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

Abstract

Transient receptor potential (TRP) channels are polymodal signal detectors that respond to a wide array of physical and chemical stimuli, making them important components of sensory systems in both vertebrate and invertebrate organisms. Mammalian TRPA1 channels are activated by chemically reactive irritants, whereas snake and Drosophila TRPA1 orthologs are preferentially activated by heat. By comparing human and rattlesnake TRPA1 channels, we have identified two portable heat-sensitive modules within the ankyrin repeat-rich aminoterminal cytoplasmic domain of the snake ortholog. Chimeric channel studies further demonstrate that sensitivity to chemical stimuli and modulation by intracellular calcium also localize to the N-terminal ankyrin repeat-rich domain, identifying this region as an integrator of diverse physiological signals that regulate sensory neuron excitability. These findings provide a framework for understanding how restricted changes in TRPA1 sequence account for evolution of physiologically diverse channels, also identifying portable modules that specify thermosensitivity.

Primary afferent (somatosensory) neurons detect a range of physical and chemical stimuli, including temperature, pressure, and noxious irritants (1). The transient receptor potential (TRP) channel family has been shown to play a predominant role in these processes, particularly in regard to thermosensitivity and chemosensitivity (2–5). TRPA1, otherwise known as the “wasabi receptor,” plays a key role in somatosensation in evolutionarily diverse phyla, including vertebrate and invertebrate species. Mammalian TRPA1 is expressed by primary afferent sensory neurons of the pain pathway, where it functions as a sensor of environmental and endogenous chemical irritants, such as allyl isothiocyanate (AITC), acrolein, and 4-hydroxynonena, and contributes to cellular mechanisms underlying inflammatory pain (6–9).

TRPA1 channels show species-specific functional variation to suit their physiological roles. For example, snakes are unique among vertebrates in that their TRPA1 channels are heat-sensitive, which some species (rattlesnakes, boas, and pythons) have exploited to detect infrared radiation (10). Similarly, insect TRPA1 channels are heat-sensitive and contribute to thermal avoidance behaviors (11–14). In these cases, however, thermosensitivity comes at the expense of chemosensitivity, such that AITC and other chemical irritants still activate these channels but with reduced potency compared with mammalian orthologs (10). Despite clear physiological differences between snake and mammalian TRPA1 channels, they share significant amino acid identity (56%), providing a unique opportunity to exploit sequence comparisons and domain swaps to pinpoint structural elements associated with stimulus detection and/or gating. Delineating elements that contribute to TRPA1 function will provide insights into the evolutionary process whereby structural changes lead to adaptive variations in physiological function. Here, we ask whether discrete structural motifs specify physical and chemical stimulus detection.

TRPA1 is a nonselective cation channel consisting of four subunits, each of which contains six transmembrane segments flanked by cytoplasmic N and C termini (15, 16). Ankyrin repeats (ARs) are ∼33-residue protein motifs consisting of two α-helices connected by a β-turn. ARs occur in tandem arrangements to form elongated domains [AR domains (ARDs)] critical for many physiological processes (17, 18). Although many TRP channel family members contain in the range of 3–6 ARs within the N-terminal region, TRPA1 is distinguished by having an unusually large number of such repeats (16–17 ARs) (19). Despite their prevalence among TRP channel subtypes, little is known about functional roles for ARs. In the case of the capsaicin receptor, TRPV1, another heat-sensitive vertebrate TRP channel (20), crystallographic and physiological studies suggest a role for AR in the binding of cytoplasmic ATP and calmodulin to regulate channel desensitization (17, 21). 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.

