1-pHluorin trafficking for a long period (20 min; n = 10 for 1 µM, and 8 for 10 µM capsaicin). (Scale bars, 10 µm.)

Fig. 3.

Interim pause facilitates the restoration of whole-cell TRPV1 currents. (A) Whole-cell recording of a TRPV1-expressing HEK 293 cell. Desensitization was induced by 10 µM capsaicin (1.8 mM Ca²⁺) over shorter periods (12 s, Top; 6 s, Bottom). (B) Dose–response curves of whole-cell currents during tachyphylaxis period. Data from briefer stimulation are compared with those obtained after normal stimulation durations (∼60 s, gray traces). (C) After tachyphylaxis induced by 1 µM capsaicin, current was hardly triggered by similar stimulation. A 5-min pause helped the recovery of whole-cell current to the pretachyphylaxis level. (D) Facilitating effect of interim pause seen for 10 μM capsaicin-evoked desensitization. The facilitation was further enhanced by a longer pause period (5 min + 5 min; E). (F) Dose–response curves of whole-cell TRPV1 currents of different conditions, normalized to their initial reference current. Solid lines are fits to the Hill equation (n = 6–10 cells for each condition). The pipette solution contained 3 mM Na₂ATP and 3 mM Mg ATP. Error bars, SEM.

Fig. 4.

Ca²⁺ signals during stimulation strength-gated TRPV1 recycling. (A) Representative images of HEK 293 cells expressing TRPV1-pHluorin and loaded with the red Ca²⁺ dye Rhod-2, AM. (B) Fluorescence time courses of TRPV1-pHluorin and Rhod-2 in response to 1 µM and 10 µM capsaicin, respectively. A single trace denotes an individual cell, and fluorescence change is expressed as dF/F₀. (C) Peak amplitude of Ca²⁺ signals and the Ca²⁺ levels at 5 min after the stimulation onset (n = 9 for 1 µM, 6 for 10 µM capsaicin). (D) Scatter plot showing the apparent loss in TRPV1 surface expression against the corresponding Ca²⁺ levels, both measured at 5min post the stimulation. (E) TRPV1-pHluorin recycling triggered by 10 µM capsaicin in different concentrations of Ca²⁺ (n = 6–8 cells for each condition). (F) Ca²⁺ signals evoked by 1 µM and 10 µM capsaicin in cells with ER Ca²⁺ ATPase inhibited by thapsigargin (0.5 µM, pretreatment 10 min, n = 8 cells for each condition). (G) Differential alteration in the declining phase of low and high capsaicin-evoked Ca²⁺ signals upon NCX inhibition (n = 6–10 cells for each condition). (Scale bar, 10 µm.)

Fig. 5.

Synaptotagmin regulation of TRPV1 recycling. (A) Interaction of Syt1 and Syt9 with TRPV1. Immunoprecipitation (IP, with anti-FLAG) and immunoblot analysis (with anti-FLAG and anti-GFP) of HEK 293 cells transfected with plasmids as indicated. Molecular weight standards (MW, in kDa) are shown on the left. (B) Whole-cell currents in HEK cells coexpressing TRPV1 and Syt1 in response to 1 μM and 10 μM capsaicin, respectively. (C) Pooled dose–response curves showing Syt1 facilitation of current recovery during 1 µM capsaicin-induced tachyphylaxis (blue vs. gray open circle, n = 6–7 cells for each condition; solid lines are fitting by Hill equation). Gray traces are without Syt1. (D) Low capsaicin-evoked TRPV1 recycling was facilitated by Syt1, but not by Syt7 (n = 8–10 cells for each condition). TRPV1-pHluorin and red fluorescent Syt1-tdTomato were coexpressed in HEK cells, with the absence of cross-talk verified (SI Appendix, Figs. S5 and S7). (E–G) Parallel experiments to examine the interaction of TRPV1 with Syt7 and Syt4, and record their impact on whole-cell TRPV1 currents (n = 6–8 cells for the pooled dose–response curves). (H) Light-sheet imaging of TRPV1 recycling upon 10 µM capsaicin stimulation, which was retarded by Syt7 expression (Syt7-tdTomato; n = 9–12 cells for each condition).