The nociceptor ion channel TRPA1 is potentiated and inactivated by permeating calcium ions

Abstract

The transient receptor potential A1 (TRPA1) channel is the molecular target for environmental irritants and pungent chemicals, such as cinnamaldehyde and mustard oil. Extracellular Ca(2+) is a key regulator of TRPA1 activity, both potentiating and subsequently inactivating it. In this report, we provide evidence that the effect of extracellular Ca(2+) on these processes is indirect and can be entirely attributed to entry through TRPA1 and subsequent elevation of intracellular calcium. Specifically, we found that in a pore mutant of TRPA1, D918A, in which Ca(2+) permeability was greatly reduced, extracellular Ca(2+) produced neither potentiation nor inactivation. Both processes were restored by reducing intracellular Ca(2+) buffering, which allowed intracellular Ca(2+) levels to become elevated upon entry through D918A channels. Application of Ca(2+) to the cytosolic face of excised patches was sufficient to produce both potentiation and inactivation of TRPA1 channels. Moreover, in whole cell recordings, elevation of intracellular Ca(2+) by UV uncaging of 1-(4,5-dimethoxy-2-nitrophenyl)-EDTA-potentiated TRPA1 currents. In addition, our data show that potentiation and inactivation are independent processes. TRPA1 currents could be inactivated by Mg(2+), Ba(2+), and Ca(2+) but potentiated only by Ba(2+) and Ca(2+). Saturating activation by cinnamaldehyde or mustard oil occluded potentiation but did not interfere with inactivation. Last, neither process was affected by mutation of a putative intracellular Ca(2+)-binding EF-hand motif. In conclusion, we have further clarified the mechanisms of potentiation and inactivation of TRPA1 using the D918A pore mutant, an important tool for investigating the contribution of Ca(2+) influx through TRPA1 to nociceptive signaling.

FIGURE 1.

TRPA1 channels are potentiated and inactivated by extracellular Ca²⁺. Currents were recorded from HEK-293 cells heterologously expressing an N-terminal YFP fusion of rat TRPA1. A and B, in the absence of extracellular Ca²⁺, brief exposure to TRPA1 agonists, as indicated, elicited a current, which was first potentiated and then decayed to base line in response to the addition of extracellular Ca²⁺ (2 mm). Inset, the I-V relationship in response to a ramp depolarization (1 V/s) at the times indicated. Agonists were menthol (30 μm) or Cin (100 μm). Note that following the introduction of Ca²⁺, no additional responses to menthol could be measured, indicating that the channels had entered a long lasting inactivated state. C, TRPA1 currents in response to prolonged exposure to Cin (100 μm) in the absence of extracellular Ca²⁺ reached a plateau after ∼100 s. The addition of 2 mm Ca²⁺ did not cause further activation but still promoted rapid inactivation of the currents. Inset, the I-V relationship in response to a ramp depolarization (1 V/s) at the times indicated. Note the linearization of the I-V curve with strong activation. D, magnitude of the TRPA1 currents at +80 mV before and after the addition of 2 mm Ca²⁺ from experiments as in A and C. The Cin-evoked currents measured with the brief exposure protocol were ∼50% of the maximal Cin-evoked currents, and only these subsaturating responses could be potentiated by Ca²⁺.

FIGURE 2.

Sensitivity and specificity of potentiation and inactivation of TRPA1. A, TRPA1 currents evoked in response to brief exposure to Cin (100 μm) and the subsequent addition of varying concentrations of Ca²⁺ (as indicated). Note that each trace represents a recording from a separate cell, and the traces have been aligned by the time at which Ca²⁺ was introduced. The currents were scaled by the magnitude of the response to Cin, and therefore the y axis is represented by an arbitrary unit. B, currents evoked in response to brief exposure to Cin (100 μm) and the subsequent addition of Ba²⁺ or Mg²⁺ (2 mm). C, average data from experiments as in A and B. Potentiation was measured as the -fold increase in the current (at +80 mV) following the addition of the divalent cation, where 1 represents no change in the current. Note the “threshold” for potentiation of the current by extracellular Ca²⁺ was ∼12 μm. The bar corresponding to the response to “0 Ca²⁺” was duplicated to allow comparison with responses to Ba²⁺ and Mg²⁺. D, inactivation was measured by the T50 or time, relative to the peak, at which the currents had decayed to 50% of their maximum value (at +80 mV). There was a continuous slowing of inactivation as the Ca²⁺ concentration was lowered. Data represent the mean ± S.E. (n = 4–10). Intracellular solution was HB-Cs⁺ (which contained 5 mm EGTA) for A and B. E, TRPA1 currents elicited in response to a saturating exposure to Cin (100 μm) with varying concentrations of free Mg²⁺ in the pipette (as indicated). HC-030031 (10 μm) was introduced at the end of each experiment to confirm that the current was entirely due to the gating of TRPA1 channels. Intracellular solution was HB-Cs⁺ with 0, 2, or 10 mm MgCl₂ added. F, magnitude of the current (+80 mV) from experiments as in E. Data represent the mean ± S.E. ^*, p < 0.05; ^**, p < 0.01; ^***, p < 0.001.