Nevertheless, the cytoplasmic N terminus is believed to play an important role in gating because many TRPA1 agonists are strong electrophiles that activate the channel through reversible covalent modification of cysteine and lysine residues within this domain (25, 26). Specifically, these highly conserved residues are located in the N-terminal region that connects the AR to the first putative transmembrane segment. How modification of these residues promotes channel opening has not been determined, but their location suggests that conformational changes affecting the N terminus are key to TRPA1 gating mechanisms. In addition to direct activation by chemical irritants, TRPA1 can function as a “receptor-operated” channel that is activated by ATP, bradykinin, or other proalgesic agents that stimulate phospholipase C signaling pathways to promote release of calcium from intracellular stores, with consequent activation of TRPA1 (6, 7). Therefore, understanding calcium-dependent modulation of TRPA1 is relevant to elucidating mechanisms underlying pain hypersensitivity and for designing novel therapeutic agents that target this channel.

Another major question in this field concerns the structural basis of thermosensitivity. Various TRP channel regions have been proposed to specify thermosensitivity or thermal activation thresholds; these include C-terminal cytoplasmic, outer pore, and turret domains. For example, swapping C-terminal domains between rat TRPV1 and TRPM8 channels has been reported to switch heat and cold sensitivity (27, 28). Deletions within the TRPV1 C terminus have been shown to alter thermal threshold (29), and substitutions or point mutations within the turret or outer pore domain of TRPV1 have been shown to alter heat sensitivity (30, 31). Recently, the linker region of TRPV1 connecting the pore-forming transmembrane core to the AR-containing N terminus has been suggested to specify heat sensitivity of the channel (32). However, a consensus view has yet to emerge regarding the mechanism(s) and region(s) that determine TRP channel thermosensitivity, and whether such domains are truly modality-specific regulators of channel function. Further insights to these issues could be provided by a system in which faithful transference of thermosensitivity to a temperature-insensitive channel is achieved.

The analysis of chimeric channels represents a classic approach to addressing such questions in cases where pharmacological or physiological properties can be transplanted among closely related family members. Unfortunately, TRP channels from different families show relatively weak protein sequence conservation; thus, most chimeras, including those formed among closely related homologs, are often nonfunctional and of limited value. However, sensory receptors are subject to rapid evolutionary adaptation that drives species-specific differences in channel properties. Thus, chimeras generated among highly conserved species orthologs more often render channels that are well-behaved and can be analyzed to identify functionally relevant protein domains. Indeed, we have successfully exploited this approach to probe structure-function relationships of sensory TRP channels. For example, the analysis of avian-mammalian or amphibian-mammalian TRPV1 chimeras led to the identification of amino acids that specify sensitivity to capsaicin or peptide toxins, respectively (33, 34). A similar approach revealed sites within the cold receptor, TRPM8 (35), that determine sensitivity to cooling compounds (36). Although this method has greatly facilitated the analysis of chemical sensitivity, it has met with limited success in delineating regions that account for thermal sensitivity because TRPV1 and TRPM8 from different species retain temperature sensitivity as a highly conserved function, save for differences in precise temperature activation threshold (34, 37). Thus, cross-species chimeric channels cannot be used to transfer heat or cold sensitivity cleanly to a thermoinsensitive ortholog. Snake and mammalian TRPA1 channels provide such an opportunity; thus, we have exploited this functional variation to delineate regions specifying thermal and chemical sensitivity.

Here, we show that the aminoterminal cytoplasmic region of TRPA1 is a portable unit that specifies stimulus detection, including sensitivity to environmental stimuli (heat and chemical irritants) as well as cellular messengers (cytoplasmic calcium). Our data support a bimodal arrangement within the N-terminal ARs in which thermal and chemical sensitivity are conferred by two independent zones, one of which includes determinants for calcium sensitivity. These findings suggest that the TRPA1 N terminus functions as a detector and integrator of physiological stimuli that regulate channel gating and excitation of somatosensory nerve fibers.

Results

Thermal Response of Snake TRPA1 Is Specified by a Portable N-Terminal Domain.

