TRP channel activation by reversible covalent modification

Abstract

Allyl isothiocyanate, the pungent principle of wasabi and other mustard oils, produces pain by activating TRPA1, an excitatory ion channel on sensory nerve endings. Isothiocyanates are membrane-permeable electrophiles that form adducts with thiols and primary amines, suggesting that covalent modification, rather than classical lock-and-key binding, accounts for their agonist properties. Indeed, we show that thiol reactive compounds of diverse structure activate TRPA1 in a manner that relies on covalent modification of cysteine residues within the cytoplasmic N terminus of the channel. These findings suggest an unusual paradigm whereby natural products activate a receptor through direct, reversible, and covalent protein modification.

Fig. 1.

Chemical reactivity, rather than structure, confers TRPA1 agonist properties. (a) Chemical structures of electrophilic TRPA1 agonists AITC (mustard oil), diallyl disulfide (DADS), and cinnamaldehyde. (b) Structural similarities between benzyl isothiocyanate (BITC, Left Upper) and benzyl thiocyanate (BTC, Left Lower) do not reflect agonist properties. Representative trace from an oocyte expressing human TRPA1 (n = 5; Right). Benzyl- and allyl isothiocyantes evoke large, reversible inward currents at –80 mV, whereas BTC does not (20 μM each). (Scale bars: 200 nA and 1 min.) (c) Precomplexed conjugates of AITC (compounds A and B, 200 μM each) do not activate TRPA1 expressed in HEK293 cells transfected with TRPA1, as measured by Fura-2/AM ratiometric calcium imaging. Traces represent normalized average response of 250 cells over two trials. Thiourea A and dithiocarbamate B are synthetic compounds designed to mimic adducts likely to form when AITC is introduced into the cellular environment. Response to AITC (200 μM) verifies expression of function channels. Mean peak response ± SEM: A, 1.04 ± 0.11; B, 1.10 ± 0.03; AITC, 4.63 ± 0.12.

Fig. 2.

Synthetic cysteine-modification reagent NMM irreversibly activates TRPA1. (a) A chemical model of reversible and irreversible agonist action by AITC and NMM, respectively. (b) Representative trace from an oocyte expressing human TRPA1, showing reversible activation by AITC and irreversible activation by NMM (50 μM each, –80 mV) (n = 9). Pore blocker ruthenium red is used to demonstrate channel specificity (RR, 10 μM). Control injected oocytes showed no response (data not shown). (Scale bars: 50 nA and 1 min.) (c) Current–voltage relationships of TRPA1 responses to AITC (red trace) and NMM (blue trace) shown in b, as assessed by 1-s voltage ramps. (d) Calcium imaging of HEK293 cells transfected with TRPA1 shows robust responses to NMM (20 μM). (e) Average traces from Fura-2 ratiometric imaging of 20 capsaicin-responsive trigeminal neurons derived from matched littermates of wild-type (+/+) and TRPA1-null (−/−) mice. Responses to NMM and AITC (20 μM each) overlap in wild-type neurons (blue trace), whereas NMM fails to activate neurons derived from TRPA1-null mice (red trace). Mean peak responses to NMM: 2.32 ± 0.2 (+/+), 1.05 ± 0.1 (−/−).

Fig. 3.

