article published online: 5 January 2014 | doi: 10.1038/nchembio.1428

Opening of an alternative ion permeation pathway in a nociceptor TRP channel

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Joris Vriens1,2*, Katharina Held1–3, Annelies Janssens1,3, Balázs István Tóth1,3, Sara Kerselaers1, Bernd Nilius1, Rudi Vennekens1 & Thomas Voets1* Sensory neurons detect chemical stimuli through projections in the skin and mucosa, where several transient receptor potential (TRP) channels act as primary chemosensors. TRP channels are tetramers, and it is generally accepted that binding of ligands causes the opening of a single central cation-conducting pore. Contrary to this view, we here provide evidence for a second permeation pathway in the TRP channel TRPM3, which can be gated by combined application of endogenous neurosteroids and exogenous chemicals such as clotrimazole or several structurally related drugs. This alternative pathway is preserved ­following desensitization, blockade, mutagenesis and chemical modification of the central pore and enables massive Na+ influx at negative voltages. Opening of this alternative pathway can enhance excitation of sensory neurons and thereby exacerbate TRPM3-dependent pain. Our findings indicate that a single sensory TRP channel can encompass two distinct ionotropic chemoreceptors, which may have important ramifications for TRP channel function and pharmacology.

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RP channels form a diverse group of cation channels involved in various physiological processes, including stimulus detection in the sensory system, transcellular cation transport and electro­genesis1. Each TRP subunit contains six transmembrane domains (S1–S6), and four TRP subunits form a tetrameric cation channel2,3. This architecture is shared with other members of the superfamily of cation channels with 24 transmembrane domains, which also includes voltage-gated K+, Na+ and Ca2+ channels; calcium-activated potassium channels; and cyclic nucleotide–gated and hyperpolarization-activated cyclic nucleotide–modulated channels4. It is generally accepted that, in members of this superfamily of cation channels, activating stimuli lead to gating of a single, central cation-conducting pore formed by S5, S6 and the interconnecting pore loop3. Nevertheless, it has recently been demonstrated that specific mutations in S4 can unmask an alternative pathway for the flux of cations in voltage-gated cation channels5,6. The resulting current, which has been termed the ‘omega current’ or ‘gating pore current’, flows through a pore that is normally occupied by voltage-sensing arginine residues5,7,8. Whether such an alternative ion permeation pathway can be uncovered in TRP channels is currently unknown. TRPM3 is a nonselective cation channel in the melastatin subfamily of TRP channels3. It is activated by ligands such as nifedipine (Nif) and the neurosteroid pregnenolone sulfate (PS) as well as by heat and membrane depolarization9,10. TRPM3 is highly expressed in nociceptor neurons, where it has a decisive role in the nocifensive response to PS and heat and in the development of heat hyperalgesia during inflammation10. Considering TRPM3 a promising target for new analgesic treatments, we tested the ability of small molecules to modulate TRPM3 activity. Notably, we discovered that specific ligand combinations induced activation of a new, TRPM3-dependent, inwardly rectifying current component. Our results suggest that this current occurs via an alternative permeation pathway distinct from the central pore and that it enhances excitation of sensory neurons, thereby exacerbating TRPM3-dependent pain.

RESULTS Clotrimazole modulates TRPM3 activity

We tested the effects of a series of potential TRPM3 modulators on whole-cell currents in HEK293 cells expressing the α2 isoform of TRPM3 (refs. 9–11). These compounds included clotrimazole (Clt), a widely used over-the-counter drug for the treatment of yeast infections. Clt also inhibits the related TRP channels TRPM2 and TRPM8 and intermediate conductance Ca2+-activated K+ channels and activates TRPV1 and TRPA1 (refs. 12–14). Brief preincubation with Clt (10 μM) caused a marked potentiation of TRPM3 currents evoked by PS (40 μM) or by the unsulfated neurosteroid pregnenolone (100 μM; Fig. 1a–d) but had no effect in nontransfected cells (Supplementary Results, Supplementary Fig. 1). Moreover, the effect of Clt was stimulus dependent, as it had no detectable effect on basal TRPM3 currents or on TRPM3-currents stimulated by heat or Nif (Fig. 1a–d). The effect of Clt on PS-activated TRPM3 currents developed over time, reaching maximal potentiation within approximately 30 s (Supplementary Fig. 1), and faded out gradually after washout, with a time constant of 25.3 ± 5.6 s (mean ± s.e.m.) (n = 8; Fig. 1a,c and Supplementary Fig. 1). This contrasts with the agonistic effect of PS itself, which was much more rapid, with kinetics largely determined by the speed of PS application and washout (Fig. 1a,c,e and Supplementary Fig. 1). The effect of Clt on PS-activated TRPM3 currents showed concentration and voltage dependence: at submicromolar concentrations, the most prominent effect was an enhancement of outward TRPM3 current, with a concentration for half-maximal potentiation of 20 ± 4 nM (n = 6) and, maximally, a threefold potentiation at +100 mV; at concentrations ≥1 μM, there was a strong enhancement of the inward current amplitude at −100 mV, with a ~70-fold potentiation at 10 μM (Fig. 1e–g). Current-voltage relations of basal TRPM3 currents and currents activated by PS, heat or Nif are consistently outwardly rectifying (Fig. 1a–c)9–11. In contrast, currents in the combined presence of PS and Clt showed double rectification, with marked inward currents at strongly hyperpolarizing potentials (Fig. 1b,f). We quantified

Laboratory of Ion Channel Research and TRP Research Platform Leuven (TRPLe), KU Leuven, Leuven, Belgium. 2Laboratory of Obstetrics and Experimental Gynaecology, KU Leuven, Leuven, Belgium. 3These authors contributed equally to this work. *e-mail: [email protected] or [email protected] 1

