Clioquinol and pyrithione activate TRPA1 by increasing intracellular Zn2+

Abstract

The antifungal and amoebicidal drug clioquinol (CQ) was withdrawn from the market when it was linked to an epidemic of subacute myelo-optico-neuropathy (SMON). Clioquinol exerts its anti-parasitic actions by acting as a Cu/Zn chelator and ionophore. Here we show that local injections of CQ produce mechanical hyperalgesia and cold hypersensitivity through a mechanism involving TRPA1 in mice. We also show that CQ activates TRPA1 in a Zn(2+)-dependent manner. Using a different Zn(2+)-ionophore, zinc pyrithione (ZnPy), we demonstrate that low, nanomolar concentrations of intracellular Zn(2+) ([Zn(2+)](i)) stimulate TRPA1. Direct application of Zn(2+) to the intracellular face of excised, inside-out patches activates TRPA1 with an EC(50) value of 7.5 +/- 1 nM. TRPA1 is expressed in a subpopulation of nociceptive dorsal root ganglion (DRG) neurons, where it acts as a sensory receptor for environmental irritants and oxidants. Using cultured DRG neurons from wild-type and TRPA1-deficient mice, we demonstrate that TRPA1 is the principal excitatory receptor for increased [Zn(2+)](i) in DRG neurons. In conclusion, we have discovered that TRPA1 acts a sensor of intracellular Zn(2+), and that Zn(2+) ionophores, such as CQ and ZnPy, activate TRPA1 by increasing [Zn(2+)](i). We also demonstrate that CQ-evoked mechanical hyperalgesia and cold allodynia require TRPA1 in vivo.

Fig. 1.

TRPA1 is required for the acute nociceptive effects of CQ. Mice (trpa1^+/+ and trpa1^−/−) received intraplantar injections of CQ (2.5 nmoles in 25 μl) and were examined for mechanical hyperalgesia (A, paw withdrawal threshold, paw pressure), cold hypersensitivity (B, paw withdrawal latency) and tactile allodynia (C, paw withdrawal threshold, von Frey). TRPA1-deficient mice had significantly higher thresholds and longer latencies than wildtype mice before administration of CQ (time = 0) in all three tests. CQ markedly reduced the withdrawal thresholds (A and C) and latency (B) in the injected paw of wild-type mice (+/+). In contrast, clioquinol only produced tactile allodynia (C) in TRPA1-deficient mice (-/-) and had no effect in the paw pressure and cold withdrawal tests. (n = 6; †, P < 0.05; ††, P < 0.01; †††, P < 0.001 compared to trpa1^+/+ before administration of CQ; *, P < 0.05; **, P < 0.01; ***, P < 0.001 compared to the contralateral paw).

Fig. 2.

CQ is a TRPA1 agonist. CQ (10 μM) activates TRPA1 currents in CHO cells in Ca²⁺-containing (A) and Ca²⁺-free external solutions (B). (C) Pretreatment of the cells with a membrane permeable (TPEN), but not an impermeable (DTPA) Zn²⁺ chelator, significantly reduced the amplitudes of CQ-induced currents (**, P < 0.01).

Fig. 3.

Zn²⁺ is a potent TRPA1 activator. In the whole-cell configuration ZnPy stimulates TRPA1 with an EC₅₀ of 2.4 μM (A and B; whole cell, −60 mV). DTPA, a membrane impermeable Zn²⁺ chelator completely prevents the ZnPy-induced activation of TRPA1 (C and D; n = 4–8). ZnPy (10 μM) rapidly evokes TRPA1 currents in cell attached patches (E). The patch pipette contained 5 mM BAPTA to remove any Zn²⁺ and Ca²⁺ from the extracellular side of the membrane. (F) FluoZin-3 measurements of [Zn²⁺]_i in untransfected CHO cells exposed to different concentrations of ZnPy for 2 min. (G) Concentration dependence of ZnPy-evoked Na⁺ influx (measured with SBFI) in untransfected CHO cells and cells expressing mouse or human TRPA1.

Fig. 4.

TRPA1 is activated by intracellular Zn²⁺. Application of Zn²⁺ to the intracellular face of excised patches concentration-dependently stimulates TRPA1. (A) No or very little TRPA1 channel activity was seen in response to 1 nM Zn²⁺, whereas 14 nM Zn²⁺ (B) rapidly evoked a marked increase in channel activity (−60 mV). (C) Concentration dependence of the agonist activity of Zn²⁺ in inside-out patches (EC₅₀ = 7.5 ± 1 nM). Each data point is the mean ± SEM. NP_o during a 60-s local Zn²⁺-application of n = 4–6 patches. Only patches without significant channel activity before application of Zn²⁺ were analyzed.

Fig. 5.

TRPA1 is necessary for ZnPy evoked [Ca²⁺]_i responses in DRG neurons. (A) ZnPy (1 μM) rapidly evokes a Ca²⁺-response in DRG neurons expressing TRPA1, whereas Zn²⁺ (10 μM) was unable to evoke responses. A brief pulse of ZnPy produced rapid elevations in [Ca²⁺]_i in 40% (76 of 188) of capsaicin-sensitive DRG neurons cultured from TRPA1^+/+ mice (B), but only evoked a small [Ca²⁺]_i increase in 1% (3 of 201) of capsaicin-sensitive neurons from TRPA1^−/− mice (C).