NCS-1 protein regulates TRPA1 channel through the PI3K pathway in breast cancer and neuronal cells

The physical and functional interaction between transient receptor potential channel ankyrin 1 (TRPA1) and neuronal calcium sensor 1 (NCS-1) was assessed. NCS-1 is a calcium (Ca2+) sensor found in many tissues, primarily neurons, and TRPA1 is a Ca2+ channel involved not only in thermal and pain sensation but also in conditions such as cancer and chemotherapy-induced peripheral neuropathy, in which NCS-1 is also a regulatory component. We explored the interactions between these two proteins by employing western blot, qRT–PCR, co-immunoprecipitation, Ca2+ transient monitoring with Fura-2 spectrophotometry, and electrophysiology assays in breast cancer cells (MDA-MB-231) with different levels of NCS-1 expression and neuroblastoma cells (SH-SY5Y). Our findings showed that the expression of TRPA1 was directly correlated with NCS-1 levels at both the protein and mRNA levels. Additionally, we found a physical and functional association between these two proteins. Physically, the NCS-1 and TRPA1 co-immunoprecipitate. Functionally, NCS-1 enhanced TRPA1-dependent Ca2+ influx, current density, open probability, and conductance, where the functional effects depended on PI3K. Conclusion: NCS-1 appears to act not only as a Ca2+ sensor but also modulates TRPA1 protein expression and channel function in a direct fashion through the PI3K pathway. These results contribute to understanding how Ca2+ homeostasis is regulated and provides a mechanism underlying conditions where Ca2+ dynamics are compromised, including breast cancer. With a cellular pathway identified, targeted treatments can be developed for breast cancer and neuropathy, among other related diseases. Supplementary Information The online version contains supplementary material available at 10.1007/s13105-024-01016-z.


Introduction
In the development from unicellular living forms to complex organisms, the ion calcium (Ca 2+ ) stands out as a crucial second messenger that plays a pivotal role in many physiological functions [13].It controls the cell cycle (death and survival), gene expression, membrane transport, and neurotransmitter release [7].Ca 2+ -binding proteins, channels, and transporters must be synchronized to transcribe these signals into specific pathways and to achieve homeostasis.Neuronal calcium sensor 1 (NCS-1) is a Ca 2+ -binding protein that was first named as frequenin [47] in the Drosophila spp.nervous system.In humans, NCS-1 is mainly found in neurons, especially in the cerebral cortex, the hippocampus [42], and dorsal root ganglion cells [51].However, this protein is also found in epithelia, adipocytes, neuroendocrine cells, and cardiac myocytes [35,42,48].
Four EF-hand motifs with high affinity for Ca 2+ integrate the molecular structure of NCS-1, which provides it with the ability to detect small changes in intracellular Ca 2+ concentration and allows it to act as an accurate Ca 2+ sensor as well as a Ca 2+ -binding protein.Under basal conditions, NCS-1 is bound to Mg 2+ , adopting a spatial configuration that reduces its hydrophobic region exposure and prevents nonspecific reactions [12].When Ca 2+ is available, it leads to a conformational shift that allows the interaction with an array of binding targets, translating intracellular Ca 2+ concentration changes into biochemical signals [8].
Over the years, many authors have studied the interaction between NCS-1 and a wide pool of target proteins, highlighting novel biological functions and pathophysiological roles in diseases.For instance, this molecular sensor modulates voltage-gated Ca 2+ channel 2.1 (CaV2.1)through its α1 subunit, inducing short-term synaptic facilitation [68].Additionally, it interacts with the inositol 1,4,5-trisphosphate receptor (InsP3R), increasing the Ca 2+ signal [56].Disturbances in this dynamic appears to be involved in several human diseases, including ataxia, seizures, Alzheimer's disease, Huntington's disease, and cerebral ischemia [52].Furthermore, NCS-1 has a role as a survival factor through indirect activation of the PI3K signaling pathway [21], which is altered in some forms of cancer [20].Experiments with overexpression of this Ca 2+ sensor in breast cancer cell lines resulted in increased invasion and motility with diminished cell-matrix adhesion [1,37].This correlates with in vivo studies, where the expression of NCS-1 is higher in breast cancer cells [5], and the level of increase is significantly correlated with shorter survival rates in breast cancer patients [37,57].
TRPA1 (transient potential receptor channel ankyrin 1) belongs to the TRP family, a versatile nonselective cation channel superfamily.This protein is mainly expressed in small diameter fibers of sensory ganglia [36] and other nonneuronal tissues, such as endothelial cells [19], enterochromaffin cells [29], airway epithelial cells [9], keratinocytes, melanocytes, and fibroblasts.It has been strongly associated with detecting thermal [26,61] and chemical nociception [76].Ca 2+ plays an important role in the modulation of this channel [46,74] where the increase in intracellular Ca 2+ activates and potentiates its response to different agonists [36].Recent evidence suggests that TRPA1 is implicated in developing chemotherapeutic-induced peripheral neuropathy (CIPN), mediating the mechanical and cold hypersensitivity provoked by platinum-based anticancer drugs [44].Furthermore, TRPA1 is overexpressed in several cancer types, including breast carcinoma.This channel promotes an oxidative stress defense in response to reactive oxygen species (ROS) and Ca 2+ influx induced by TRPA1 activation reduces chemosensitivity [64].
In this study, we explored the physical and functional interaction between TRPA1 and NCS-1 and investigated how NCS-1 can modulate the expression and electrophysiological activity of TRPA1.These findings provide new insights into the dynamics of these two molecules, which may contribute to understanding cancer and pathophysiological mechanisms.