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Figure 1 | Activation of a new inwardly rectifying TRPM3 current component. (a) Time course of whole-cell currents at ±80 mV upon stimulation with PS (40 μM) and Clt (10 μM) in HEK 293 cells stably expressing TRPM3. (b) I-V relations obtained at time points indicated in a. The inset shows the I-V relationship of the Clt-induced current obtained as the difference between traces c and b. (c) Time course of whole-cell currents at ±80 mV upon stimulation with PS (40 μM), Nif (50 μM) and Clt (10 μM) in HEK 293 cells stably expressing TRPM3. (d) Relative potentiation by Clt (10 μM) of the inward (−150 mV, black bars) and outward (+150 mV, gray bars) current upon stimulation with Nif (n = 5), heat (42 °C; n = 4), pregnenolone, P (100 μM; n = 5) and PS (40 μM, n > 50). (e) Time course of whole-cell currents at ±80 mV upon stimulation with PS (40 μM) and different concentrations of Clt in HEK 293 cells stably expressing TRPM3. (f) I-V relations obtained from time points indicated in e. (g) Clt concentration dependence (n = 6) of the potentiation of PS-induced TRPM3 currents for the outward (+100 mV, top) and inward current (−100 mV, bottom). Solid line in upper part represents a fit with a modified Hill function of the form I/Icontrol = 1 + R/(1 + EC50/C), where R + 1 represents the maximal potentiation, EC50 is the concentration for half-maximal potentiation, and C is the concentration of Clt. (h) Maximal inward currents at −150 mV plotted versus outward currents at +150 mV activated by PS (40 μM) in the absence or presence of Clt (10 μM) (n > 75).

r­ ectification as the ratio of absolute current amplitude at −150 mV and +150 mV (ratio–150/+150), which increased from 0.11 ± 0.01 in PS to 0.76 ± 0.03 in the combined presence of PS and Clt. The amplitudes of inward and outward current components were strongly linearly correlated (R > 0.94; Fig. 1h), indicating that they both depend on the level of TRPM3 expression. The effect of Clt was observed in different mammalian expression systems (HEK293 and F11 cells) as well as in the amphibian A6 cell line (Supplementary Fig. 2), suggesting that it does not require cell type–specific components. Taken together, these data indicate that Clt potentiates

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PS-induced currents through TRPM3, which includes activation of a distinct, inwardly rectifying current component.

The inwardly rectifying current component is cationic

Strong ligand stimulation causes pore dilation of TRPV1 and TRPA1, which can be detected as a positive shift of the reversal potential (Erev) when using large monovalent cations as sole extracellular charge carriers15–17. When N-methyl-D-glucamine (NMDG+; diameter (a) = 6.8 Å) was substituted for Na+ in the extracellular solution, TRPM3 currents evoked by PS in the absence or presence of Clt lacked an inward component (Supplementary Fig. 3). This indicates that the inward current is exclusively carried by cations and that NMDG+ is too large to permeate the channel under these circumstances. However, in both the absence and the presence of Clt, PS-activated inward currents were carried by substituted ammonium ions as large as tetramethylammonium (TMA+; a = 5.8 Å), and no detectable differences in relative permeability were found between the two conditions (Supplementary Fig. 3). These data indicate that, at least at Erev, Clt does not affect the selectivity of the current.

Clt acts independently of P450-dependent enzymes

As Clt inhibits many members of the cytochrome P450 (CYP) superfamily of enzymes12, we considered that Clt could modulate TRPM3 via its effects on one or more CYP enzymes. In particular, Clt could increase the PS concentration at the channel by inhibiting CYP-dependent steps in steroid metabolism. However, two findings demonstrate that Clt does not act by altering local PS concentration. First, inward rectification of TRPM3 currents was not observed at PS concentrations approaching the solubility limits (Fig. 2a,b), with a mean ratio–150/+150 of 0.08 ± 0.03 at a PS concentration of 500 μM (n = 8). At −100 mV, the mean amplitude of the inward current in the combined presence of 250 μM PS and 10 μM Clt (4.12 ± 2.32 nA) was one order of magnitude larger than that evoked by 500 μM PS (240 ± 95 pA; n = 9). Second, PS and Clt acted from opposite sides of the plasma membrane in inside-out patches. In line with published results9, we found that PS works strictly from the extracellular side of the membrane. In contrast, Clt caused clear current potentiation, including activation of an inwardly rectifying component, when applied to the cytosolic side of an inside-out patch (Fig. 2c,d; 8.9 ± 2.1-fold and 2.5 ± 0.8-fold current increase at −100 mV and +100 mV, respectively; n = 6). In contrast, there was no detectable inwardly rectifying component when Clt was only supplemented in the extracellular pipette solution, suggesting that the Clt that permeates through the inside-out patch is effectively washed away by the bath solution before it can act on the channel. Clt was also effective when included in the intracellular solution during whole-cell recordings, as evidenced by the double rectification of PS-evoked currents (Supplementary Fig. 4; ratio−150/+150 = 0.68 ± 0.13; n = 6). Alternatively, the Clt-induced inwardly rectifying current component could reflect disturbances of the lipid environment through modulation of CYPs involved in sterol metabolism, phospholipid metabolism or both. In contrast to this view, econazole, a related imidazole antifungal drug with Clt-like inhibitory effects on CYP enzymes, was unable to activate the inwardly rectifying current component and caused an inhibition of PS-induced TRPM3 currents (Supplementary Fig. 4; half-maximum inhibitory concentration = 6 ± 2 μM; n = 7). Oppositely, TRAM-34 and senicapoc, two Clt structural analogs that lack activity against most CYP enzymes12,18, and tamoxifen, an estrogen receptor antagonist with some structural similarity to Clt19, did activate the inwardly rectifying current component (Supplementary Fig. 4). Taken together, these results demonstrate that activation of the inwardly rectifying current component is unrelated to CYP enzyme activity.

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Next, we considered that Clt could induce the inwardly rectifying current component by affecting the voltage-dependent gating of TRPM3. At room temperature (23 °C) and in the absence of chemical ligands, substantial TRPM3 currents can only be measured at potentials ≥+50 mV10. During voltage-steps, whole-cell conductance (G) increased with depolarization but did not saturate at voltages that are reliably attainable in patch-clamp recordings (~+250 mV; Supplementary Fig. 5). Activation by PS involved a shift of the steady-state activation (G-V) curve toward more negative voltages (Supplementary Fig. 5), as has been previously shown for TRPM8 and TRPV1 (refs. 20–22). Notably, G-V curves in the presence of PS and Clt were biphasic, showing a minimal conductance around −50 mV and increasing conductance at more hyperpolarizing potentials, which saturated at approximately −250 mV (Supplementary Fig. 5). This indicates that the inward TRPM3 current component is not simply due to a parallel shift of the G-V curve toward very negative voltages and suggests activation of an additional inward conductance component at hyperpolarizing potentials. Realizing that the voltage dependence of this conductance resembles that of the omega current described in voltage-gated K+ and Na+ channels5,6, we hypothesized that the combination of PS and Clt opens a comparable b