In initial experiments, we generated chimeras between two closely related snake species, rattlesnake (Crotalus atrox) and rat snake (Elaphe obsoleta lindheimeri), whose TRPA1 channels share 81% identity but exhibit substantially different thermal activation thresholds (28 °C and 38 °C, respectively) and sensitivity to electrophilic agonists (low and high, respectively). We found that exchanging the whole aminoterminal cytoplasmic domain (chimeras 1 and 2, ∼725–726 amino acids) was sufficient to switch both temperature activation threshold and relative chemical sensitivity precisely between these two species orthologs (Fig. 1). This result suggests that the N-terminal cytoplasmic domain specifies both thermal and chemical response properties of the channel. To determine whether this is more generally applicable to vertebrate TRPA1 orthologs, we extended this approach to the analysis of chimeric snake-mammalian channels.

Fig. 1.

N-terminal domain of snake TRPA1 determines thermal and chemical sensitivity. (A) Schematic representation of chimeras between rattlesnake and rat snake TRPA1 channels. (B) Heat-evoked response profiles of rattlesnake and rat snake chimeras expressed in oocytes. Data represent mean ± SD. I/I, the current at each temperature/the current at 45 °C. (C–F) Current-voltage relationships determined by two-electrode voltage clamp recording from oocytes expressing rattlesnake, rat snake, and chimeras challenged with heat (42 °C, red trace) or AITC (1 mM, orange trace).

Human and rattlesnake TRPA1 channels show even greater functional disparity in that the former is completely insensitive to heat; thus, we asked whether this difference can also be mapped to a discrete domain. Remarkably, a human TRPA1 chimera containing a portion of the rattlesnake N terminus (chimera 3, 1–423 amino acids containing the first 10 ARs) gained substantial heat sensitivity (Fig. 2 A and B and Fig. S1A) while retaining robust AITC sensitivity characteristic of the human channel (see below). At the same time, heat-evoked currents produced by the human-rattlesnake chimera 3 were attenuated by HC 030031, a selective antagonist that also abolishes responses to chemical agonists (38), or by the broad-spectrum pore-blocker Ruthenium Red, demonstrating that the observed responses were mediated through heterologously expressed chimeric channels (Fig. 2C and Fig. S1B, respectively). These results further support the hypothesis that the N-terminal cytoplasmic domain specifies heat sensitivity that is both necessary and sufficient to confer thermosensitivity to a related, albeit heat-insensitive, ortholog. Furthermore, elimination of key electrophile modification sites (3 cysteines and 1 lysine) within the N-terminal linker region of chimera 3 abolished responses to AITC but not to heat (Fig. 2D), suggesting that activation by thermal and chemical stimuli requires different structural elements within this N-terminal region. Taken together, these data demonstrate that thermal and chemical sensitivities are specified by a portable N-terminal AR-rich domains of the channel.

Fig. 2.

Portability of the heat-sensitive domain. (A) Schematic representation of a chimera between rattlesnake and human TRPA1 channels. (B) Heat-evoked response profile of human-rattlesnake chimera 3 expressed in oocytes. A single heat-evoked response trace from the heat-insensitive human TRPA1 is shown (gray) for comparison. Data represent mean ± SD. I/I, the current at each temperature/the current at 42 °C. (C) Heat-evoked currents of chimera 3 were inhibited with the selective TRPA1 antagonist HC 030031 (20 μM). (D) Chimera 3 lacking electrophile modification sites (3C-A/K-Q) is not sensitive to AITC (1 mM, orange trace) but retains heat (42 °C, red trace) sensitivity. The 3C-A/K-Q construct corresponds to a quadruple human TRPA1 mutant in which four sites (C621A, C641A, C665A, and K710Q) essential for AITC activation have been altered to nonreactive amino acids.

Mammalian TRPA1 channels can also be activated by intracellular calcium (8, 22–24), 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. S1C), consistent with intrinsic sensitivity of the chimera to heat rather than an indirect action via intracellular calcium. Importantly, exchange of equivalent regions between two heat-insensitive TRPA1 orthologs (human and zebrafish) (10) did not produce a heat-sensitive channel (chimera 4, Fig. S1D), showing that thermal sensitivity is not simply a default state associated with a chimeric TRPA1 protein.

