Abstract
Human transient receptor potential ankyrin channel 1 (TRPA1) is a polymodal sensor implicated in pain, inflammation and itching. An important locus for TRPA1 regulation is the cytoplasmic N-terminal domain, through which various exogenous electrophilic compounds such as allyl-isothiocyanate from mustard oil or cinnamaldehyde from cinnamon activate primary afferent nociceptors. This major region is comprised of a tandem set of 17 ankyrin repeats (AR1-AR17), five of them contain a strictly conserved T/SPLH tetrapeptide motif, a hallmark of an important and evolutionarily conserved contribution to conformational stability. Here, we characterize the functional consequences of putatively stabilizing and destabilizing mutations in these important structural units and identify AR2, AR6, and AR11-13 to be distinctly involved in the allosteric activation of TRPA1 by chemical irritants, cytoplasmic calcium, and membrane voltage. Considering the potential involvement of the T/SP motifs as putative phosphorylation sites, we also show that proline-directed Ser/Thr kinase CDK5 modulates the activity of TRPA1, and that T673 outside the AR-domain is its only possible target. Our data suggest that the most strictly conserved N-terminal ARs define the energetics of the TRPA1 channel gate and contribute to chemical-, calcium- and voltage-dependence.
Highlights
Human transient receptor potential (TRP) subtype A1 (TRPA1) is an intrinsically cold and chemosensitive ion channel whose physiological role has been implicated in nociception, inflammatory pain, and itching[1,2,3,4,5]
To examine how the conserved TPLH motif in ankyrin repeat 2 contributes to the functioning of the transient receptor potential ankyrin channel 1 (TRPA1) channel, we constructed three mutants: T100A, T100D, and M131G/A133T, that were intended to either destabilize or stabilize the conformation of the ankyrin repeat AR2 (Fig. 2A)
The N-terminus is likely to be dispensable for a functional channel[25], it is hard to believe that evolution has allocated such a large and structurally organized proportion of a protein merely for auxiliary functions such as trafficking, subcellular localization, homotetramerization, or a number of poly-specific interactions
Summary
Human transient receptor potential (TRP) subtype A1 (TRPA1) is an intrinsically cold and chemosensitive ion channel whose physiological role has been implicated in nociception, inflammatory pain, and itching[1,2,3,4,5]. This channel is one of the key sensors for various pungent and irritant compounds, being activated by thiol-reactive electrophiles and oxidants, and by a number of nonelectrophilic chemicals, including menthol, nicotine, carvacrol, clotrimazole, and certain cannabinoids (for a review, see[6]). Proline initiates the first α-helix, whereas the pair of threonine and histidine forms intraand inter-repeat hydrogen bonds (see Fig. 2A)
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