Cells
The MDA-MB-231 human breast cancer cell line (RRID:CVCL_0062) was obtained from ATCC (American Type Culture Collection, Manassas, VA).This cell line was maintained at 37 °C with 5% CO 2 in Dulbecco's modified essential medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 1% glutamine, and 1% penicillin/streptomycin.The cells were stably transfected with a previously modified lentivirus and then replicated to generate knockdown (KD) and overexpression (OE) NCS-1 cell lines.The level of NCS1 expression was corroborated, and all cells were passaged for use no more than three months after being thawed and maintained until the start of the experiments.
The SH-SY5Y human cell line (RRID:CVCL_0019) was obtained from ATCC and maintained at 37 °C with 5% CO 2 in DMEM supplemented with 10% FBS, 1% penicillin/streptomycin, and 1% essential amino acids until the experiments were performed.

Cell viability assay
Cell viability was determined by the 4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT) assay.Briefly, SH-SY5Y cells were seeded at a density of 2 × 10 4 cells/ well in 96-well plates and incubated in a 37 °C, 5% CO 2 incubator.After overnight incubation and subsequent reagent exposure, MTT solution (0.5 mg/ml) was added to each well and incubated for 4 h.After incubation, the MTT solution was removed and 100 µl DMSO was added to dissolve the formazan crystals.Absorbance at 570 nm was measured with a microplate reader (ELX800; BioTek Instruments, Inc., Winooski, VT, USA).

Reverse Transcription-Quantitative Polymerase Chain Reaction (qRT-PCR)
Total RNA was extracted from cultured cells using an RNeasy kit (QIAGEN Science, Hilden, Germany) and treated with 55 U RNase-free DNase (QIAGEN Science, Hilden, Germany) following the manufacturer's instructions.A Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific Inc., Waltham, MA) was employed to determine the purity of the RNA by the 260 nm/280 nm absorbance ratio.RT-qPCR was developed by a StepOnePlus™ Real-Time PCR System (Applied Biosystems), transforming RNA in DNAc using a TaqMan® RNA-to-CT TM 1-Step Kit (Applied Biosystems) following the manufacturer's instructions.The genes evaluated were TRPA1 (Hs00175798_m1) and β-actin (Hs01060665_g1), which were used as controls.The following protocol was applied: initial denaturation at -50 °C (30 min) and 95 °C (15 min) and 40 cycles of 95 °C (15 s) and 60 °C (60 s).The results were analyzed employing the 2 −ΔΔCt method and reported as relative gene expression normalized to the average cycle threshold for the β-actin gene.