To identify a minimal portable module that confers thermal sensitivity, we constructed an extensive series of human-rattlesnake TRPA1 chimeras (Fig. S2) and measured heat-evoked currents in Xenopus oocytes expressing these channels. In this way, we identified two adjacent regions from the rattlesnake channel (chimera 16, AR3–8 and chimera 29, AR10–15), each of which was sufficient to confer heat sensitivity to the human ortholog (Fig. 3 A–C and Fig. S3). Thus, rattlesnake TRPA1 contains two independent and portable heat-sensitive regions, each consisting of six ARs. Importantly, transfer of other ARs (e.g., AR1–6, AR4–9, or AR6–11, corresponding to chimeras 8, 18, and 19, respectively) did not confer heat sensitivity, demonstrating a requirement for a specific ARs rather than just a stretch of minimum size. Moreover, although chimeras containing AR1–10, AR3–8, or AR10–15 (chimeras 3, 16, and 29, respectively) showed robust heat-evoked currents, their average temperature coefficients of activation (Q₁₀s) were different (3.9 ± 0.7, 4.1 ± 0.8, and 10 ± 1.6, respectively) (Fig. 3C and Fig. S1A). Thus, although each module is sufficient to confer heat sensitivity, both likely contribute to the overall thermal response properties of native rattlesnake TRPA1 channel (Q₁₀ = 13.9) (10).

Fig. 3.

Rattlesnake and Drosophila TRPA1 channels contain portable heat-sensitive domains. (A) Chimeras generated within ARs of human (yellow), rattlesnake (blue), or Drosophila (red) TRPA1 channels. All chimeras shown responded to a saturating concentration (1 mM) of AITC. ND, Q₁₀ not determined. (B) Thermal response profiles of chimera 16 and chimera 29 show the presence of two heat-sensitive modules in the rattlesnake TRPA1, each consisting of six specific ARs. I/I (38 °C), the current at each temperature/the current at 38 °C; I/I (45 °C), the current at each temperature/the current at 45 °C. (C) Representative Arrhenius plots show thermal thresholds and Q₁₀ values for baseline and evoked responses for chimera 16 (4.7) and chimera 29 (11.2). Data represent mean ± SD. (D) Comparison of heat- and AITC-evoked response profiles of WT Drosophila TRPA1, chimera 40, and chimera 41 highlights the presence of one heat-sensitive module in the Drosophila channel corresponding to a region containing AR10–15. I/I (max), the current at each temperature/the current reached at the maximal temperature. Chimera 40 contains residues 400–612 from Drosophila TRPA1, and chimera 41 contains residues 1–372 (holding potential = −80 mV, n ≥ 6).

Drosophila TRPA1 (dTRPA1) is also a heat-sensitive channel; thus, we asked whether transfer of the cognate ARs to human TRPA1 would similarly confer temperature sensitivity. In general, human-fly TRPA1 chimeras exhibited high basal activity, likely reflecting the low sequence identity (<30%) between these two orthologs. Nonetheless, we found that substitution of AR10–15 of the human channel with that of the fly ortholog produced a channel (chimera 40) in which robust heat-evoked responses were clearly observed over baseline current (Fig. 3 A and D). Interestingly, this chimera exhibited reduced AITC sensitivity resembling that of WT dTRPA1. However, transfer of a region containing AR1–8 produced channels (chimera 41) with the converse phenotype, namely, meager heat sensitivity and robust AITC sensitivity compared with chimera 40 or WT dTRPA1. These results demonstrate portability of a heat-sensitive module (AR10–15) that is functionally conserved between flies and snakes. Interestingly, this region represents the most highly conserved area (41% identity) when comparing the N-terminal AR-rich domain of fly and snake TRPA1.

AR-Containing Region of the N Terminus Modulates Chemical Sensitivity.