Three cysteine residues in the intracellular N terminus of TRPA1 confer sensitivity to NMM. (a) Cysteine mutations conferring sensitivity to NMM cluster within a 50-aa stretch between the cluster of 18 ankyrin repeats and the first putative transmembrane region. Red dots denote residues required for full response to sulfhydryl modification reagents. Blue dots denote sites where mutations had no effect. (b) Activation of wild-type hTRPA1 (black trace) and triple mutant hTRPA1-3C (green trace) by sulfhydryl-selective reagents show expected properties for an intracellular reactive site. Membrane-impermeable reagent MTSET (200 μM) fails to activate either channel, whereas membrane-permeant MTSEA (200 μM) activates wild-type hTRPA1 only. AITC (200 μM) is used as a positive control. Traces represent normalized average calcium response of transfected HEK293 cells (n = 350) as assessed by Fura-2/AM ratiometric calcium imaging. Mean peak responses ± SEM: MTSET, 1.09 ± 0.01 (WT), 1.02 ± 0.0003 (3C); MTSEA, 2.34 ± 0.07 (WT), 1.03 ± 0.01 (3C); AITC, 4.73 ± 0.05 (WT), 4.87 ± 0.50 (3C). (c) Representative traces (–80 mV) from an oocyte expressing wild-type hTRPA1 (black) compared with NMM-insensitive triple mutant hTRPA1-3C (green). THC (400 μM, with 200 μM β-cyclodextran carrier) elicits robust, reversible inward currents in both cases, whereas NMM (50 μM) does not appreciably activate the triple mutant (n = 5 for each trace). (Scale bars: 200 nA and 1 min.) (d) NMM (50 μM, trace 1) activates human TRPA1 in a similar manner to AITC (20 μM, trace 2). For traces 1 and 2, HEK293 cells stably expressing hTRPA1 were recorded under cell-attached configuration at a holding potential of –60 mV, with sampling frequency of 5 kHz, filtering at 1 kHZ. Single-channel conductances (±SD) were as follows: 102.7 ± 12.9 for NMM (n = 4) and 101.8 ± 10.9 pS (n = 4) for AITC. Agonists were delivered by backfilling pipet with drug. For traces 3 and 4, oocytes injected with TRPA1-3C were recorded under similar conditions. Activation of TRPA1 by NMM (50 μM) is specifically abrogated in triple mutant hTRPA1-3C (trace 3), whereas AITC (500 μM) still can activate this current (trace 4). Single-channel conductance (±SD) was 107.7 ± 10.7 pS (n = 6) with 500 μM AITC backfilled in the pipet. (Scale bars: 10 pA and 200 msec.) (e) Triple mutant TRPA1-3C retains receptor-operated channel function. Average traces of calcium response from transfected HEK 293 cells (n = 50, two trials), as assessed by Fura-2/AM imaging. Upon application of bradykinin (BK) (100 nM), cells cotransfected with BK2R and wild-type hTRPA1 (black trace) or triple mutant hTRPA1-3C (green trace) show sustained elevated calcium levels in excess of cells transfected with BK2R alone (red trace). Mean peak responses: 2.52 ± 0.18 (WT + BK2R), 2.80 ± 0.23 (3C + BK2R), and 1.29 ± 0.20 (BK2R alone). (f) Triple mutant hTRPA1-3C is not responsive to diallyl disulfide (DADS) (600 μM). Representative trace from oocytes expressing triple mutant hTRPA1-3C (n = 5). Response to AITC (200 μM) is used as a positive control. (Scale bars: 100 nA and 1 min.)

Fig. 4.

Residual AITC response of triple mutant TPPA1-3C is mediated by irreversible modification of lysine-708. (a) AITC dose–response curves for TRPA1 and TRPA1-3C expressed in oocytes (n = 5 for each data point). Points were obtained by measuring response to 30-sec pulse of test dose at +80 mV and normalized to maximum response elicited by 5 mM AITC. EC₅₀ values were obtained by using curves fit with the least-squares approximation method. (b) Representative traces (–80 mV) from oocytes expressing wild-type hTRPA1 (black trace), triple mutant hTRPA1-3C (green trace), and quadruple mutant TRPA1-3C–K708Q (red trace) (n = 5 each). Response to AITC (200 μM) is weaker and irreversible in triple mutant and completely ablated in quadruple mutant. Response to THC (400 μM) and blockade by ruthenium red (10 μM) are used as positive controls. (Scale bars: 200 nA and 1 min.) (c) Average traces of calcium response from transfected HEK293 cells (n = 250, two trials), as assessed by ratiometric Fura-2/AM imaging. Cells transfected with quadruple mutant hTRPA1-3C–K708R (red trace) are not sensitive to AITC (150 μM) but retain capacity to respond to 2-APB (200 μM), whereas cells transfected with triple mutant TRPA1-3C (green trace) or wild-type TRPA1 (black trace) respond to both drugs. Mean peak responses: AITC, 4.58 ± 0.04 (WT), 3.54 ± 0.01 (3C), 1.17 ± 0.04 (K708R); 2-APB, 4.43 ± 0.04 (WT), 5.19 ± 0.05 (3C), 5.30 ± 0.05 (K708R). (d) A chemical model for irreversible activation of TRPA1-3C involving the formation of a stable thiourea linkage between AITC and a lysine side chain.