Physiol. Res. 66: 425-439, 2017 https://doi.org/10.33549/physiolres.933553 REVIEW Molecular Basis of TRPA1 Regulation in Nociceptive Neurons. A Review A. KÁDKOVÁ1, V. SYNYTSYA1,2, J. KRUSEK1, L. ZÍMOVÁ1, V. VLACHOVÁ1 1Department of Cellular Neurophysiology, Institute of Physiology of the Czech Academy of Sciences, Prague, Czech Republic, 2Department of Physical and Macromolecular Chemistry, Faculty of Science, Charles University in Prague, Prague, Czech Republic Received November 8, 2016 Accepted February 27, 2017 Summary Introduction Transient receptor potential A1 (TRPA1) is an excitatory ion channel that functions as a cellular sensor, detecting a wide The Transient Receptor Potential Ankyrin 1 range of proalgesic agents such as environmental irritants and (TRPA1) channel, originally called ANKTM1 (Story et al. endogenous products of inflammation and oxidative stress. 2003), is an ion channel dominantly expressed in a subset Topical application of TRPA1 agonists produces an acute of nociceptive somatosensory neurons where it acts as nociceptive response through peripheral release of a polymodal sensor for diverse physical and chemical neuropeptides, purines and other transmitters from activated stimuli of extracellular or intracellular origin. This channel sensory nerve endings. This, in turn, further regulates TRPA1 is characterized by a marked activating agent promiscuity activity downstream of G-protein and phospholipase C-coupled towards painful or potentially harming stimuli, including signaling cascades. Despite the important physiological relevance natural and exogenous electrophilic compounds, ultraviolet of such regulation leading to nociceptor sensitization and radiation, oxygen, extreme temperatures, chemical irritants consequent pain hypersensitivity, the specific domains through and endogenous inflammatory mediators (Nilius et al. which TRPA1 undergoes post-translational modifications that 2011, Nilius et al. 2012, Laursen et al. 2014, Zygmunt and affect its activation properties are yet to be determined at Hogestatt 2014, Chen and Hackos 2015, Viana 2016). a molecular level. This review aims at providing an account of Studies using TRPA1 knock-out mice and specific our current knowledge on molecular basis of regulation by antagonists have linked the function of TRPA1 to the neuronal inflammatory signaling pathways that converge on the regulation of temperature perception, inflammation, TRPA1 channel protein and through modification of its specific mechanosensation, pain, itching, but also the homeostatic residues influence the extent to which this channel may balance between the nociceptive and the immune system contribute to pain. (Bandell et al. 2004, Kwan et al. 2006, Viana 2016). In peptidergic nociceptors, TRPA1 is extensively Key words co-expressed with the structurally related vanilloid receptor • • Transient receptor potential ankyrin 1 Bradykinin Structure- subtype 1 channel TRPV1. These two channels • • • function Nociception Post-translational modifications functionally interact and share multiple and overlapping Signaling pathways signaling pathways that lead to their post-translational modifications regulating their activity and thus the Corresponding authors nociceptor excitability (Story et al. 2003, Bandell et al. A. Kádková and V. Vlachová, Department of Cellular 2004, Jordt et al. 2004, Staruschenko et al. 2010). A clear Neurophysiology, Institute of Physiology AS CR, Videnska 1083, demonstration of functional interaction between TRPV1 142 20 Prague 4, Czech Republic. Fax. 420-29644-2488. E-mail: and TRPA1 in the bradykinin signaling pathways was first [email protected] or [email protected] PHYSIOLOGICAL RESEARCH • ISSN 0862-8408 (print) • ISSN 1802-9973 (online) 2017 Institute of Physiology of the Czech Academy of Sciences, Prague, Czech Republic Fax +420 241 062 164, e-mail: [email protected], www.biomed.cas.cz/physiolres 426 Kádková et al. Vol. 66 shown in 2006 when the TRPA1-deficient mice were receptors and phospholipase C-coupled signaling generated (Bautista et al. 2006). The authors demonstrated cascades (Fig. 1). These mediators can directly activate that the magnitude of bradykinin-evoked responses in TRPA1 (Taylor-Clark et al. 2008) or stimulate protein trigeminal neurons from control mice correlated with the kinase A (PKA), protein kinase C (PKC), and size of TRPA1-mediated responses induced by the phospholipase C (PLC) pathways to induce selective agonist allyl isothiocyanate but not with those phosphorylation of TRPA1 (Dai et al. 2007, Wang et al. induced by the specific TRPV1 agonist capsaicin. The 2008a, Andrade et al. 2012, Petho and Reeh 2012). bradykinin-evoked responses were significantly attenuated Phosphorylation, in turn, significantly affects