Requirement of a soluble intracellular factor for activation of transient receptor potential A1 by pungent chemicals: Role of inorganic polyphosphates

Pungent chemicals such as allyl isothiocyanate ( AITC), cinnamaldehyde, and allicin, produce nociceptive sensation by directly activating transient receptor potential A1 ( TRPA1) expressed in sensory afferent neurons. In this study, we found that pungent chemicals added to the pipette or bath solution easily activated TRPA1 in cell-attached patches but failed to do so in inside-out or outside-out patches. Thus, a soluble cytosolic factor was required to activate TRPA1. N-Ethylmaleimide, ( 2-aminoethyl)-methane thiosulfonate, 2-aminoethoxydiphneyl borate, and trinitrophenol, compounds that are known to activate TRPA1, also failed to activate it in inside-out patches. To identify a factor that supports activation of TRPA1 by pungent chemicals, we screened similar to 30 intracellular molecules known to modulate ion channels. Among them, pyrophosphate ( PPi) and polytriphosphate ( PPPi) were found to support activation of TRPA1 by pungent chemicals. Structure-function studies showed that inorganic polyphosphates ( polyP(n), where n = number of phosphates) with at least four phosphate groups were highly effective ( polyP4 approximate to polyP65 approximate to polyP45 approximate to polyP25 > PPPi > PPi), with K-1/2 values ranging from 0.2 to 2.8 mM. Inositol-trisphosphate and inositol-hexaphosphate also partially supported activation of TRPA1 by AITC. ATP, GTP, and phosphatidylinositol-4,5-bisphosphate that have three phosphate groups did not support TRPA1 activation. TRPA1 recorded from cell bodies of trigeminal ganglion neurons showed similar behavior with respect to sensitivity to pungent chemicals; no activation was observed in inside-out patches unless a polyphosphate was present. These results show that TRPA1 requires an intracellular factor to adopt a functional conformation that is sensitive to pungent chemicals and suggest that polyphosphates may partly act as such a factor.