Lipid Raft Destabilization Impairs Mouse TRPA1 Responses to Cold and Bacterial Lipopolysaccharides

Abstract

The Transient Receptor Potential ankyrin 1 cation channel (TRPA1) is expressed in nociceptive sensory neurons and epithelial cells, where it plays key roles in the detection of noxious stimuli. Recent reports showed that mouse TRPA1 (mTRPA1) localizes in lipid rafts and that its sensitivity to electrophilic and non-electrophilic agonists is reduced by cholesterol depletion from the plasma membrane. Since effects of manipulating membrane cholesterol levels on other TRP channels are known to vary across different stimuli we here tested whether the disruption of lipid rafts also affects mTRPA1 activation by cold or bacterial lipopolysaccharides (LPS). Cooling to 12 °C, E. coli LPS and allyl isothiocyanate (AITC) induced robust Ca²⁺ responses in CHO-K1 cells stably transfected with mTRPA1. The amplitudes of the responses to these stimuli were significantly lower in cells treated with the cholesterol scavenger methyl β-cyclodextrin (MCD) or with the sphingolipids hydrolyzer sphingomyelinase (SMase). This effect was more prominent with higher concentrations of the raft destabilizers. Our data also indicate that reduction of cholesterol does not alter the expression of mTRPA1 in the plasma membrane in the CHO-K1 stable expression system, and that the most salient effect is that on the channel gating. Our findings further indicate that the function of mTRPA1 is regulated by the local lipid environment and suggest that targeting lipid-TRPA1 interactions may be a strategy for the treatment of pain and neurogenic inflammation.

Keywords: LPS; cholesterol; cold; lipid rafts; mouse TRPA1; sphingolipids.

Figure 1

Effects of cholesterol depletion on cold-induced responses of mTRPA1. (a and b) Representative traces of [Ca²⁺] change induced by cooling (upper panels) in the control condition (a) and after pretreatment with 10 mM MCD (b). Application of AITC (100 µM) was used as control for mTRPA1 activation. (c and d) Amplitudes of [Ca²⁺] responses evoked by cold (c) and AITC (d) in control condition (n = 118) or after pretreatment with 1 (n = 149), 5 (n = 202) or 10 mM (n =130) of MCD. (e and f) Maximal amplitudes of the first time derivative of the intracellular Ca²⁺ signal elicited by cold (e) and AITC (f) in control condition or after treatment with 1, 5 or 10 mM of MCD (the n numbers are the same as in panels c and d). The symbols *, ** and *** indicate p < 0.05, p < 0.01 and p < 0.001, respectively; Kolmogorov–Smirnov test.

Figure 2

Effects of SMase pretreatment on mTRPA1 responses to cold. (a and b) Representative traces of [Ca²⁺] change induced by cold in the control condition (a) and after pretreatment with 50 mUN SMase (b). Application of AITC (100 µM) was used as control for mTRPA1 activation. (c and d) Amplitudes of [Ca²⁺] responses evoked by cold (c) and AITC (d) in control condition (n = 183) or after pretreatment with 1 (n = 126), 10 (n = 164), 20 (n = 135) or 50 mUN (n = 111) of SMase. (e and f) Maximal amplitudes of the first time derivative of the intracellular Ca²⁺ signal elicited by cold (e) and AITC (f) in control condition or after treatment with 1, 10, 20 or 50 mUN of MCD (the n numbers are the same as in panels c and d). The symbols *, ** and *** indicate p < 0.05, p < 0.01 and p < 0.001, respectively; Kolmogorov–Smirnov test.

Figure 3

Effects of cholesterol depletion using MCD on mTRPA1 responses to LPS. (a and b) Representative [Ca²⁺] traces showing the effects of E. coli LPS (20 µg/mL) in control condition (a) and after pretreatment with 10 mM MCD (b). Application of AITC (100 µM) was used as control for mTRPA1 activation. (c and d) Amplitudes of [Ca²⁺] responses to LPS (c) and AITC (d) in control (n = 102) or after pretreatment with 1 (n = 72), 5 (n = 64) or 10 mM (n = 90) of MCD. Maximal amplitude of the first time derivative of the intracellular Ca²⁺ signal elicited by LPS (e) and AITC (f) in control condition or after treatment with 1, 5 or 10 mM of MCD (the n numbers are the same as in panels c and d). The symbols *, ** and *** indicate p < 0.05, p < 0.01 and p < 0.001, respectively; Kolmogorov–Smirnov test.

Figure 4

Effects of SMase pretreatment on mTRPA1 responses to LPS. (a and b) Representative [Ca²⁺] traces showing the effects of E. coli LPS (20 µg/mL) in control condition (a) and after pretreatment with 50 mUN SMase (b). Application of AITC (100 µM) was used as control for mTRPA1 activation. (c and d) Average amplitude of [Ca²⁺] responses to LPS (c) and AITC (d) in control condition (n = 108) or after pretreatment with 1 (n = 83), 10 (n = 75), 20 (n = 60) or 50 mUN (n = 64) of SMase. Maximal amplitude of the first time derivative of the intracellular Ca²⁺ signal elicited by LPS (e) and AITC (f) in control condition or after treatment with 1, 10, 20 or 50 mUN of SMase (the n numbers are the same as in panels c and d). The symbols *, ** and *** indicate p < 0.05, p < 0.01 and p < 0.001, respectively; Kolmogorov–Smirnov test.

Figure 5

Effects of cholesterol depletion using MCD on mTRPA1 activation by AITC in stably transfected CHO-K1 cells. (a) Mean traces of Ca²⁺ responses to different concentrations of AITC in control (left panel) or after treatment with 10 mM MCD (right panel; n = 63–178). (b) Dose-response curves of the amplitude of the Ca²⁺ responses to AITC averaged over different cells. The lines represent fits with Hill functions with parameters EC₅₀ = 4.6 ± 1.1 µM and 70 ± 20 µM, Δ[Ca²⁺]_MAX = 1.32 ± 0.09 µM and 1.4 ± 0.3 µM and H = 1.4 ± 0.3 and 1.5 ± 0.5, for control and MCD 10 mM, respectively.