the and the correlation was completely lost in both TRPA1- activation threshold of TRPA1, rendering nociceptors and TRPV1-deficient neurons. more sensitive to noxious stimulation. The putative roles The activation of TRPA1 at nerve terminals of the PKA- and PLC-dependent signaling pathways in results in membrane depolarization mainly due to TRPA1-mediated nociception have recently been very Na+ influx and induces a local calcium influx through the well reviewed by previous authors (Andrade et al. 2012, channel which further releases neuropeptides such as Nilius et al. 2012, Bautista et al. 2013, Zygmunt and calcitonin gene-related peptide (CGRP), substance P, or Hogestatt 2014, Viana 2016). Despite the presumed neurokinin A from large dense-core vesicles via physiological importance of TRPA1 post-translational Ca2+-dependent exocytosis (Gustavsson et al. 2012). The modifications, little is known about the specific domains presence of neuropeptides causes a further amplification and residues through which the TRPA1 protein of nociception, recruitment of immune cells, undergoes conformational changes that affect the vasodilatation and neurogenic inflammation (Geppetti et activation properties of this channel (Voolstra and Huber al. 2008). Accordingly, TRPA1 has been functionally 2014). This review aims at providing an overview of the linked to a number of chronic pain conditions associated currently available results of site-directed mutagenesis with inflammation, including visceral pain, arthritic pain, studies on TRPA1 and summarizes the data on the cancer pain, and migraine. It is generally assumed that functionally important amino acids which are involved in proinflammatory agents released at the site of injury, such TRPA1 activation and may represent the residues through as prostaglandins, bradykinin, serotonin and extracellular which this channel is regulated by phosphorylation or proteases, modulate TRPA1 via G-protein-coupled other post-translational modifications. Fig. 1. Different but interconnected signaling pathways contribute to the sensitization of TRPA1 (adapted from Andrade et al. 2012). The binding of an agonist to the bradykinin receptor 2 (B2) triggers phospholipase Cβ (PLCβ) through Gαq protein. PLCβ cleaves 2+ phosphatidylinositol-4,5-bisphosphate (PIP2) into diacylglycerol (DAG) and inositol triphosphate (IP3). Ca release from the endoplasmic reticulum (ER) induced by binding of IP3 to the inositol triphosphate receptor (IP3R) probably directly sensitizes TRPA1. The activation of protein kinase Cε (PKCε) by DAG results into the binding of the kinase to the anchoring protein AKAP79/150 (AKAP) bound to the C-terminus of TRPV1 and into phosphorylation of TRPV1 which, in turn, sensitizes this channel to an agonist, promoting Ca2+ influx. AKAP79/150 binds also to TRPA1 (Zhang et al. 2008). The B2 receptor can also stimulate the Gαs protein that activates the synthesis of cyclic adenosine monophosphate (cAMP) by adenylate cyclase (AC). The activation of protein kinase A (PKA) by cAMP or the activation of protein kinase C (PKC) by DAG sensitizes TRPA1 (putative phosphorylatable serine/threonine residues are indicated as yellow circles). In addition, PKA can also bind to the AKAP79/150 protein on TRPV1 together with PKCε and the phosphatase calcineurin (PP2B) to either phosphorylate or dephosphorylate the receptor at serine S502, depicted as a yellow circle. 2017 Structural Basis of TRPA1 Regulation 427 Structural characteristics of TRPA1 trypsin, acting on G protein-coupled receptors, stimulate the production of intracellular mediators that directly or All TRP channels share a similar general indirectly activate TRPA1. Oxygen, reactive oxygen topology consisting of four pore-forming subunits, each species and reactive nitrogen species directly activate of which contains six transmembrane segments (S1-S6) TRPA1. Molecular mechanism underlying O2-sensing comprised of a sensor module S1-S4 and an ion involves prolyl hydroxylation of conserved proline P394 conduction pore-forming region S5-S6. Both amino- and within the N-terminal ankyrin repeat 10 and oxidation of carboxyl-terminal domains are located cytoplasmically cysteines C633 and/or C856 (Miyamoto et al. 2009, (Owsianik et al. 2006). Among mammalian Takahashi et al. 2011). TRP channels, TRPA1 is unique in bearing an extensive The origin of the exogenous chemicals cytoplasmic amino terminus (720 of 1119 amino acids), activating TRPA1 is either natural, or synthetic. Pungent comprised of a prominent ankyrin repeat domain (ARD; compounds from cinnamon (cinnamaldehyde), amino acids 1-639) and a linker (amino acids 640-720) horseradish and wasabi (allyl isothiocyanate) or garlic consisting of a β-hairpin loop followed by two α-helices (allicin)