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alternative pathway for inward ion flux through TRPM3, distinct from the central pore23. Several lines of evidence are in support of such a mechanism. First, Clt altered the relative contribution of Ca2+ and Na+ to the PS-activated inward current. When performing intracellular Ca2+ imaging in TRPM3-expressing HEK293 cells, the Clt-induced potentiation of PS-induced Ca2+ responses was relatively mild (1.4 ± 0.3 fold potentiation (n = 276); Fig. 3a), which seemed inconsistent with the greater than tenfold increase in inward currents observed in whole-cell recordings (Figs. 1 and 2). However, measurements of intracellular Na+ revealed that Clt strongly augmented PS-induced Na+ influx (20.4 ± 4.4 fold potentiation; n = 33; Fig. 3a and Supplementary Fig. 8). These data indicate that, in intact HEK293 cells, Clt alters the ionic nature of the PS-induced cation influx, promoting the influx of Na+ with respect to Ca2+. Likewise, combined patchclamp and Fura-2 fluorescence recordings revealed that the fraction of the PS-induced inward current at −60 mV carried by Ca2+ drops from ~40% in the absence to ~20% in the presence of Clt (Supplementary Fig. 6). Second, the inward and outward current components exhibited distinct sensitivity to desensitization (Fig. 3b–d). In most wholecell recordings, we used Ca2+-free extracellular solutions, thereby

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Evidence for an alternative permeation pathway

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g­ enerally limiting current decay of PS-activated TRPM3 currents to less than 10% per minute. However, with 1 mM Ca2+ in the extracellular solution, we observed a much more pronounced, timedependent decay of TRPM3 currents activated by PS or combined PS and Clt, similar to the Ca2+-dependent desensitization of many other sensory TRP channels24–26. Markedly, the inwardly rectifying current component activated by the combined application of PS and Clt was much more resistant to desensitization than the outward current component (Fig. 3b), and during prolonged application of PS and Clt in the presence of extracellular Ca2+, the shape of the current-voltage relation gradually changed from doubly rectifying to strongly inward rectifying (Fig. 3c). The ratio–150/+150 increased from 0.95 ± 0.04 to 4.8 ± 1.8 (Fig. 3d). Third, the nonspecific cation channel pore blocker27 La3+ isolated the inwardly rectifying current component. Following stimulation with PS and Clt, application of 10–100 μM La3+ caused a rapid and reversible inhibition of the outward TRPM3 current but left the inward current largely intact (Fig. 3e,f and Supplementary Fig. 2). La3+ did not measurably permeate the channel (Supplementary Fig. 6). The La3+-insensitive current exhibited several properties of the omega current of voltage-gated K+ and Na+ channels5,6, including (i) strong inward rectification, (ii) minimal inactivation during 1.5-s hyperpolarization to −200 mV, (iii) inhibition by millimolar concentrations of Mg2+ and (iv) larger inward currents when extracellular Na+ was substituted by guanidinium (Fig. 3f–i). We also analyzed the ability of substituted ammonium cations to carry the La3+-insensitive inward current and compared it with results obtained for the outwardly rectifying, PS-activated TRPM3 current. As the La3+-insensitive current (like the omega current in voltage-gated K+ and Na+ channels5,6) only becomes prominent at hyperpolarizing potentials, its selectivity cannot be reliably be measured from Erev. We therefore quantified the amplitude of the inward currents carried by the different cations at a strongly hyperpolarizing potential (−150 mV), normalized to the current carried by Na+ (Supplementary Fig. 7)5,6. This analysis indicated that the La3+-insensitive inwardly rectifying current is less efficiently carried by mono-, di- and trimethylammonium (Supplementary Fig. 7). Fourth, combined application of PS and Clt gave rise to two clearly distinct types of single-channel current. In cell-attached patch-clamp recordings, inclusion of PS in the patch pipette resulted in the regular appearance of channel activity (Fig. 4). Whereas these cell-attached patches mostly contained multiple channels, leading to macroscopic currents, we obtained six patches containing apparently only one active channel. At −150 mV, singlechannel currents measured in these patches had an average amplitude of −7.1 ± 0.6 pA and an estimated single-channel conductance of approximately 50 pS (47 ± 3 pS; n = 6; Fig. 4b–d). Application of Clt to the bath solution reversibly potentiated the net inward current at −150 mV by 8.6 ± 1.0-fold (n = 6; Fig. 4a–d). Notably, Clt not only increased the open probability of the 50-pS channel but also evoked a second type of channel activity with readily discernible properties (Fig. 4b–d). In particular, the new Clt-induced channel current exhibited a lower current amplitude of 1.9 ± 0.7 pA (n = 5) at −150 mV and longer openings (mean open time = 20.3 ± 5.9 ms; n = 5; Fig. 4e) compared to the 50-pS channel, whose mean open time increased from 1.4 ± 0.5 ms in the absence to 5.6 ± 2.0 ms in the presence of Clt (n = 5; Fig. 4e). Neither the 50-pS channel nor the Clt-induced lower conductance channel were observed in cell-attached patches (at −150 mV) when PS was not included in the pipette (n = 15) or when using nontransfected HEK293 cells (n = 12). Taken together, these results suggest that the inwardly rectifying current component activated by the combined application of Clt and PS is functionally distinct and discernible from the ‘canonical’, outwardly rectifying current through the central pore.

