Molecular determinants of species-specific activation or blockade of TRPA1 channels

Abstract

TRPA1 is an excitatory, nonselective cation channel implicated in somatosensory function, pain, and neurogenic inflammation. Through covalent modification of cysteine and lysine residues, TRPA1 can be activated by electrophilic compounds, including active ingredients of pungent natural products (e.g., allyl isothiocyanate), environmental irritants (e.g., acrolein), and endogenous ligands (4-hydroxynonenal). However, how covalent modification leads to channel opening is not understood. Here, we report that electrophilic, thioaminal-containing compounds [e.g., CMP1 (4-methyl-N-[2,2,2-trichloro-1-(4-nitro-phenylsulfanyl)-ethyl]-benzamide)] covalently modify cysteine residues but produce striking species-specific effects [i.e., activation of rat TRPA1 (rTRPA1) and blockade of human TRPA1 (hTRPA1) activation by reactive and nonreactive agonists]. Through characterizing rTRPA1 and hTRPA1 chimeric channels and point mutations, we identified several residues in the upper portion of the S6 transmembrane domains as critical determinants of the opposite channel gating: Ala-946 and Met-949 of rTRPA1 determine channel activation, whereas equivalent residues of hTRPA1 (Ser-943 and Ile-946) determine channel block. Furthermore, side-chain replacements at these critical residues profoundly affect channel function. Therefore, our findings reveal a molecular basis of species-specific channel gating and provide novel insights into how TRPA1 respond to stimuli.

Figure 1.

Thioaminal compounds and their species-specific effects on rTRPA1 and hTRPA1 expressed in HEK293F cells. A, Chemical structures, activities, and potencies on rTRPA1 (EC₅₀) and hTRPA1 (IC₅₀). CMP1–3 contain thioaminal groups with a reactive sulfur atom labeled by an asterisk. The sulfur atom is replaced by a nitrogen in CMP4. Compound nomenclature is described in Materials and Methods. To obtain the IC₅₀ value for CMP1–3, 30 μm AITC (∼EC₈₀) was used to activate hTRPA1 channel. B, C, In cells expressing rTRPA1, URB597, AITC, and CMP1 evoked increases of intracellular Ca²⁺ as represented by the changes of fluorescence signals (RFU) in the FLIPR-based Ca²⁺ assay. D, Concentration–effect relationship for rTRPA1 activation by AITC, URB597, and CMP1. E, F, In cells expressing hTRPA1, CMP1 did not induce Ca²⁺ increases but blocked URB597 and AITC-evoked responses. G, Concentration–effect relationship of CMP1 inhibition of AITC (30 μm) or URB597 (100 μm) evoked Ca²⁺ responses in hTRPA1 cells.

Figure 2.

CMP1 activated rTRPA1 and blocked hTRPA1, as assessed in whole-cell and cell-attached single-channel recordings. A, In a representative cell expressing rTRPA1 (held at −60 mV), CMP1 evoked whole-cell currents in a reversible manner. A follow-up application of AITC also evoked inward currents. The dotted line indicates zero current level. B, CMP1 blocked AITC-induced hTRPA1 currents. C, CMP1 evoked rTRPA1 single-channel activities (−60 mV). D, CMP1 blocked AITC-induced hTRPA1 single-channel activities. The concentrations for CMP1 and AITC were 100 μm.

Figure 3.

CMP1 activities on TRPA1 depend on its chemical reactivity. A, Schematic of the chemical reaction between CMP1 and a cysteine residue with a free SH group. B, Reactivity in CMP1 but not in CMP4, as assessed by La antigen-based ALARM NMR. Each panel contains an overlay of the [¹³C-¹H] HSQC with the leucine and valine methyl proton chemical shifts (protein alone, red contours; protein with compound, black contours). Compounds were tested with and without 20 mm DTT. C, Concentration-effect relationships of rC622S and rTRPA1 activation by AITC, CMP1, and TNP, as determined in the Ca²⁺ assay. AITC EC₅₀, 7.5 ± 1.0 μm (rTRPA1), 40.3 ± 3.6 μm (rC622S); CMP1 EC₅₀, 4.0 ± 0.4 μm (rTRPA1), 10.7 ± 0.9 μm (rC622S); TNP EC₅₀, 108.0 ± 6.6 μm (rTRPA1), 103.5 ± 5.7 μm (rC622S). Note reductions in sensitivity to AITC and CMP1 (arrows) but not to TNP. D, hC621S reduced the activation sensitivity to AITC but not TNP. AITC EC₅₀, 11.3 ± 2.3 μm (hTRPA1), 62.9 ± 3.1 μm (hC611S); TNP EC₅₀, 259 ± 19 μm (hTRPA1), 245 ± 16 μm (hC611S). E, hC621S reduced inhibition potency of CMP1 but not RR. CMP1 IC₅₀, 0.36 ± 0.00 μm (hTRPA1), 2.6 ± 0.1 μm (hC611S); IC₅₀ RR, 18.9 ± 0.4 μm (hTRPA1), 19.9 ± 0.1 μm (hC611S). TNP (300 μm) was used to activate channels. n = ∼4–12.

Figure 4.

S6 domains of rTRPA1 and hTRPA1 dictate CMP1-mediated channel gating. A, Schematic representations of chimeras and their responses to CMP1, as measured in the Ca²⁺ assays. The amino acid compositions of chimeras are included in the supplemental material (available at www.jneurosci.org) and also indicated by arrows in supplemental Figure 5A (available at www.jneurosci.org as supplemental material). B, h-rS6 was insensitive to block or activation by CMP1 (in μm). C, r-hS6 was blocked by CMP1. Asterisk, In r-hS5L56, 300 μm CMP1 evoked 54.0 ± 4.9% of Ca²⁺ signals compared with AITC.

Figure 5.

S6 residues critical for CMP1-mediated channel gating. A, A sequence alignment around the S6 domains of rTRPA1 and hTRPA1. Arrows indicate junction sites for S6 swaps. Positions with residue differences are marked in bold, and critical residues are marked by an asterisk. B, Single-residue substitutions and their responses to CMP1. act, Activation; no effect, no activation or block at concentration up to 300 μm. IC₅₀ and EC₅₀ values are in μm (n = ∼4–12). C, rM949I was blocked by CMP2 and CMP3 (the thioaminal containing analogs of CMP1), as accessed in the Ca²⁺ assay. Representative traces were taken from four trials.