The stress protein heat shock cognate 70 (Hsc70) inhibits the Transient Receptor Potential Vanilloid type 1 (TRPV1) channel

Abstract

Background: Specialized cellular defense mechanisms prevent damage from chemical, biological, and physical hazards. The heat shock proteins have been recognized as key chaperones that maintain cell survival against a variety of exogenous and endogenous stress signals including noxious temperature. However, the role of heat shock proteins in nociception remains poorly understood. We carried out an expression analysis of the constitutively expressed 70 kDa heat-shock cognate protein, a member of the stress-induced HSP70 family in lumbar dorsal root ganglia from a mouse model of Complete Freund's Adjuvant-induced chronic inflammatory pain. We used immunolabeling of dorsal root ganglion neurons, behavioral analysis and patch clamp electrophysiology in both dorsal root ganglion neurons and HEK cells transfected with Hsc70 and Transient Receptor Potential Channels to examine their functional interaction in heat shock stress condition.

Results: We report an increase in protein levels of Hsc70 in mouse dorsal root ganglia, 3 days post Complete Freund's Adjuvant injection in the hind paw. Immunostaining of Hsc70 was observed in most of the dorsal root ganglion neurons, including the small size nociceptors immunoreactive to the TRPV1 channel. Standard whole-cell patch-clamp technique was used to record Transient Receptor Potential Vanilloid type 1 current after exposure to heat shock. We found that capsaicin-evoked currents are inhibited by heat shock in dorsal root ganglion neurons and transfected HEK cells expressing Hsc70 and TRPV1. Blocking Hsc70 with matrine or spergualin compounds prevented heat shock-induced inhibition of the channel. We also found that, in contrast to TRPV1, both the cold sensor channels TRPA1 and TRPM8 were unresponsive to heat shock stress. Finally, we show that inhibition of TRPV1 depends on the ATPase activity of Hsc70 and involves the rho-associated protein kinase.

Conclusions: Our work identified Hsc70 and its ATPase activity as a central cofactor of TRPV1 channel function and points to the role of this stress protein in pain associated with neurodegenerative and/or metabolic disorders, including aging.

Keywords: ATPase; cell stress; heat shock cognate 70; rho-associated protein kinase; transient receptor potential vanilloid type 1 channel.

Figure 1.

CFA-induced thermal and mechanical hypersensitivity coincides with an increase in Hsc70. (a) Mechanical sensitivity measured in the ipsilateral versus contralateral hind paw using the dynamic plantar aesthesiometer, before and after intraplantar injection of CFA. (b) Thermal sensitivity in the ipsilateral versus contralateral paw, measured using the Hargreaves test in mice injected with CFA. Data are expressed as mean values ± SEM (n = 7 mice per group); ***P < 0.001 (two-way ANOVA analysis of variance). (c) Hsc70 protein level in DRGs (L4–L5) was increased following CFA injection in the ipsilateral paw (Ipsi) versus control (contra). (d) Densitometry analysis of Hsc70 protein level normalized to tubulin from n = 7 mice between day 3 and 7 post CFA injection, *P < 0.05 (paired t test). (e) Representative immunostaining for TRPV1 (green) and Hsc70 (red) in lumbar DRG section. Note the presence of both TRPV1 and Hsc70 in small (<20µm diameter) DRG neurons (scale bar = 20 µm).

Figure 2.

Effect of heat shock on capsaicin-evoked current in DRG neurons. (a) Representative whole-cell patch clamp capsaicin-induced currents recorded from a DRG neuron exposed to either 37℃ (control) or 42℃ (heat shock) for 1 h in calcium free solution. Voltage ramp protocol from −100 mV to +100 mV was applied from a holding potential of 0 mV. (b) Representative time-course of a capsaicin-evoked current in control (black) and heat shock treated (gray) DRG neurons. (c) Voltage dependence of activation (G-V curves) in the absence and presence of heat shock normalized to the respective Gmax values. Superimposed are best fits of a Boltzmann function with the following V_a values: 60.12 ± 5.45 (n = 11) vs 66.37 ± 5.93 mV (n = 8) (inset). (d) The Hsc70 blocker spergualin (5 µg/ml, n = 5) completely reverses the heat shock-induced inhibition of TRPV1 current elicited by capsaicin. Asterisks indicate a significant difference, *P < 0.05. Data are expressed as means ± SEM.

Figure 3.

Heat shock inhibition of TRPV1 requires Hsc70. (a) Representative whole-cell TRPV1 current recorded from HEK cells transfected with TRPV1 and Hsc70 exposed to either 37℃ (control) or 42℃ (heat shock). Voltage steps were applied from a holding potential of 0 mV to various membrane potentials from −100 mV to +100 mV, in 20 mV intervals. (b) Peak capsaicin-evoked TRPV1 current density at +100 mV. The effect of heat shock is completely reversed by pretreatment with either of the Hsc70 blockers: matrine (200 μM, n = 5) or spergualin (5 µg/ml, n = 5) and not observed in the absence of cotransfected Hsc70 (right panel).