Electrophysiology
Whole-cell or inside-out patch-clamp techniques were used to record membrane currents (voltage clamp) in MDA-MB-231 and SH-SY5Y cells.Cells were placed in a recording chamber attached to an inverted microscope (TE2000U, Nikon, Tokyo, Japan).Patch pipettes (Sutter Instruments, Novato, CA) were pulled to resistances of 5-8 MΩ (P-97, Sutter Instruments, Novato, CA) and then polished.After a seal of resistance greater than 5 GΩ was obtained, recordings of membrane currents were made using the whole cell or the inside-out mode.An Axopatch 200B amplifier with a CV203BU headstage (Molecular Devices, Union City, CA) was used.Voltage clamp signals were generated by a Digidata 1440A interface (Molecular Devices, Union City, CA).Acquisition and analysis of signals were performed using pCLAMP 10.0 (Molecular Devices, Union City, CA).A ramp protocol where the membrane potential stepped from a holding potential of − 40 mV to + 100 mV and then ramped to − 100 mV was employed.Open probability (Po) was calculated as the proportion of the total recording time that an ion channel spends in its open state in comparison to its closed state.

Spectrophotometric measurement of Ca. 2+ by Fura-2
MDA-MB-231 and SH-SY5Y cells in suspension were loaded with Fura-2 AM (5 µmol/l) through incubation in HEPES buffer solution (HBS) for 30 min at 20 °C and then for 15 min at 37 °C.A spectrophotometer was used to measure fluorescence oscillations for 300 s (FP-6500 spectrophotometer, Jasco, Tokyo, Japan).The temperature was maintained at 37 °C, and magnetic stirring was implemented during the measurements with alternating excitation wavelengths of 340 and 380 nm; the fluorescence emission was detected at 510 nm before each experiment.The 380 nm/340 nm signal was calibrated using the Grynkiewicz et al. method [23] before each experiment.Briefly, the fluorescence ratio was measured in HBS lacking Ca 2+ supplemented with EGTA (1 mM) and in HBS supplemented with ionomycin (300 nM) containing 2 mM Ca 2+ , the Ca 2+ concentration at which Fura-2 is saturated.Maximal and minimal ratios (Rmax and Rmin) were obtained under these two conditions, and the [Ca 2+ ] i values were derived using the following equation: R is the experimentally measured ratio, Sf2 is the fluorescence measured at 380 nm in Ca 2+ -free conditions, and Sb2 is the fluorescence measured at 380 nm with saturating Ca 2+ (2 mM).

Isolation and purification of Ca 2+ -free NCS-1 protein
NCS-1 was produced by overexpression of rat NCS-1 in Stratagene BL21(DE3) Codon Plus RIL competent E. coli cells transformed with a pET21-a + bacterial expression vector subcloned with rat NCS-1 cDNA.The published purification protocol (Zozulya et al., 1995) was slightly modified.Briefly, cells were grown at 37 °C in 2 L baffled flasks with 1 L LB Broth (Miller) plus ampicillin (100 µg/ mL) and chloramphenicol (30 µg/mL).At an OD595 nm of 0.5-0.7,overexpression was induced with 1 mM isopropyldithiogalactoside (IPTG) and incubated for 3 h.Cells were harvested by centrifugation at 3000 rpm for 3 min at 4 °C and resuspended in 10 mL of 50 mM HEPES, 100 mM KCl, 1 mM tris (2-carboxyethyl)phosphine, TCEP), 1 mM MgCl 2 , and 10 mM CaCl 2 at pH 7.5.Bacteria expressing recombinant NCS-1 were lysed in a buffer containing lysozyme (Sigma Aldrich, 2 mg/mL) and DNase I (from bovine pancreas, Sigma Aldrich, 2 µL/1 mL of 2 mg/mL stock) and subjected to three freeze-thaw cycles using ethanol and dry ice.The lysate was homogenized by sonication for 2 min on ice using a 50% duty cycle and an output level of 5.The lysate was then clarified by centrifugation at 40,000 × g (20,000 rpm, 1 h, 4 °C) and sonicated again to reduce sample viscosity.The supernatant was then filtered with a 0.22 µm Steriflip filter unit before hydrophobic interaction chromatography (HIC).HIC was performed using a GE Health care HiTrap Phenyl HP 5 mL column equilibrated with 50 mM HEPES, 100 mM KCl, 1 mM TCEP, 1 mM MgCl 2 , and 10 mM CaCl 2 at pH 7.5.After the application of the lysate three times through the column, the column was washed with ten volumes of the same buffer used to equilibrate the column.The recombinant protein was eluted using 50 mM HEPES, 100 mM KCl, 1 mM TCEP, 1 mM MgCl 2 , and 50 mM EDTA at pH 7.5.The protein was collected in 25 × 1 mL fractions and evaluated for purity by SDS-PAGE and Coomassie staining.Recombinant protein fractions were pooled to be desalted using a Bio-Rad Econo-Pac 10DG column with 50 mM HEPES and 100 mM KCl at pH 7.5 as the exchange buffer.NCS-1 was then dialyzed through a series of buffers in a Pierce Slide-A-Lyzer 7 K MWCO cassette: 1 L 10 mM EDTA at pH 2 for 1.5 h; 1 L Milli-Q water for 1.5 h; 1 L 10 mM HEPES at pH 7.4 for 1.5 h; and finally, 1 L 50 mM HEPES, 100 mM KCl, and 0.5 mM TCEP at pH 7.2 overnight.Dialysis was performed using only plastic containers to prevent Ca 2+ contamination from glass.The protein was concentrated to the desired concentration using a Millipore Ultracel 3 K Amicon Ultra15 centrifugal filter device.