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Separation of two permeation pathways using mutagenesis

To further substantiate the hypothesis of two distinct cation permeation pathways, we searched for mutations that differentially affect the two current components. Mutating a glutamate in the putative pore region to cysteine (mutant E1057C) resulted in a functionally expressed channel that responded poorly to PS or Nif (Fig. 5a,b and Supplementary Fig. 8) and was insensitive to blockade by La3+ (Supplementary Figs. 8 and 9). However, combined application of PS and Clt caused robust activation of an inwardly rectifying, La3+-insensitive current (Fig. 5a–c and Supplementary Figs. 8 and 9). Intracellular Ca2+ and Na+ imaging experiments further revealed that E1057C did not generate Ca2+ signals but provoked Na+ influx in response to combined application of PS and Clt (Supplementary Fig. 8). During combined application of PS and Clt, replacing extracellular Na+ by Ca2+, Sr2+ or Ba2+ abolished the inward current in E1057C-transfected HEK cells (Supplementary Fig. 8). These findings indicate that the E1057C mutation has a strong inhibitory effect on the central, La3+-sensitive, Ca2+permeable pore but leaves the alternative, La3+-insensitive and Ca2+-impermeable pore largely unaffected. To further establish that Glu1057 is located in the central pore, we used methanethiosulphonate (MTS) reagents, which covalently link a positively charged (2-(trimethylammonium) ethyl methanethiosulfonate (MTSET)) or a negatively charged (2-sulfonatoethyl methanethiosulfonate (MTSES))) moiety to the thiol group of water-exposed cysteines. MTSET and MTSES (1 mM) had a small and reversible inhibitory effect on the wild-type channel (Supplementary Fig. 9), indicating that the 27 endogenous cysteines are either not accessible to MTS reagents or not critical for normal channel function. MTSET was also without effect on the E1057C mutant (Supplementary Fig. 9). However, application of MTSES to the E1057C mutant evoked a gradual increase

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Figure 5 | Mutations in TRPM3 differentially affect the two current components. (a) Time course of whole-cell currents at ± 80 mV in HEK293 cells transfected with the E1057C mutant upon stimulation with PS (40 μM), Clt (10 μM) and PS + Clt. (b) I-V relations obtained at time points indicated in a. (c) Clt-induced current, obtained as the difference between two traces in b. (d) Time course of whole-cell currents at ± 80 mV in HEK293 cells transfected with the E1057C showing the effect of treatment with MTSES (1 mM). (e) I-V relations of the PS-activated current (IPS) of mutant E1057C in basal conditions and after treatment with MTSES. (f) I-V relations of the current activated by combined application of PS and Clt (IPS + Clt) of mutant E1057C in basal conditions and after treatment with MTSES. (g) Normalized effect of MTSES treatment on inward and outward currents activated by PS (n = 11) and PS + Clt (mean ± s.e.m.; n = 6). (h) Time course of whole-cell currents in W982R-transfected HEK293 cells at ± 80 mV upon stimulation with PS (100 μM), Clt (10 μM) and PS + Clt. (i) I-V relations obtained at time points indicated in h. (j) I-V relationship of the Clt-induced current obtained as the difference between two traces in i.

of the outward component of the PS-induced current, yielding an approximately fourfold increase of the current at +80 mV (Fig. 5d,e,g), thereby restoring the typical outward rectification of the wild-type channel (Supplementary Fig. 9). Notably, MTSES treatment had little or no effect on the inward current component in response to PS or to the combined application of PS and Clt (Fig. 5f,g), but it restored La3+ sensitivity of the PS-activated outward current (Supplementary Fig. 9). Taken together, these results indicate that Glu1057 is located in the central pore and that cysteine modification at this position affects the current via the central pore without affecting the alternative permeation pathway. In voltage-gated K+ and Na+ channels, the omega current is not detectable in the wild-type channel but can be uncovered by specific mutations in the voltage sensor domain. In particular, mutating the outermost arginine in the S4 segment of the Shaker K+ channel to a noncharged residue leads to the appearance of the omega current at hyperpolarized potentials. TRPM3 lacks the high density of positively charged arginines in the S4 domain, which is a defining feature of the voltage sensor of voltage-gated K+ and Na+ channels. Nevertheless, alignment of a 15-amino-acid region encompassing the voltage-sensing arginines of the Shaker K+ channel with the entire TRPM3 sequence revealed the highest degree of similarity in the putative S4 region of TRPM3 (40% identity; 60% similarity) and suggest that TRPM3 may contain a tryptophan at the position corresponding to the outermost arginine in Shaker (Supplementary Fig. 8). Mutating this tryptophan to arginine (mutant W982R) resulted in a channel that was expressed at lower levels than wild-type TRPM3 but generated substantial outwardly rectifying, La3+-sensitive whole-cell currents and intracellular Ca2+ transients in response to PS (Fig. 5h,i and Supplementary Figs. 8 and 9). Co-application of Clt caused an approximately threefold potentiation of the outward PS-activated current through W982R but failed to induce an inwardly rectifying current component (Fig. 5i,j and Supplementary Fig. 8). Moreover, PS-induced increases in intracellular Na+ and Ca2+ in cells expressing the W982R mutant were not potentiated by Clt (Supplementary Fig. 8). 1 92

These findings suggest that introducing an arginine in the S4 domain at a position that may correspond to the outermost voltage-sensing arginine in the Shaker K+ channel prevents opening of pathway responsible for the inwardly rectifying current component.

Activation of the alternative pathway in sensory neurons

To investigate the presence of the alternative cation permeation pathway in endogenous TRPM3 channels and its potential implications in vivo, we focused on the role of TRPM3 in nociceptors10. We used mice with a TRPV1-deficient background to avoid potential interference of TRPV1-mediated Clt responses14. In line with our previous results10, we observed that about half of dorsal root ganglion (DRG) neurons from Trpv1−/− mice (53 out of 102) responded to 10 μM PS with a prominent rise in intra­cellular Ca2+. Preapplication of Clt resulted in a greater than fivefold potentiation of intracellular Ca2+ responses to PS (Fig. 6a,b). Ca2+ responses to consecutive PS applications in the absence of Clt did not show such potentiation (data not shown; see also ref. 10). Responses to PS or PS plus Clt were not detected in neurons from Trpv1−/− Trpm3−/− mice (n = 120; Fig. 6a,b). In whole-cell current recordings in DRG neurons from Trpv1−/− mice, pretreatment with Clt reversibly enhanced the PS-evoked inward currents (Fig. 6c,d). Currents evoked by Clt plus PS exhibited dual rectification (Fig. 6e), similar to TRPM3 expressed in cell lines. Clt induced an 8.2 ± 1.0-fold and 2.0 ± 0.4-fold increase (n = 7) of the PS-activated currents at −120 mV and +80 mV, respectively. An inwardly rectifying current component was preserved upon addition of La3+ (Fig. 6e). Notably, Clt caused a strong potentiation of both intracellular Ca2+ (Fig. 6a,b) and current (Fig. 6c,d) responses in sensory neurons, whereas in TRPM3-expressing HEK293 cells intracellular Ca2+ responses were largely unaffected by Clt (Fig. 3a). This difference indicates that, in sensory neurons, Na+ influx via the alternative pathway may facilitate action potential generation and concomitant Ca2+ influx via voltage-gated Ca2+ channels. In line herewith, Clt caused a greater than fivefold increase in the