Figure 4.

Desensitization of TRPV1 current is unchanged by heat shock. (a) Conductance–temperature relationship in HEK cells transfected with TRPV1 (black) or TRPV1 + Hsc70 (gray) (n = 4 for each data point). (b) Sample recording responses to three consecutive applications of capsaicin (100 nM, 20 sec) separated by a 5 min wash in HEK cells expressing TRPV1 and Hsc70 at a holding potential of −80 mV. (c) Histogram representing the peak of TRPV1 current density obtained at −80 mV for each of the three consecutive applications of capsaicin (100 nM) in control and heat shock condition. Data are expressed as means ± SEM. n = 5 for each column. *P < 0.05. One way ANOVA followed by the Bonferroni post hoc test.

Figure 5.

Voltage and capsaicin sensitivity of TRPV1 upon heat shock treatment. (a) Voltage dependence of TRPV1 activation (G-V) in capsaicin-stimulated HEK cells cotransfected with TRPV1 and Hsc70 in the absence (black) and presence (gray) of heat shock. Superimposed are best fits of a Boltzmann function and the Va values are: 65.54 ± 5.41 mV (n = 11) and 67.15 ± 6.34 mV without (black) and with (gray) heat shock, respectively (n = 8). (b) Capsaicin dose-response curves from HEK cells cotransfected with TRPV1 and Hsc70 with (gray) or without (black) heat shock (n = 5 for each data point). Curves represent fits of a Hill equation. (c) Time-course of the capsaicin-evoked peak current density at +100 mV, recorded during and after exposure to heat shock (n = 6 for each column). Data are expressed as mean values ± SEM.

Figure 6.

Hsc70-mediated TRPV1 current inhibition occurs via suppression of TRPV1 channel incorporation at the cell surface. (a) Western blot analysis of TRPV1 and HA-tagged Hsc70 in heat shock treated HEK cells; note that heat shock does not alter either TRPV1 or Hsc70 protein level. (b) Confocal images of HEK cells transfected with TRPV1-pHluorin or TRPV1-pHluorin + Hsc70 with or without heat shock treatment. Non permeabilized cells were immunostained with a GFP antibody (red) to detect TRPV1 at the cell surface. (c) Quantification of membrane versus total expression of TRPV1-pHluorin for different condition of heat shock treatment. Data are expressed as mean values ± SEM (n = 18). ***P < 0.001. Two-way ANOVA followed by Tukey post test.

Figure 7.

Hsc70-mediated current inhibition upon heat shock is specific to TRPV1 channel. (a) I–V curves of MO (100 µM)-evoked TRPA1 current in HEK cells cotransfected with TRPA1 and Hsc70 upon heat shock treatment. (b) Menthol (50 µM)-evoked TRPM8 current in HEK cells cotransfected with TRPM8 and Hsc70 upon heat shock treatment.

Figure 8.

ADP-bound Hsc70 mediates channel inhibition. (a) TRPV1 current in transfected HEK cells elicited by a ramp protocol from −100 to +100 mV upon application of capsaicin (100 nM) in the presence of either 2 mM ATP or 2 mM ADP in the pipette solution. (b) TRPV1 current, in DRG neurons, elicited by a ramp protocol from −100 to +100 mV upon application of capsaicin (100 nM) in the presence of either 2 mM ATP or 2 mM ADP in the pipette solution. (c) peak of TRPV1 current density at +100 mV for different experimental conditions shown in (a) and (b). *P < 0.05. Paired t test.

Figure 9.

Hsc70-mediated decrease in TRPV1 current is independent of PKC or MAPK but acts through ROCK inhibition. Peak of TRPV1 current density at +100 mV measured in HEK cells transfected with TRPV1 + Hsc70 upon application of inhibitors of PKC (bisindolylmaleimide, GFX), MAPK (UO126), or ROCK (Y27632). Data are expressed as mean values ± SEM (n = 6 to 8 cells per group). *P < 0.05.

Figure 10.

Hsc70-dependent inhibition of ROCK phosphorylation on TRPV1 S502 site. (a) Capsaicin-evoked peak current (at +100 mV) in HEK cells transfected with Hsc70 and each of the following: TRPV1wt (n = 6), S502A (n = 6), and S800G (n = 5) mutant, in the absence and presence of heat shock and in the presence of the ROCK inhibitor Y27632 (10 μM). (b) Representative WB showing the changes, after heat shock, in levels of phosphorylated ROCK (top), ROCK2 (middle) and ROCK1 (bottom) in HEK cells expressing the indicated constructs (n = 4 independent experiments). (c) Densitometry analysis of phosphorylated-ROCK2 Western blot illustrated in (b). Note that a significant decrease in phosphorylated ROCK2 level appears only when HEK cells are cotransfected with TRPV1 + Hsc70 and treated with heat shock. *P < 0.05. Unpaired t test.