Rattlesnake and rat snake channels exhibit markedly different sensitivities to AITC (low and high, respectively). Indeed, swapping their whole N-terminal domains (chimeras 1 and 2) switched not only heat but AITC sensitivity (Fig. 1 C–F), focusing our attention on this region of the channel as a major determinant of chemical responsiveness. The cysteines and lysine critical for AITC activation are conserved in all vertebrate TRPA1 channels (10); thus, other as yet unidentified regions must account for the difference in chemical sensitivity. Substantial differences in chemical sensitivity are also observed when comparing human and rattlesnake channels. Thus, whereas human TRPA1 responds to AITC with a half-maximum effective concentration (EC₅₀) of 62 μM, the rattlesnake ortholog is substantially less sensitive, with an EC₅₀ of >2 mM. Additionally, chemical agonists activate the rattlesnake channel to ≤10% of maximal heat-evoked responses (Fig. 4 C and G and Fig. S4A), further illustrating its preferential sensitivity to heat.

Fig. 4.

Contribution of the ARs to chemical sensitivity. (A) Diagram depicting chimeras generated between human and rattlesnake TRPA1. (B–F) Representative current-voltage relationships determined by two-electrode voltage clamp recording from oocytes expressing human and rattlesnake chimeras following exposure to AITC (1 mM, orange trace) or heat (42 °C, red trace). (G) AITC dose–response curves for human, rattlesnake, chimera 3, and chimera 32 TRPA1 channels expressed in oocytes (n = 5 for each data point). Points were obtained by measuring response to a 1-min pulse of test dose at +80 mV and normalized to the maximum response elicited by 6 mM AITC. The dose–response curve was fit to the data. EC₅₀s for human (62 μM), rattlesnake (>2 mM), chimera 3 (36 μM), and chimera 32 (>700 μM) are shown.

We next used our series of human-rattlesnake chimeras to dissect this N-terminal locus further. Indeed, we found that replacement of the first 10 ARs in the human channel with those of rattlesnake (chimera 3) retained substantial chemical sensitivity (Fig. 4 A, D, and G) resembling that of the parental human ortholog. The inverse chimera 32 (in which the first 10 ARs from rattlesnake are replaced with the first 10 human ARs) also exhibited AITC sensitivity, as reflected by the large rightward shift of the dose–response profile (Fig. 4G). Moreover, the rate of AITC activation for chimera 32 was substantially slower compared with human or chimera 3 channels (Fig. S4B). Finally, we examined the importance of the linker segment connecting the ARD to the transmembrane core, which contains conserved cysteine and lysine modification sites. When this region of the rattlesnake channel was replaced with that from the human channel (chimera 33), we observed no improvement in AITC sensitivity (Fig. 4 A and F and Fig. S4A). We reached a similar conclusion by examining analogous chimeras between rattlesnake and rat snake channels (Fig. S4C). Taken together, these findings suggest that the AR-containing region of the N terminus (distinct from the conserved downstream cysteines or lysine) tunes chemical sensitivity. Furthermore, although chemical sensitivity can be conferred by distinct ARs, those adjacent to the linker region (AR11–16) appear to be most critical in setting AITC sensitivity.

Calcium-Dependent Desensitization Is Specified by AR11 and Adjacent ARs.

Several studies have shown that calcium potentiates TRPA1 activity, followed by a process of calcium-dependent desensitization or inactivation (8, 22–24). 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. S5A), as it was shown for human TRPA1 (8), whereas only the human channel exhibits robust calcium-dependent desensitization (with a time constant of 9 s; Fig. 5 A–C), consistent with the notion that calcium-mediated potentiation and desensitization represent independent modulatory processes (24). These differential properties of human and snake channels enabled us to focus in on a region required for calcium-dependent desensitization.

Fig. 5.