FIGURE 3.

Pore region mutations reduce Ca²⁺ permeability of TRPA1. A, whole cell patch clamp recording of wild-type or mutant TRPA1 currents activated by Cin (100 μm). Plots show the current in response to a ramp depolarization (1 V/s) in the presence of the indicated extracellular cation (Na⁺, NMDG⁺, or Ca²⁺). The extracellular solution was rapidly exchanged to minimize changes in the magnitude of the currents during the recording. The arrow indicates the reversal of the current with 128 mm Ca²⁺ in the bath. Intracellular solution was HB-Cs⁺. B, ion selectivity was calculated based on the reversal potential of the current as measured from experiments as in A. D918A (n = 3) and D918N (n = 6) showed dramatically reduced Ca²⁺ permeability, whereas D918E (n = 8) had increased Ca²⁺ permeability as compared with wild type (n = 6). C, the S5-S6 region of TRPA1 aligned with that of related cation-permeable channels. Conserved acidic residues important for ion permeability are shown in red. Gray shading highlights the conserved GXG sequence in the selectivity filter. Note that Asp⁹¹⁸ is at the same position as the Tyr in the GYG motif of the K⁺ channel. Pore region alignment from multiple channels was based partly on Ref. . D, alignment of the putative selectivity filters of TRPA1 from multiple species. Data represent the mean ± S.E. Significance was determined by two-tailed Student's t test and was Bonferroni-corrected. ^*, p < 0.05; ^**, p < 0.01; ^***, p < 0.001. Ca2+I and Na+I, the first transmembrane domains of the Ca²⁺ and Na⁺ channel, respectively. Accession numbers are as follows: POA334 (KcsA), ZP_02523257 (NaKbc), ZP_02523257 (CNGA1), NP_997491 (TRPA1), NP_076460 (TRPV4), NP_446239 (TRPV5), Q13936 (Ca²⁺I), NP_062139 (Na⁺I), NP_015628 (human TRPA1), NP_997491 (rat TRPA1), NP_808449 (mouse TRPA1), XP_544123 (dog TRPA1), XP_581588 (cow TRPA1), NP_001007066 (fish TRPA1), NP_001097554 (fly TRPA1).

FIGURE 4.

Pore region mutations that reduce Ca²⁺ permeability disrupt Ca²⁺ potentiation and inactivation of TRPA1. A–D, effect of extracellular Ca²⁺ (2 mm) on wild-type and mutant TRPA1 currents activated by brief exposure to Cin (100 μm). B, extracellular Ca²⁺ caused no significant potentiation and only slow inactivation of the mutant D918A, which is poorly permeable to Ca²⁺. C, currents carried by the D918N mutant, which has an intermediate Ca²⁺ permeability, were strongly potentiation by Ca²⁺, but potentiation was slowed as compared with wild type. D, currents carried by the D918E mutant, which has an enhanced Ca²⁺ permeability, were strongly activated and rapidly inactivated by extracellular Ca²⁺. E and F, average data from experiments as in A–D, quantified as in Fig. 1. Wild-type data are reproduced from Fig. 1. Significance was determined by two-tailed Student's t test with Bonferroni correction for multiple samples. ^***, p < 0.001. Internal solution was HB-Cs⁺, which contained 5 mm EGTA.

FIGURE 5.