Statistical analysis
The results are presented as the mean ± standard deviation (SD), or as the median and maximal and minimal values in figures.n represents the number of cells tested in the electrophysiological experiments or the number of different cell batches employed in all other assays.Analysis was performed employing GraphPad 9.0 software.Statistically significant differences were determined using one-or two-way ANOVA according to the number of variables, employing Bonferroni post hoc test, where applicable.A p < 0.05 was considered significant, but the exact value of p was reported in each analysis.

Cell survival
To assess the effects of the chemicals to be used on cell survival, cell viability assessment of MDA-MB231 CTR cells and SH-SY5Y cells in the presence of AITC (300 µM), HC030031 (50 nM), wortmannin (1 µM), H89 (10 µM) and chelerythrine (10 µM) for 6 and 12 h was performed.Any of the chemicals was able to induce a significant cell death (Supplementary Fig. 1).

NCS-1 modulates the expression of TRPA1
We evaluated the expression of TRPA1 employing qRT-PCR and Western blot in MDA-MB-231 cells with different levels of NCS-1 expression.TRPA1 was increased in cells that overexpressed NCS-1 (OE, mRNA relative expression 2.401 ± 0.443 p = 0.001, protein relative expression 2.089 ± 0.25 p = 0.003) and lower in cells that underexpressed this protein (KD, mRNA relative expression 0.199 ± 0.112 p = 0.0001, protein relative expression 0.334 ± 0.138 p = 0.002) in comparison with controls (CTR, mRNA relative expression 1.075 ± 0.074, protein relative expression 0.997 ± 0.169) when measured as either mRNA (Fig. 1A) or protein bands (Fig. 1B and C, Supplemenary Fig. 2A).In Western blot experiments, the expression of TRPA1 was confirmed by identifying a band at 128 kDa, as expected for TRPA1.The expression of β-actin was used as a loading control with a band at 42 kDa.The expression of TRPA1 [53] and NCS-1 [65] in SHSY5Y cells was previously reported.

NCS-1 immunoprecipitates with the ion channel TRPA1
We explored the molecular interaction between NCS-1 and TRPA1 using co-immunoprecipitation assays in nonmodified MDA-MB-231 cells (CTR).TRPA1 was detected by Western blot in immunoprecipitates pulled down by anti-NCS-1, and NCS-1 was detected by Western blot in immunoprecipitates pulled down by anti-TRPA1, showing the existence of a stable protein-protein interaction (Fig. 1D, Supplementary Fig. 2B, n = 4).