Calcium-dependent desensitization is specified by AR11 and adjacent ARs. (A) Diagram depicting chimeras generated between human and rattlesnake TRPA1. The right column denotes desensitization to 2 mM Ca²⁺. ND, no Ca²⁺-evoked desensitization. (B) Representative traces showing peak AITC-evoked currents with (Left) or without (Right) extracellular Ca²⁺ (2 mM) from oocytes expressing human TRPA1. (C) Representative trace showing peak heat-evoked current in the presence of extracellular Ca²⁺ (2 mM) from an oocyte expressing rattlesnake TRPA1, recorded as above. (D–F) Representative traces of chimera 23 (AR7–11), chimera 38 (AR11–14), and chimera 37 (AR11), respectively. Representative traces of peak AITC or heat-evoked currents were extracted from voltage-clamp ramp protocols at ±80 mV. Chimera 37 contains residues 416–457 from rattlesnake, chimera 38 contains residues 416–563, and chimera 39 contains residues 460–563.

Specifically, we found that human-rattlesnake chimeras 23 and 38 containing rattlesnake AR7–11 or AR11–14, respectively, did not undergo calcium-dependent desensitization (Fig. 5 D and E). A more restricted chimera (construct 37) containing just rattlesnake AR11 exhibited clear calcium-dependent desensitization, albeit with a reduced rate compared with the WT human channel (τ = 45 ± 12 s vs. 9.0 ± 1.7 s, respectively) (Fig. 5F). Moreover, chimera 23 resembled WT rattlesnake TRPA1 in regard to calcium-dependent desensitization (Fig. 5D), consistent with previous mutagenesis data (24) suggesting that a postulated EF hand-like motif located in AR12 is not, by itself, necessary or sufficient to mediate this process. Taken together, these and other results (Fig. S5 B–E) suggest that calcium-dependent desensitization is specified by an AR cluster centered around AR11.

To determine whether calcium binds directly to negatively charged residues present in AR7–11 and/or AR11–14 of human TRPA1 (but absent from the rattlesnake channel), we converted all such aspartate residues to alanine (8 in all), expressed these mutants in oocytes, and measured their extent of calcium-dependent desensitization. We found that these mutations, when introduced singly or together, had minimal effect on AITC-evoked activation or desensitization (Fig. S6), ruling out a simple model in which calcium binds directly to negative charges differentially present in this region of the human channel. Irrespective of the calcium-binding site, our data identify AR7–11 and/or AR11–14 of human TRPA1 as an important determinant of calcium regulation.

Discussion

We show that the N-terminal domain of TRPA1 is a portable unit that specifies stimulus detection, including sensitivity to heat, chemical irritants, and cytoplasmic calcium. Other domains of thermosensitive TRP channels (e.g., C terminus, pore region) have been implicated in stimulus detection or threshold modulation (27, 29–31, 39, 40), but our findings with TRPA1 represent, to our knowledge, a previously undescribed functional demonstration that temperature sensitivity can be faithfully and precisely transferred to a temperature-insensitive but otherwise functional ortholog. Mammalian TRPA1 has been suggested to function as a cold-activated channel (41, 42); thus, one could argue that we have interchanged properties between two thermally sensitive channel orthologs, albeit in opposite directions (cold-sensitive vs. heat-sensitive). However, under our experimental conditions, oocytes expressing human TRPA1 showed no cold-evoked currents with cooling down to 10–12 °C, consistent with recent reports that cold produces slight or no activation of transfected cells or sensory neurons expressing TRPA1 (43, 44).

Aminoterminal ARs represent a structural hallmark of many members of the extended family of TRP channels, but their functional roles remain largely unknown. Recent studies of the capsaicin receptor, TRPV1, suggest that the N-terminal ARD constitutes a binding site for cytoplasmic regulatory factors, such as ATP and calmodulin (17, 45), consistent with our findings that the TRPA1 ARD contributes to channel modulation by environmental and endogenous agents.

TRPA1 Amino Terminus as a Modular Polymodal Signal Detector.