Lowering Ca²⁺ buffering rescues potentiation and inactivation of D918A currents. A, D918A currents evoked by Cin (100 μm) with 5 mm EGTA in the pipette were not potentiated or inactivated by either 2 mm (left) or 10 mm (right) extracellular Ca²⁺ (gray lines). Lowering the Ca²⁺ buffer in the pipette to 20 μm EGTA rescued potentiation and inactivation (black lines). Traces are from separate cells. To allow direct comparison of the currents, the responses were normalized to the amplitude of the Cin-evoked current and aligned by the time at which Ca²⁺ was added to the bath. B, average data from experiments as in A. C, simultaneous recordings of the current magnitude (top) and intracellular Ca²⁺ level (bottom) in D918A and wild type-expressing cells loaded through the pipette with the Ca²⁺ indicator Fura-4F (20 μm) with or without 5 mm EGTA. In the absence of intracellular Ca²⁺ buffering (no EGTA; left trace), D918A currents were potentiated and inactivated by bath application of 2 mm Ca²⁺, which produced a large rise in intracellular Ca²⁺. Including EGTA in the pipette (middle trace) under identical conditions eliminated the rise in intracellular Ca²⁺, and the currents showed no potentiation or inactivation. Wild-type currents (right trace) supported a large Ca²⁺ influx that produced a significant elevation of intracellular Ca²⁺, despite the presence of 5 mm EGTA in the pipette. D, peak intracellular Ca²⁺ concentration after the addition of extracellular Ca²⁺ from experiments as shown in C. In D918A-expressing cells loaded with no Ca²⁺ buffer, Ca²⁺ levels rose to the micromolar range. This elevation was completely blocked by 5 mm EGTA in the pipette. Data represent the mean ± S.E. A.U., arbitrary units.

FIGURE 6.

TRPA1 currents can be potentiated by elevation of intracellular Ca²⁺ through UV uncaging of DMNP-EDTA. A, potentiation of heterologously expressed TRPA1 currents in response to flash photolysis of caged Ca²⁺ (DMNP-EDTA loaded with Ca²⁺). TRPA1 currents were activated by brief exposure to Cin (100 μm), and following stabilization of the current magnitude, UV uncaging (100 ms) potentiated the currents. Subsequent bath application of 2 mm Ca²⁺ produced nearly complete inactivation of the current. B, UV light had little effect on the TRPA1 currents when cells were loaded with DMNP-EDTA in the absence of intracellular Ca²⁺. C, average data from experiments as in A and B. D, simultaneous recording of current magnitude (top) and intracellular Ca²⁺ (bottom) in HEK-293 cells expressing wild-type TRPA1 channels. The Ca²⁺ indicator Fluo-5F (10 μm) was loaded into the cells through the patch pipette. Cin (100 μm) in the absence of extracellular Ca²⁺ activated TRPA1 currents, and UV uncaging of Ca²⁺ potentiated the currents. The addition of extracellular Ca²⁺ (2 mm) caused a significantly higher Ca²⁺ elevation compared with that achieved by Ca²⁺ uncaging. E, mean data from experiments as in D. In the same experiments, the change in fluorescent emission from the Ca²⁺ indicator fluo-5F (ΔF) was measured immediately after UV uncaging and following the introduction of 2 mm Ca²⁺ to the bath. Data represent the mean ± S.E. ^**, p < 0.01; ^***, p < 0.001. a.u., arbitrary units.

FIGURE 7.

Potentiation and inactivation of TRPA1 channels by extracellular Ca²⁺ in cell-attached patches. A, TRPA1 channel activity in cell-attached patch clamp recording (V_m =-80 mV) in response to the addition of agonists outside the patch. B, conductance of spontaneously active channels measured from step depolarizations prior to exposure to Cin. The slope conductance was 139 ± 2 pS (n = 3). C–E, single channel activity at an expanded time scale (top panel) before and after bath application of Cin (100 μm) and following the subsequent addition of 2 mm Ca²⁺. All points histograms (bottom panel) fitted with Gaussians (gray line) indicate that the single channel conductance was not altered upon activation by Cin or potentiation by Ca²⁺. The pipette solution was HB-Cs⁺, which contained 5 mm EGTA and 100 nm Ca²⁺.

FIGURE 8.

Ca²⁺ potentiates and inactivates TRPA1 channels in excised patches. A, current activation (V_m = -80 mV) in response to increasing concentrations of Ca²⁺ in inside-out patches from TRPA1-expressing HEK-293 cells. Currents were activated by brief exposure to Cin (100 μm) in cell-attached recording mode, and all measurements from excised patches were obtained in the presence of polyP3 (1 mm). B, average Ca²⁺ dose-response curve from experiments as shown in A (data were normalized to the peak current in each patch). Fit was with V_max = 1, K_½ = 0.225 μm, n = 1.83. Data are represented by the mean ± S.E., n = 6. C and D, currents activated by 12 μm Ca²⁺ (C) or 200 μm Ca²⁺ (D) applied to the cytoplasmic side of inside-out patches (V_m =-80 mV). E, average data for the rate of current decay from experiments as in C and D. At lower cytoplasmic Ca²⁺ concentrations, the currents decayed more slowly. Data are represented by the mean ± S.E., n = 4–5.