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Figure 6 | Clt potentiates TRPM3-mediated neuronal responses and pain. (a) Representative changes in intracellular Ca2+ concentration in DRG neurons derived from Trpv1−/− (upper) and Trpv1−/−Trpm3−/− (lower) mice in response to PS (10 μM), Clt (10 μM), MO (100 μM) and high K+ (50 mM). (b) Average increase in [Ca2+]i in response to PS and PS + Clt (n = 102 neurons for Trpv1−/− and n = 120 neurons for Trpv1−/−Trpm3−/− mice, from four independent isolations). **P = 0.003; paired t-test. (c,d) Whole-cell currents at −40 mV in neurons from Trpv1−/−Trpa1−/− mice upon repetitive stimulation with PS (20 μM), in the absence or presence of Clt (10 μM) (n = 12; **P = 0.007; paired t-test). (e) I-V relations of background-subtracted whole-cell currents in a Trpv1−/− DRG neuron stimulated with Clt (10 μM) and PS (40 μM), before and during addition of La3+ (10 μM) to the extracellular solution. Background currents before stimulation with PS were subtracted to isolate the TRPM3-like current. (f) Cell-attached spike activity recorded from DRG neurons derived from Trpv1−/− (top) and Trpv1−/−Trpm3−/− (bottom) knockout mice upon stimulation with PS (40 μM), Clt (10 μM) or high K+ (50 mM). (g) Changes in spike frequency upon stimulation of Trpv1−/− DRG neurons with PS and PS + Clt. (h) Number of behavioral responses (paw licks and lifts) following intraplantar injection of vehicle (Vhc), Clt, PS, PS + Clt or mustard oil (MO) (n = 8 for each genotype; **P = 0.008; paired t-test).

frequency of PS-induced spike activity in cell-attached recordings on intact neurons from Trpv1−/− mice (0.74 ± 0.49 Hz in PS to 5.08 ± 2.04 Hz in PS plus Clt; Fig. 6f,g). PS-induced spike activity, both in the absence and in the presence of Clt, was never observed in neurons derived from Trpv1−/− Trpm3−/− mice (n = 13; Fig. 6f). To evaluate the potential in vivo consequences of activation of the alternative pathway, we used intraplantar injection of PS in mice as a model of TRPM3-dependent, chemically induced pain10. Intraplantar injection of Clt in Trpv1−/− mice did not evoke a pain response by itself, but it enhanced the PS-induced pain response (Fig. 6h and Supplementary Fig. 10). The nocifensive responses to PS, either alone or in combination with Clt, were not different from responses to vehicle control in Trpv1−/− Trpm3−/− mice (Fig. 6h and Supplementary Fig. 10). We also evaluated the effect on mouse nocifensive behavior of co-injection of PS and diphenylamine (DPA). This compound, like Clt, has no effect on its own but potentiates PS-induced TRPM3 responses in HEK293 cells. However, DPA did not alter the rectification of the channel, indicating that it is unable to activate the alternative pathway, and it did not enhance the nocifensive response when injected together with PS (Supplementary Fig. 11). Taken together, Clt exacerbates PS-induced pain in a TRPM3-dependent manner, which is at least partly due to its ability to open the alternative cation pathway. At 23 °C, we were unable to detect TRPM3-mediated responses in HEK293 cells or sensory neurons at submicromolar concentrations of PS, irrespective of the presence of Clt (data not shown; see also refs. 9 and 10.). In contrast, at 37 °C, Clt induced activation of the characteristic inwardly rectifying current component at a PS concentration as low as 500 nM (Supplementary Fig. 12), which is well within the concentration range (100–800 nM) measured in

the plasma of healthy human adults28,29. Intracellular Ca2+ imaging experiments at 37 °C in sensory neurons revealed robust TRPM3dependent responses to Clt when combined with 500 nM PS (Supplementary Fig. 12). These data suggest that, at physiological temperature, circulating levels of PS may be sufficient to support activation of the alternative cation pathway in TRPM3 upon application of Clt (or related drugs).

DISCUSSION

It was generally accepted that the TRP channels are tetramers with a single, central, cation-permeable pore. In contrast to this view, our present data indicate the existence of an alternative ion permeation pathway in TRPM3, distinct from the central pore, that can be ­specifically activated by the combined application of the neurosteroid PS and the widely used antifungal Clt (Supplementary Fig. 13). The current through this alternative pathway can be discriminated from the canonical TRPM3 current on the basis of its (i) strong inward rectification, (ii) low permeability to Ca2+, (iii) resistance to Ca2+-dependent desensitization, (iv) low sensitivity to block by La3+ and (v) resistance to mutagenesis and cysteine modification of the central pore region. Ion selectivity, conductance and pore block are often envisaged as invariable properties of the open channel pore. Nevertheless, there are several examples of mammalian cation channels exhibiting marked variations in such pore properties, for instance, in response to prolonged stimulation (for example, TRP channels15–17 or P2X receptors30) or in the function of the expression of accessory subunits (for example, Orai1-Stim1 (ref. 31) or minK-KCNQ1 (ref. 32). Although we cannot entirely exclude that the inwardly rectifying component represents ion flux through an alternative