Several TRP channels act as polymodal signal detectors (5, 46), but TRPA1 is unique in that different species use this channel for distinct physiological purposes, generally favoring one modality (thermal or chemical) over the other (10, 12). This may reflect evolutionary pressure to reduce background noise from environmental irritants in organisms in which the channel is used primarily for thermoreception, and vice versa. Organization of the N-terminal AR region into functionally redundant and independent modular domains (Fig. 6) may facilitate evolutionary tuning of thermal or chemical sensitivity while retaining a high degree of sequence similarity across species. For example, ancient and modern infrared-sensing snakes (boas/pythons and pit vipers, respectively) likely gained heat sensitivity independently through changes to one or both modules of non–infrared-sensing ancestors. Furthermore, the bimodal nature of the TRPA1 amino terminus AR may account for modality competition. Thus, although it is possible to construct channels that exhibit both heat and chemical sensitivity, this may come at some expense to the detection of one or both stimuli, as suggested by a decreased Q₁₀ for human-rattlesnake chimeras (Fig. 3C) or properties of native rat snake TRPA1, which exhibits low heat and moderate AITC sensitivity (10). Indeed, this inverse relationship between thermosensitivity and chemosensitivity also pertains to invertebrate (insect) orthologs (10, 12), suggesting an ancient arrangement in TRPA1 structures that has been maintained over a long evolutionary period and in the face of extensive drift in primary amino acid sequence (Drosophila and human TRPA1 share <30% sequence identity). Unlike TRPA1, TRPV1 is heat-sensitive in all vertebrate species thus far examined. As such, thermosensation appears to be its core physiological function, whereas chemosensitivity plays primarily a modulatory role whereby detection of endogenous inflammatory agents enhances the channel's sensitivity to heat to produce thermal hyperalgesia (1). Interestingly, and in contrast to TRPA1, these factors target numerous sites on the channel, including the transmembrane core, outer pore region, and C terminus, illustrating two different strategies for tuning TRP channel stimulus sensitivity.

Fig. 6.

Schematic representation of the TRPA1 N-terminal region. ARs in red and light red specify heat sensitivity for the primary and enhancer modules, respectively; stars denote location of cysteine residues required for activation by electrophiles.

Structural Framework for Bimodality in Vertebrate TRPA1.

Our extensive chimeric analysis indicates that there are two spatially distinct AR modules (AR3–8 and AR10–15) that are each capable of conferring heat or robust chemical sensitivity in snake and mammalian TRPA1, respectively. Biochemical analyses of canonical AR proteins provide a molecular framework for understanding how large stretches of AR can exhibit distinct functional properties. For example, a 12-AR region from the ankyrinR protein has been shown to consist of two modules having distinct thermodynamic properties, such that one module exhibits reduced stability compared with the other (47). This arrangement provides directionality and cooperativity in the kinetics of protein unfolding when this AR model peptide is challenged with chaotropic agents; the aminoterminal ARs unravel first as an intermediate to subsequent unfolding of more distal C-terminal ARs. This is reminiscent of our finding that the primary module (AR10–15) predominates in regard to specifying stimulus sensitivity, as illustrated by relative Q₁₀ for heat or EC₅₀ for chemical irritants (Figs. 3C and 4G). This suggests a model in which two independent AR modules establish a combinatorial code in which the one closest to the transmembrane core establishes stimulus sensitivity (thermal or chemical), whereas the one closest to the N terminus serves as an enhancer (Fig. 6). In the case of flies, our initial chimeric analysis suggests a more rudimentary arrangement in which AR10–15 represents the only module conferring both heat and AITC sensitivity. Later in evolution, snakes may have acquired the enhancer module to augment thermosensitivity. With regard to the actual gating mechanism, thermal or chemical stimuli could induce conformational changes in the ARD that directly gate the channel, or they could alter interaction with another cellular factor, consequently triggering channel opening. Distinguishing between these possibilities will require biophysical studies of TRPA1 function in fully defined reconstituted systems.

