Transient Receptor Potential Channel A1 Is Directly Gated by Calcium Ions

Abstract

Members of the superfamily of transient receptor potential (TRP) channels are proposed to play important roles in sensory physiology. As an excitatory ion channel TRPA1 is robustly activated by pungent irritants in mustard and garlic and is suggested to mediate the inflammatory actions of environmental irritants and proalgesic agents. Here, we demonstrate that, in addition to pungent natural compounds, Ca 2؉ directly gates heterologously expressed TRPA1 in whole-cell and excised-patch recordings with an apparent EC 50 of 905 nM. Pharmacological experiments and site-directed mutagenesis indicate that the N-terminal EFhand calcium-binding domain of the channel is involved in Ca 2؉ -dependent activation. Furthermore, we determine Ca 2؉ as prerequisite for icilin activity on TRPA1. The transient receptor potential channel A1 (TRPA1)2 is a member of the superfamily of TRP channels. In mammals, A1 forms its own subfamily and is distinguished from other TRP channels by the presence of ϳ14 ankyrin repeats in its N terminus (1). TRPA1 was initially described as a cold sensitive nonselective cation channel (2), but it also functions as a ligand-gated channel in heterologous expression systems and sensory neurons (3). The pungent ingredients in mustard (allyl isothiocyanate, AITC) and garlic (allicin) robustly activate TRPA1 currents (4 -6). In addition, TRPA1 appears to be regulated by phospholipase C (PLC)-coupled receptors, suggesting that channel opening can be mediated by second messengers (4,7,8).TRPA1 expression was first described in a subset of sensory neurons of dorsal root and trigeminal ganglia that contribute to nociception and co-express calcitonin gene related peptide, substance P and TRPV1 (2, 4). Recent studies on TRPA1 deficient mice support a role of the channel in inflammatory pain and sensation of noxious cold (8,9). A model suggests TRPA1 activation by bradykinin, a potent algogenic substance released due to tissue injury and inflammation, in two possible ways: through PLC-mediated increases in intracellular Ca 2ϩ or other metabolites (e.g. diacylglycerol) and via Ca 2ϩ influx through TRPV1 (9). Whether an increase in intracellular Ca 2ϩ is sufficient to activate TRPA1 is still debated (4, 7), but several findings indicate a role of Ca 2ϩ on TRPA1 function. It was shown that extracellular Ca 2ϩ enhances the current rate and magnitude of AITC-induced currents (4, 10). Furthermore, Ca 2ϩ is thought to be responsible for fast channel closure (10). In addition, single channel recordings of heterologously expressed TRPA1 revealed an AITC-induced conductance, which is reduced in the presence of Ca 2ϩ (10). Together these reports emphasize the importance of Ca 2ϩ for TRPA1 function. Very recently the existence of a putative EF-hand calciumbinding domain (EF-hand CBD) at the N terminus of TRPA1 was reported (11). The EF-hand CBD is the most common motif among Ca 2ϩ -binding sites of a large number of Ca 2ϩ -interacting proteins (12). The classical EF-hand is a helix-loophelix motif that coordinates the Ca 2ϩ...

Cytoplasmic ankyrin repeats of transient receptor potential A1 (TRPA1) dictate sensitivity to thermal and chemical stimuli

“…Taken together, these data demonstrate that thermal and chemical sensitivities are specified by a portable N-terminal ARrich domains of the channel. Mammalian TRPA1 channels can also be activated by intracellular calcium (8,(22)(23)(24), and we therefore asked whether heat-evoked responses of the human-rattlesnake chimera 3 were still observed when oocytes were loaded with the calcium chelator 1,2-bis(o aminophenoxy) ethane-N,N,N′,N′-tetraacetic acid (BAPTA) and perfused with calcium-free medium. Chelation of cytoplasmic calcium did not eliminate heat-evoked currents (Fig.…”

“…Several studies have shown that calcium potentiates TRPA1 activity, followed by a process of calcium-dependent desensitization or inactivation (8,(22)(23)(24). Despite the potential relevance of these phenomena to nociceptor excitability and inflammatory pain (7, 16), mechanisms underlying calcium-dependent TRPA1 modulation remain controversial, with some groups favoring direct binding of calcium to an EF hand-like motif within the N-terminal domain (22,23) and others advocating a calmodulin-or EF hand-independent process of unknown nature (24). Interestingly, we found that rattlesnake TRPA1 channels show calcium-dependent potentiation (52 ± 6%, n = 3; Fig.…”

“…Molecular dynamic simulations of the extended AR of TRPA1 have led to the hypothesis that this region of the channel functions as a molecular spring, perhaps tethering the channel to the cytoskeletal matrix as part of a mechanical gating mechanism (18). Moreover, mutagenesis studies suggest that cytoplasmic calcium interacts with residues within the AR of TRPA1 to regulate channel function; however, the mechanism remains controversial, with some groups favoring a direct binding of calcium to an EF hand-like motif (22,23), and others suggesting an indirect calmodulin-independent mechanism of an unknown nature (24). Aside from these examples, relatively little is known about the contribution of ARs to TRP channel function.…”

Pinacidil, a Katp channel opener, identified as a novel agonist for TRPA1

“…It has been reported that TRPA1 is often desensitized in the presence of extracellular Ca 2+ [10,30]. It has even been reported that calcium ions alone can directly gate heterologously expressed TRPA1 [31,32]. Therefore, many researchers choose to use a calcium-free solution in the patch clamp experiment to avoid desensitization.…”

“…Therefore, many researchers choose to use a calcium-free solution in the patch clamp experiment to avoid desensitization. However, some reported TRPA1 agonists such as icilin, which uses a different activation mechanism to other TRPA1 agonists, display a requirement for calcium ions to elicit an agonist response [31,33]. Calcium ions were shown to potentiate the agonist-mediated activation of TRPA1 without changing the property of the agonists.…”