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conformation of the central pore of TRPM3, several experimental findings are difficult to reconcile with a single-pore model. First, the inwardly rectifying current exhibited voltage-dependence opposite from that of the depolarization-activated current through the canonical pore. Assuming a single pore, this would imply that the activation gate of the central pore can be controlled by two activation mechanisms with opposite voltage sensitivity, a property that, to our knowledge, has never been observed in the superfamily of cation channels with 24 transmembrane domains. The opposite voltage dependence does, however, fit well with a model in which the voltage sensors need to be in a resting state for the alternative pathway to be active, as is the case in voltage-gated channels5,6. Second, the inward current was resistant to Ca2+-dependent ­desensitization, which leads to a rapid decay of the outwardly rectifying TRPM3 current. Whereas Ca2+-dependent desensitization or deactivation has been described for several related voltage-gated TRPM channels, this was invariably associated with a more prominent reduction of inward than outward currents26,33, attributable to a rightward shift of the voltage-dependent activation curve33. Third, La3+ caused a rapid blockade of the outward current while leaving the inwardly rectifying current largely unaltered. Assuming a single pore, this would imply that La3+ is more potent at blocking outward currents than inward currents through this pore, which is opposite to the voltage dependence of La3+ block of related TRPM channels34 and most other cation channels. Fourth, the outwardly rectifying, La3+-sensitive PS-activated current was largely eliminated by the E1057C mutation, whereas the inwardly rectifying current activated by PS plus Clt was preserved. Most notably, reaction of this mutant with MTSES, which recreated a glutamate-like negative charge at this residue, restored the outwardly rectifying, La3+-sensitive PS-activated current without affecting the inwardly rectifying component activated by PS plus Clt. These findings indicate that Glu1057 contributes to the pore that mediates the outwardly rectifying current but not to the pathway that generates the inwardly rectifying component. In the Shaker potassium channel, there is evidence that the omega pore is lined by residues from S1–S4 (ref. 8). The lack of knowledge of the structure of TRP channels, combined with our observation that many mutations in the S4 region resulted in nonfunctional and/or nonexpressing channels, impeded a rational strategy to identify the location of the inwardly rectifying pathway in TRPM3. Nevertheless, the results obtained with the W982R mutant, which introduces an arginine at a site that may correspond to the outermost arginine in the voltage sensor of Shaker, are consistent with an alternative ion permeation pathway in the S1–S4 region in TRPM3. However, it cannot be excluded that this mutation has an allosteric effect on a distal permeation pathway, influences the binding of Clt or both. Whereas the omega pore in classical voltage-gated cation channels is uncovered by artificial or disease-related mutations in the voltage sensor domain5–7, the inwardly rectifying TRPM3 current component exists in the wild-type channel. Activation of this pathway may thus have physiological consequences in TRPM3expressing cells such as sensory neurons, pancreatic β cells or synovial cells9,10,35. Our findings indicate that the alternative pathway is operating in sensory neurons and exacerbates TRPM3-mediated pain. Clt is widely used for the topical treatment of yeast infections of skin and mucous membranes, requiring micro­molar concentrations for its fungicidal or fungistatic effects36,37. This may lead to activation of the alternative cation pathway in sensory nerves, which could contribute to common side effects such as irritation and burning pain36,38,39. Our results with TRAM34, senicapoc and tamoxifen suggest that opening of the alternative pathway in TRPM3 may be a more frequent side effect of various clinically or pharmacologically relevant drugs. 1 94

In conclusion, we presented evidence for the coexistence within a single TRP channel of two separate permeation pathways with distinct permeability, gating and desensitization properties. Our findings expand the repertoire of potential TRP channel functions in vivo and represent a potential new challenge in the development of TRP channel–specific drugs, as blockade of the central pore may not always suffice to inhibit the channel’s physiological (or pathophysiological) action. Further work is required to clarify whether the alternative cation permeability pathway can be gated by other stimuli and whether other TRP channels exhibit similar properties. Received 21 August 2013; accepted 4 November 2013; published online 5 January 2014

Methods

Methods and any associated references are available in the online version of the paper.

References

1. Nilius, B., Owsianik, G., Voets, T. & Peters, J.A. Transient receptor potential cation channels in disease. Physiol. Rev. 87, 165–217 (2007). 2. Hoenderop, J.G. et al. Homo- and heterotetrameric architecture of the epithelial Ca2+ channels TRPV5 and TRPV6. EMBO J. 22, 776–785 (2003). 3. Wu, L.J., Sweet, T.B. & Clapham, D.E. International Union of Basic and Clinical Pharmacology. LXXVI. Current progress in the mammalian TRP ion channel family. Pharmacol. Rev. 62, 381–404 (2010). 4. Yu, F.H., Yarov-Yarovoy, V., Gutman, G.A. & Catterall, W.A. Overview of molecular relationships in the voltage-gated ion channel superfamily. Pharmacol. Rev. 57, 387–395 (2005). 5. Tombola, F., Pathak, M.M. & Isacoff, E.Y. Voltage-sensing arginines in a potassium channel permeate and occlude cation-selective pores. Neuron 45, 379–388 (2005). 6. Sokolov, S., Scheuer, T. & Catterall, W.A. Ion permeation through a voltage-sensitive gating pore in brain sodium channels having voltage sensor mutations. Neuron 47, 183–189 (2005). 7. Sokolov, S., Scheuer, T. & Catterall, W.A. Gating pore current in an inherited ion channelopathy. Nature 446, 76–78 (2007). 8. Tombola, F., Pathak, M.M., Gorostiza, P. & Isacoff, E.Y. The twisted ion-permeation pathway of a resting voltage-sensing domain. Nature 445, 546–549 (2007). 9. Wagner, T.F. et al. Transient receptor potential M3 channels are ionotropic steroid receptors in pancreatic beta cells. Nat. Cell Biol. 10, 1421–1430 (2008). 10. Vriens, J. et al. TRPM3 is a nociceptor channel involved in the detection of noxious heat. Neuron 70, 482–494 (2011). 11. Oberwinkler, J., Lis, A., Giehl, K.M., Flockerzi, V. & Philipp, S.E. Alternative splicing switches the divalent cation selectivity of TRPM3 channels. J. Biol. Chem. 280, 22540–22548 (2005). 12. Wulff, H. et al. Design of a potent and selective inhibitor of the intermediateconductance Ca2+-activated K+ channel, IKCa1: a potential immunosuppressant. Proc. Natl. Acad. Sci. USA 97, 8151–8156 (2000). 13. Hill, K., McNulty, S. & Randall, A.D. Inhibition of TRPM2 channels by the antifungal agents clotrimazole and econazole. Naunyn Schmiedebergs Arch. Pharmacol. 370, 227–237 (2004). 14. Meseguer, V. et al. Transient receptor potential channels in sensory neurons are targets of the antimycotic agent clotrimazole. J. Neurosci. 28, 576–586 (2008). 15. Chung, M.K., Guler, A.D. & Caterina, M.J. TRPV1 shows dynamic ionic selectivity during agonist stimulation. Nat. Neurosci. 11, 555–564 (2008). 16. Banke, T.G., Chaplan, S.R. & Wickenden, A.D. Dynamic changes in the TRPA1 selectivity filter lead to progressive but reversible pore dilation. Am. J. Physiol. Cell Physiol. 298, C1457–C1468 (2010). 17. Karashima, Y. et al. Agonist-induced changes in Ca2+ permeation through the nociceptor cation channel TRPA1. Biophys. J. 98, 773–783 (2010). 18. McNaughton-Smith, G.A. et al. Novel inhibitors of the Gardos channel for the treatment of sickle cell disease. J. Med. Chem. 51, 976–982 (2008). 19. Jordan, V.C. Tamoxifen: a most unlikely pioneering medicine. Nat. Rev. Drug Discov. 2, 205–213 (2003). 20. Janssens, A. & Voets, T. Ligand stoichiometry of the cold- and mentholactivated channel TRPM8. J. Physiol. (Lond.) 589, 4827–4835 (2011). 21. Voets, T. et al. The principle of temperature-dependent gating in cold- and heat-sensitive TRP channels. Nature 430, 748–754 (2004). 22. Voets, T., Owsianik, G., Janssens, A., Talavera, K. & Nilius, B. TRPM8 voltage sensor mutants reveal a mechanism for integrating thermal and chemical stimuli. Nat. Chem. Biol. 3, 174–182 (2007).