Interestingly, our chimeric analysis also suggests that the predominant locus of calcium-dependent desensitization (AR11) overlaps substantially with the primary AR module. This is consistent with our findings that robust chemical sensitivity and calcium-dependent desensitization are functionally linked in the human TRPA1 channel, whereas neither heat- nor AITC-evoked responses show appreciable desensitization in cells expressing snake channels. Such an arrangement is perhaps ideal for evolutionary coadaptation of traits. The capsaicin receptor, TRPV1, contains six ARs within its cytoplasmic N terminus, equivalent in size to a single module as defined by our analysis in TRPA1 (17). The implication is that an AR consisting of six repeats represents a minimal functional unit in heat-sensitive TRP channels. Does this also apply to other thermosensitive TRP channel family members? A recent study (32) of chimeras between TRPV1 and the high-threshold heat-sensitive channel TRPV2 concluded that heat sensitivity is specified by the linker region connecting the ARD to the transmembrane core. Moreover, the two ARs (AR5 and AR6) most adjacent to this linker region were found to enhance heat sensitivity. Our observations diverge with this recent study, since our data suggest that in the case of TRPA1, the linker region does not specify heat sensitivity per se, but rather the ARD predominate in this regard, similar to AR5 and 6 of TRV1.

Taken together, our findings show that the TRPA1 cytoplasmic N terminus serves as a key integration site for multiple physiological signals that activate or modulate the channel. Our function-based studies identify regions and potential domains that specify sensitivity to thermal and chemical stimuli, but a deep mechanistic understanding of how these modules promote TRPA1 gating will require further biophysical and structural analysis of the N-terminal domain.

Materials and Methods

Mutagenesis and Chimera Constructs.

Point mutations and chimeras of human, rattlesnake, rat snake, and zebrafish TRPA1 were generated using standard overlapping PCR or site-directed mutagenesis-based methods (catalog no. 250519; Stratagene). In all cases, conserved amino acid sequences were used for chimeric junctions. For oocyte expression, genes were subcloned into the combined mammalian/oocyte expression vector pMO. All constructs were verified by DNA sequencing.

Oocyte Electrophysiology.

cRNA transcripts were synthesized and injected into Xenopus laevis oocytes using Ambion mMessage machine kits according to the manufacturer's protocol. Surgically extracted oocytes (Nasco) were cultured and analyzed 1–3 d postinjection using a two-electrode voltage clamp as previously described by Jordt et al. (8). Briefly, oocytes were injected with 0.05–5 ng per cell of RNA and whole-cell currents were measured after 24–72 h using a Geneclamp 500 amplifier (Axon Instruments, Inc.). Microelectrodes were pulled from borosilicate glass capillary tubes to obtain resistances of 0.5 MΩ. Bath solution contained 10 mM Hepes, 120 mM NaCl, 2 mM KCl, 0.2 mM EGTA, 2 mM MgCl₂, and 1 mM CaCl₂ to a final pH of 7.4. For experiments in the absence of Ca²⁺, we used the same bath solution containing 5 mM EGTA. For BAPTA/acetoxymethyl ester (AM) experiments, currents were induced by increasing the temperature of bath solution after a 3-h incubation of oocytes with 10–50 μM BAPTA/AM (Sigma). BAPTA/AM permeates into the cytoplasm and, after hydrolysis, chelates Ca²⁺ ions. Data were analyzed using pCLAMP10 software (Molecular Devices).

Determination of Thermal Threshold.

Response at each temperature was normalized to the response at the maximal applied temperature (holding potential = −80 mV, n ≥ 8). All data are shown as mean ± SD. Temperature thresholds represent the point of intersection between linear fits to baseline and the steepest component of the Arrhenius profile. Values are derived from averages of individual curves (n ≥ 10). Arrhenius curves were obtained by plotting the current on a log-scale against the reciprocal of the absolute temperature. Q₁₀ was used to characterize temperature dependence of ionic currents as calculated by the following equation:

where R₂ is the current at the higher temperature, T₂, and R₁ is the current at the lower temperature, T₁ (48).

Determination of Time Constant.

Desensitization time constants were fitted to a single exponential function from peaks of AITC-evoked currents as calculated using Origin 7.1 (OriginLab) with the following equation: y = A1⋅exp(−x/τ1) + ψ0.