Activation of TRPA1 by membrane permeable local anesthetics

Abstract

Background: Low concentrations of local anesthetics (LAs) suppress cellular excitability by inhibiting voltage-gated Na⁺ channels. In contrast, LAs at high concentrations can be excitatory and neurotoxic. We recently demonstrated that LA-evoked activation of sensory neurons is mediated by the capsaicin receptor TRPV1, and, to a lesser extent by the irritant receptor TRPA1. LA-induced activation and sensitization of TRPV1 involves a domain that is similar, but not identical to the vanilloid-binding domain. Additionally, activation of TRPV1 by LAs involves PLC and PI(4,5)P₂-signalling. In the present study we aimed to characterize essential structural determinants for LA-evoked activation of TRPA1.

Results: Recombinant rodent and human TRPA1 were expressed in HEK293t cells and investigated by means of whole-cell patch clamp recordings. The LA lidocaine activates TRPA1 in a concentration-dependent manner. The membrane impermeable lidocaine-derivative QX-314 is inactive when applied extracellularly. Lidocaine-activated TRPA1-currents are blocked by the TRPA1-antagonist HC-030031. Lidocaine is also an inhibitor of TRPA1, an effect that is more obvious in rodent than in human TRPA1. This species-specific difference is linked to the pore region (transmembrane domain 5 and 6) as described for activation of TRPA1 by menthol. Unlike menthol-sensitivity however, lidocaine-sensitivity is not similarly determined by serine- and threonine-residues within TM5. Instead, intracellular cysteine residues known to be covalently bound by reactive TRPA1-agonists seem to mediate activation of TRPA1 by LAs.

Conclusions: The structural determinants involved in activation of TRPA1 by LAs are disparate from those involved in activation by menthol or those involved in activation of TRPV1 by LAs.

Figure 1

Lidocaine activates and blocks rat TRPA1. A. Representative inward currents in HEK293t cells expressing rTRPA1 activated by 30 mM lidocaine and 100 μM mustard oil. Cells were held at -60 mV. B- C. Concentration dependent activation of rTRPA1 by lidocaine. Only one concentration was applied on each cell. The Hill-equation was applied to calculate the EC₅₀ value. D. A typical example of a lidocaine-evoked current blocked by the selective TRPA1-anatagonist HC-030331 (100 μM). E. Ramp currents of rTRPA1 in control solution and during application of 30 mM lidocaine. Cells were held at -60 mV and currents were measured during 500 ms long voltage-ramps from -100 to +100 mV. Note the lack of an outward rectification of the lidocaine-evoked current F. Representative effects on rTRPA1 30 mM lidocaine in cells held at +60 mV..G. Typical MO-evoked inward current blocked by 100 mM lidocaine. Increasing concentrations of lidocaine were co-applied with MO during the steady-state phase of MO-evoked currents. Experiments were performed in a Ca²⁺-free extracellular solution to minimize desensitization. The fractional block was plotted against the lidocaine concentration. The line represents the fit of the data to the Hill equation. H. Representative currents of TRPA1-currents activated by three consecutive applications of 30 mM lidocaine in standard extracellular solution containing 2 mM Ca²⁺. Lidocaine was applied in intervals of 2 min. I. Mean current amplitudes of rTRPA1 currents evoked by repeated applications of lidocaine in standard extracellular solution containing 2 mM Ca²⁺ or in a Ca²⁺-free solution. Current amplitudes are normalized to the value obtained with the first application of lidocaine (dotted line).

Figure 2

Lidocaine activates human TRPA1. A. Representative inward currents in HEK293t cells expressing hTRPA1 activated by 3, 10, 30 and 100 mM lidocaine. Cells were held at -60 mV and only one concentration was applied on each cell. B. Concentration-dependent activation of hTRPV1 by lidocaine. The Hill-equation was applied to calculate the EC₅₀ value. Transmembrane domain 5 is a determinant for species different activation of TRPA1 by lidocaine C. Representative inward currents evoked by 30 mM lidocaine on mTRPA1-WT, and the chimeras mTRPA1-hTM5/6 and hTRPA1-mTM5/6. Note the lack of a resurging current for mTRPA1-hTM5/6. D. Typical experiments for lidocaine-induced block of MO-evoked inward currents on hTRPA1-WT, mTRPA1-hTM5/6, mTRPA1-WT and hTRPA1-mTM5/6. Lidocaine was co-applied with MO during the steady-state phase of MO-evoked currents and experiments were performed in a Ca²⁺-free extracellular solution to minimize desensitization. Note the slow onset of lidocaine-induced block for hTRPA1-WT and mTRPA1-hTM5/6 as compared to mTRPA1-WT and hTRPA1-mTM5/6. Cells were held at -60 mV.

Figure 3

Lidocaine blocks but does not activate TRPM8. A. Typical recordings on rTRPM8 with a completely lacking response to 30 mM lidocaine but with a large inward current evoked by 100 μM menthol. Cells were held -60 mV B. Representative current trace displaying lidocaine-induced block of a menthol-evoked inward current on rTRPM8. Lidocaine was co-applied with menthol during the steady-state phase of menthol-evoked currents. The fractional block was plotted against the lidocaine concentration. The line represents the fit of the data to the Hill equation. C. Cold-induced inward currents of rTRPM8 in control solution (black) or in presence of 30 mM lidocaine (grey). The applied solution was cooled from room temperature (~ 24°C) to ~12°C within 10 seconds. Cells were held at -60 mV.

Figure 4

Intracellular interaction sites of lidocaine on TRPA1. A. Current traces of wildtype rTRPA1 activated by 3 mM lidocaine at pH 6.9, 7.4 or 7.9. Lidocaine-solutions with different pH-values were applied on the same cells in intervals of 1 min. B. Relative current amplitudes of rTRPA1 activated with 3 mM at different pH-values. The absolute current amplitudes were normalized to the amplitude determined for pH 7.4. C. Typical effect of the impermeable lidocaine-derivative QX-314 on rTRPA1. 30 mM QX-314 did not evoke any current responses in TRPA1-expressing HEK293t cells in which 30 mM lidocaine evoked large inward currents. Cells were held at -60 mV. D. Representative experiment performed on the acrolein-insensitive mutant construct hTRPA1-C621S/C641S/C665S (hTRPA1-3C). The cells were treated with 30 mM lidocaine, 250 μM carvacrol and 50 μM acrolein in intervals of 1 min. E. Relative current amplitudes of lidocaine-evoked currents on hTRPA1-WT and the hTRPA1-3C construct. Peak current amplitudes determined for lidocaine-evoked currents were normalized with the amplitudes of carvacrol-evoked currents in the same cells. Lidocaine (30 mM) and carvacrol (250 μM) were applied in intervals of 1 min. Cells held at -60 mV. F. Ramp currents of hTRPA1-wildtype (WT) and hTRPA1-3C (3C) in control solution and during application of 1 mM 2-APB. Cells were held at -60 mV and currents were measured during 500 ms long voltage-ramps from -100 to +100 mV.