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23. Clapham, D.E. TRP channels as cellular sensors. Nature 426, 517–524 (2003). 24. Caterina, M.J. et al. The capsaicin receptor: a heat-activated ion channel in the pain pathway. Nature 389, 816–824 (1997). 25. Nagata, K., Duggan, A., Kumar, G. & Garcia-Anoveros, J. Nociceptor and hair cell transducer properties of TRPA1, a channel for pain and hearing. J. Neurosci. 25, 4052–4061 (2005). 26. McKemy, D.D., Neuhausser, W.M. & Julius, D. Identification of a cold receptor reveals a general role for TRP channels in thermosensation. Nature 416, 52–58 (2002). 27. Grimm, C., Kraft, R., Sauerbruch, S., Schultz, G. & Harteneck, C. Molecular and functional characterization of the melastatin-related cation channel TRPM3. J. Biol. Chem. 278, 21493–21501 (2003). 28. Havliková, H., Hill, M., Hampl, R. & Starka, L. Sex- and age-related changes in epitestosterone in relation to pregnenolone sulfate and testosterone in normal subjects. J. Clin. Endocrinol. Metab. 87, 2225–2231 (2002). 29. Morley, J.E. et al. Potentially predictive and manipulable blood serum correlates of aging in the healthy human male: progressive decreases in bioavailable testosterone, dehydroepiandrosterone sulfate, and the ratio of insulin-like growth factor 1 to growth hormone. Proc. Natl. Acad. Sci. USA 94, 7537–7542 (1997). 30. Virginio, C., MacKenzie, A., Rassendren, F.A., North, R.A. & Surprenant, A. Pore dilation of neuronal P2X receptor channels. Nat. Neurosci. 2, 315–321 (1999). 31. McNally, B.A., Somasundaram, A., Yamashita, M. & Prakriya, M. Gated regulation of CRAC channel ion selectivity by STIM1. Nature 482, 241–245 (2012). 32. Tai, K.K. & Goldstein, S.A. The conduction pore of a cardiac potassium channel. Nature 391, 605–608 (1998). 33. Nilius, B. et al. The Ca2+-activated cation channel TRPM4 is regulated by phosphatidylinositol 4,5-biphosphate. EMBO J. 25, 467–478 (2006). 34. Runnels, L.W., Yue, L. & Clapham, D.E. TRP-PLIK, a bifunctional protein with kinase and ion channel activities. Science 291, 1043–1047 (2001). 35. Ciurtin, C. et al. TRPM3 channel stimulated by pregnenolone sulphate in synovial fibroblasts and negatively coupled to hyaluronan. BMC Musculoskelet. Disord. 11, 111 (2010). 36. Czerninski, R. et al. A novel sustained-release clotrimazole varnish for local treatment of oral candidiasis. Clin. Oral Investig. 14, 71–78 (2010).

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37. Torres-Rodríguez, J.M. et al. In vitro susceptibilities of clinical yeast isolates to the new antifungal eberconazole compared with their susceptibilities to clotrimazole and ketoconazole. Antimicrob. Agents Chemother. 43, 1258–1259 (1999). 38. Binet, O. et al. Flutrimazole 1% dermal cream in the treatment of dermatomycoses: a randomized, multicentre, double-blind, comparative clinical trial with 1% clotrimazole cream. Flutrimazole Study Group. Mycoses 37, 455–459 (1994). 39. del Palacio, A. et al. A double-blind randomized comparative trial: eberconazole 1% cream versus clotrimazole 1% cream twice daily in Candida and dermatophyte skin infections. Mycoses 44, 173–180 (2001).

Acknowledgments

We thank all members of the Laboratory of Ion Channel Research for helpful discussions. We thank the Cell Banking Facility of the Physiology Department at the Institute of Functional Genomics (Montpellier, France) for the F11 cell line. This work was supported by grants from the Belgian Federal Government (IUAP P7/13 to T.V.), the Research Foundation-Flanders (G.0565.07 and G.0825.11 to T.V. and J.V.), the Research Council of the KU Leuven (GOA 2009/07 and PF-TRPLe to T.V. and R.V.), the Planckaert-De Waele fund (to J.V.) and by a Marie Curie Intra-European Fellowship within the Seventh European Community Framework Programme (to B.I.T.).

Author contributions

J.V. and T.V. designed the project strategy and wrote the manuscript. J.V., K.H., A.J., B.I.T., S.K. and T.V. conducted and analyzed experiments. J.V., B.N., R.V. and T.V. supervised the project.

Competing financial interests

The authors declare no competing financial interests.

Additional information

Supplementary information is available in the online version of the paper. Reprints and permissions information is available online at http://www.nature.com/reprints/index. html. Correspondence and requests for materials should be addressed to T.V. or J.V.

