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 membranepermeable 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.chemical modification ͉ irritants ͉ natural products ͉ pain P lants have evolved ingenious defensive strategies to ward off herbivorous predators. Such protective mechanisms often involve the production of chemical irritants that activate sensory nerve fibers of offending creatures to produce discomfort or pain (1, 2). For example, plants of the genus Brassica (mustard), Allium (onion), and Cinnamomum (cinnamon) produce pungent isothiocyanate, thiosulfinate, and ␣,␤-unsaturated aldehyde compounds, respectively, which elicit acute pain and neurogenic inflammation by activating TRPA1, a nonselective cation channel expressed by sensory neurons of the pain pathway (3-7). Each of these compounds is capable of forming covalent adducts with thiols, primary amines, and to a lesser extent, hydroxyl groups (8), raising questions as to how electrophiles with such indiscriminate reactivity function as reversible and specific ionchannel agonists.Here we provide evidence to support a model whereby a variety of structurally distinct environmental irritants activate TRPA1 through a mechanism primarily involving covalent modification of specific cysteine side chains located within the putative cytoplasmic N-terminal domain of the channel. Pharmacological analysis of various sulfhydryl reactive agents provides a biochemical rationale for the reversible properties of mustard oil and other naturally occurring TRPA1 agonists. Importantly, TRPA1 mutants that are insensitive to these electrophilic agonists retain their ability to function as ''receptor operated'' channels, indicating that distinct biochemical pathways can contribute to TRPA1 activation by environmental or endogenous stimuli. These studies provide a mechanistic framework to further elucidate biochemical and structural parameters underlying activation of this member of the TRP ion channel family. ResultsThe diverse chemical nature of TRPA1-activating irritants (Fig. 1a) suggests that reactivity, rather than structure per se, constitutes the critical determinant of TRPA1 agonist activity. To clarify this issue, we first asked whether benzyl thiocyanate (BTC) is able to activate TRPA1. BTC is isosteric with the TRPA1 agonist benzyl isothiocyanate (BITC), and, thus, whereas both compounds possess thiocyanate functional groups of similar size, th...

A primer on ankyrin repeat function in TRP channels and beyond

“…3A-B). 22,23 These results implicate the TRPA1 ankyrin repeats in the channel's sensing and gating functions, although how the covalent attachments lead to channel activation is still unclear. TRPA1 is also directly gated by calcium, and residues in the finger loop of predicted ankyrin repeat 12 have been implicated in this mechanism (Fig.…”

“…The EF-hand-like sequence motif is boxed. Colored cysteines are important in activation of TRPA1 by electrophiles as identified by the Patapoutian (red) 22 or Julius group (cyan) 23 or both (magenta). (B) The structure of four canonical ankyrin repeats is used to illustrate the position of important TRPA1 cysteines either in internal repeat 11 (red) or terminal repeat 17 (magenta and cyan).…”

“…21 These compounds have very different structures, but two groups noted that they all are electrophiles and determined that the compounds covalently modify intracellular cysteines in the ankyrin repeat domain and adjacent linker region of TRPA1. 22,23 Two sets of cysteines were identified as important in the response to these agents -cysteines 414 and 421 on the predicted palm surface of repeat 11, and cysteines 621, 641 and 665 on the most C-terminal predicted repeat and adjacent linker (numbering follows the human sequence; Fig. 3A-B).…”

Nociceptors: the sensors of the pain pathway

“…MA, mechanically activated; RA, rapidly adapting; IA, intermediate adapting; SA, slowly adapting. (88,89). Importantly, endogenous reactive chemicals are also effective agonists of TRPA1 (90).…”

Transient Receptor Potential Channels in Chemotherapy-Induced Neuropathy

“…TRPA1 channel can be gated by distinct mechanisms. Most of TRPA1 activators are characterized by the presence of a highly reactive electrophilic group that, via a Michael-addition reaction, form covalent bonds with nucleophilic groups, such as cysteine and lysine residues located in the N-terminal cytoplasmic domain of the channel, hence inducing modifications of TRPA1 N-terminal that lead to dilation of the channel permeation pore [141,142]. As in the case of electrophilic agonists, HNE provokes TRPA1 gating by covalent modification of cysteine and lysine residues located within the N-terminal cytoplasmic domain of the channel [138].…”