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.

Species Comparison and Pharmacological Characterization of Human, Monkey, Rat, and Mouse TRPA1 Channels

“…Species differences between human and rodent TRPA1 have previously been identified by us (Chen et al, 2008;Chen and Kym, 2009) ) were recorded in a FLIPR-based Ca 2ϩ influx assay. Cells were treated with different concentrations of CMP1 for a 3.5-min period, and MO was added 3.5 min later (30 M for human, rat, and mouse TRPA1; 120 M for rhesus monkey TRPA1).…”

“…In this position, rTRPA1 and mTRPA1 have the identical residue (i.e., Gly878 in rTRPA1 and mTRPA1), whereas hTRPA1 and rhTRPA1 have the identical residue (i.e., Val875 in hTRPA1 and rhTRPA1). The opposite gating effects of CMP1 on hTRPA1 and rTRPA1 were previously attributed to two S6 transmembrane domain residues (Ser943 and Ile946 in hTRPA1; Ala946 and Met949 in rTRPA1) (Chen et al, 2008). In these two positions, mTRPA1 has the same residues as rTRPA1, whereas rhTRPA1 has one identical residue (Ile946-rhTRPA1) and one conserved, serine-to-cysteine substation (Cys943-rhTRPA1) compared with hTRPA1.…”

“…Despite this progress, drug discovery targeting the TRPA1 channel has been hampered by differences between human and rodent species. For instance, a series of structurally related thioaminals have been reported to have antagonist activity at human TRPA1 (hTRPA1), but when tested at rat TRPA1 (rTRPA1), they were either inactive [4-bromo-N-[2,2,2-trichloro-1-(4-chloro-phenylsulfanyl)-ethyl]-benzamide (AMG7160) and 4-nitro-N-[2,2,2- (Klionsky et al, 2007;Chen et al, 2008). Such compounds have to be excluded from the drug development process because conventional preclinical studies using rodents would not yield useful information.…”

Roles of TRPA1 in Pain Pathophysiology and Implications for the Development of a New Class of Analgesic Drugs

“…It is well documented that some compounds identified as antagonists at the human isoform show very different pharmacology at the rat receptor; this highlights the critical importance of surrogate model species in assessing the in vivo pharmacology [36,37]. Several electrophilic compounds blocking human TRPA1 receptor have been shown to either lack activity or even activate the rat receptor (AMG7160, AMG2504, AMG9090, AMG5445, CMP1, CMP2 and CMP3) [36,38]. Analogous discrepancies have been seen for non-reactive ligands like caffeine and menthol [39,40].…”

Intracellular Signaling Pathways Integrating the Pore Associated with P2X7R Receptor with Other Large Pores

“…Recently, it was described that TRPA1 activation induces dye uptake, which is blocked by selective TRPA1 antagonists. In addition, outside-out patch recordings using N-methyl-Dglucamine (NMDG + ) as the sole external cation and Na + as the internal cation, TRPA1 activation results in dynamic changes in permeability to NMDG + (Chen et al, 2008). Other groups have reproduced this data (Banke et al, 2010(Banke et al, , 2011, but in every cell studied the intracellular signaling was not investigated.…”