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Cell culture and transfection. Culture of HEK293, F11 and A6 cells was as described previously10,33,40,41. Transfection of wild-type or mutant TRPM3 constructs was performed using TransIT transfection reagents (Mirus). TG and DRG neurons were isolated as described previously10. Animals. Trpv1 mice were obtained from The Jackson Laboratory (http:// jaxmice.jax.org/strain/003770.html). Trpm3−/− and Trpv1−/− Trpa1−/− mice have been described earlier10,42,43. Trpv1−/− mice were mated with Trpm3−/− mice to obtain Trpv1−/− Trpm3−/− double knockout mice. All knockout strains were backcrossed at least six times into the C57BL/6J background, and C57BL/6J mice were used as wild-type controls. Mice of all genotypes were housed under identical conditions, with a maximum of four animals per cage on a 12-h lightdark cycle and with food and water ad libitum. Ten- to twelve-week-old male mice were used in all experiments.

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Fluorescence imaging and electrophysiology. Fura-2–based ratiometric intracellular Ca2+ measurements and SBFI-based ratiometric Na+ measurements were performed as described previously10. Whole-cell membrane currents were measured with an EPC-10 (HEKA Elektronik, Lambrecht, Germany). The sampling rate was 20 kHz, and currents were digitally filtered at 2.9 kHz. For whole-cell recordings on HEK293 cells, the standard extracellular solution contained 150 mM NaCl, 1 mM MgCl2 and 10 mM HEPES (pH 7.4 with NaOH), and the pipette solution contained 100 mM CsAsp, 45 mM CsCl, 10 mM EGTA, 10 mM HEPES and 1 mM MgCl2 (pH 7.2 with CsOH). When filled with this solution, patch pipettes had resistance between 2 MΩ and 3 MΩ. Between 70% and 90% of the series resistance was compensated. Recordings with estimated voltage clamp errors >10 mV were excluded from analysis. The MTS reagents MTSET and MTSES (Toronto Research Chemicals) were added to the standard extracellular solution at 1 mM from 100-mM stocks, which were prepared daily and kept on ice until use. In experiments allowing Ca2+-dependent desensitization of TRPM3, Mg2+ was replaced by Ca2+ in the extracellular solution, and Cs+ was replaced by Na+ in the pipette solution. Combined patch-clamp and Fura-2 recordings were performed as described earlier17. For whole-cell current recordings on sensory neurons, the extracellular solution contained 140 mM NaCl, 4 mM KCl, 2 mM MgCl2, 100 nM TTX, 10 mM TRIS (pH 7.4 with HCl), and the pipette solution contained 140 mM CsCl, 0.6 mM MgCl2, 1 mM EGTA, 10 mM HEPES and 5 mM TEA (pH 7.2 with CsOH). For cell-attached recordings from sensory neurons, the bath and pipette solution contained 145 mM NaCl, 4 mM KCl, 2 mM MgCl2, 1.2 mM CaCl2, 10 mM glucose and 10 mM Hepes, pH 7.4, with NaOH. To estimate the central pore diameter, the permeability of substituted ammonium ions of increasing diameter was determined as described earlier44. Briefly, all Na+ ions in the standard extracellular solution were replaced by the respective substituted ammonium ions, and the relative permeability (PX/PNa) was calculated from the shift in Erev of the whole-cell current (ΔErev), after correction for liquid junction potentials44, using the following equation:  F ΔErev    RT 

PX / PNa = e 

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Pore diameter was estimated by fitting the data with the excluded-volume function: PX / PNa = k × (1 − a / d )2

where a is the diameter of the permeant cation, d is the estimated pore diameter, and k is a scaling constant. Behavior. PS (15 nmol/paw), Clt (1 nmol/paw), DPA (1 nmol/paw) and MO (5 nmol/paw) were dissolved in PBS + 0.1% DMSO. An intraplantar injection of 50 μl was performed using a 30G needle coupled to a Hamilton syringe, and behavior was video-taped. Experiments were performed during the light cycle. Nocifensive behavior was quantified during the first 2 min after injection10. Sample sizes were consistent with those reported in similar studies10,42,43. Execution and analysis of behavioral experiments was done blinded, i.e., without knowledge of the mouse genotype. All of the animal experiments were carried out in accordance with the European Union Community Council guidelines and approved by the local ethics committee. Data analysis. Electrophysiological data were analyzed using FITMASTER (HEKA Elektronik, Germany) and WinASCD software (Guy Droogmans, Leuven). Origin 7.1 (OriginLab Corporation, Northampton, USA) was used for statistical analysis and data display. Pooled data of continuous parameters are expressed as mean ± s.e.m. from n biological replicates. Student’s paired or unpaired, two-tailed t-test was used for statistical comparison between two data sets. When required, the Shapiro-Wilk test and Levene’s test were used to verify the normality and equal variance of the compared data sets. Conductance-voltage (G-V) curves were fitted with a Boltzmann function of the form: G(V ) =

Gmax ,  (V1/ 2 − V )   1 + exp  zF   RT  

where z is the apparent gating charge, V1/2 is the potential for half-maximal activation, Gmax is the maximal conductance, F is the Faraday constant, R is the gas constant, and T is the absolute temperature. Unless mentioned otherwise, experiments were performed at room temperature (23 ± 1 °C). 40. Gaudioso, C., Hao, J., Martin-Eauclaire, M.F., Gabriac, M. & Delmas, P. Menthol pain relief through cumulative inactivation of voltage-gated sodium channels. Pain 153, 473–484 (2012). 41. Erlij, D., De Smet, P. & Van Driessche, W. Effect of insulin on area and Na+ channel density of apical membrane of cultured toad kidney cells. J. Physiol. (Lond.) 481, 533–542 (1994). 42. Karashima, Y. et al. TRPA1 acts as a cold sensor in vitro and in vivo. Proc. Natl. Acad. Sci. USA 106, 1273–1278 (2009). 43. Kwan, K.Y. et al. TRPA1 contributes to cold, mechanical, and chemical nociception but is not essential for hair-cell transduction. Neuron 50, 277–289 (2006). 44. Voets, T., Janssens, A., Droogmans, G. & Nilius, B. Outer pore architecture of a Ca2+-selective TRP channel. J. Biol. Chem. 279, 15223–15230 (2004).

doi:10.1038/nchembio.1428

Opening of an alternative ion permeation pathway in a nociceptor TRP channel.

Sensory neurons detect chemical stimuli through projections in the skin and mucosa, where several transient receptor potential (TRP) channels act as p...
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