NCS-1 enhances I TRPA1 density, open probability (Po), and conductance (G)
The voltage patch-clamp technique was employed to determine whole-cell currents in MDA-MB-231 CTR and SH-SY5Y cells.The specific TRPA1 agonist AITC (300 µM) elicited a predominantly inward current with a characteristic voltage dependence in both MDA-MB-231 CTR (Fig. 2A and B) and SH-SY5Y cells (Fig. 2C and  D).These currents significantly decreased in the presence of the TRPA1 antagonist HC-030031 (HC030, 50 µM), which confirmed its identity as I TRPA1 .
Inside-out excised patches were generated from MDA-MB-231 CTR and SH-SY5Y cells to assess the direct effect of NCS-1 on I TRPA1 .This configuration is necessary because the NCS-1 protein cannot cross the plasma membrane.To determine the NCS-1 concentration to be used, a concentration-response curve was constructed, assessing the effect at -60 mV and + 60 mV, and the minimal concentration that evoked the maximal response was employed (data not shown).NCS-1 (10 μM) was added to the extracellular solution 1 min before recording the currents.AITC (300 µM), alone and in the presence of HC030031 (50 nM) or NCS-1 (10 μM), was added to activate TRPA1 channels and the currents were recorded for 1 min; the corresponding histograms indicating the time that the channel spent in the open and closed states were constructed (Fig. 3A, B and C) and Powas derived in each case.Using MDA-MB-231 CTR cells, TRPA1 Po was significantly increased by NCS-1 (0.883 ± 0.105 vs 0.575 ± 0.07, p = 0.0006, Fig. 3D).

NCS-1 increases TRPA1-dependent Ca 2+ influx and this effect is dependent on PI3K pathway activation, but not on PKA or PKC pathways
The effects of AITC on intracellular Ca 2+ concentrations were recorded using suspensions of MDA-MB-231 KD, CTR, and OE cells.This agent significantly increased the intracellular Ca 2+ concentrations in all three cell lines.However, the increase was higher in OE cells and markedly lower in KD cells than in CTR cells (Fig. 6A and B).AITC also elicited an increase in intracellular Ca 2+ in SH-SY5Y cells (Fig. 6B).This increase was inhibited by HC030 in all three MDA-MB-231 cell lines and SH-SY5Y cells (Fig. 6A and B), confirming that TRPA1 was the pathway responsible for Ca 2+ .The TRPA1dependent Ca 2+ increase in OE and CTR cells and SH-SY5Y cells was lower in the presence of wortmannin but unaffected by treatment with H-89 or chelerythrine.This effect of wortmannin was not observed in KD cells (Fig. 6B).

Discussion
The present study shows that TRPA1 is expressed in MDA-MB and SH-SY5Y cell lines.Furthermore, our results support the existence of a functional association between this ion channel and NCS-1.We observed a regulatory relationship in which the expression and electrophysiological properties of TRPA1 are regulated by the levels of NCS-1, probably through PI3K pathway activation.
NCS-1 interacts with several proteins and modulates their signaling pathways, executing roles in neurotransmission [28,50], neuroprotection [40], and nonneuronal processes [41,72].However, to date, the TRPA1 channel has not been linked with NCS-1, although some of the most essential functions of TRPA1 appear to require a close relationship with this Ca 2+ sensor, such as memory [51], neuronal survival [30,31,58,59], cell death [15,70], Parkinson's disease [17,43], CIPN [2,44], and cancer [18,55,63].There is little knowledge of the mechanisms that lead to transcriptional regulation of TRPA1 [25].Our results show that the expression of TRPA1 directly correlates with NCS-1 levels.This finding suggests a regulatory role of the NCS-1 protein in the gene expression of the channel.We know that this Ca 2+ sensor is present in nuclear and perinuclear regions, where it modulates Ca 2+ levels in cardiomyocytes [69].Furthermore, NCS-1 regulates gene transcription/stabilization of mRNAs in neurons [33], making this hypothesis reasonable.Nonetheless, NCS-1 might also control gene expression by indirect mechanisms.
TRPA1 is activated by an extensive variety of factors and molecules [36], where many of these factors are irritants [76].TRPA1 is also strongly regulated by intracellular Ca 2+ levels, eliciting a bell-shaped response: small and localized increases activate the channel and enhance its response to different agonists [27,66], whereas higher Ca 2+ concentrations inactivate it in a process named desensitization or tachyphylaxis [25].There are several hypotheses by which Ca 2+ modulates the activity of the TRPA1 channel, either by the presence of direct binding sites in the cytosolic S2-S3 junction [6,62] or through the intervention of Ca 2+ sensor proteins such as calmodulin (CaM), where EF3 and EF4 of the C-lobe trigger different degrees of TRPA1 binding and regulation [24].We hypothesize that NCS-1, as a Ca 2+ sensor, modulates the activation of the TRPA1 channel in similar ways.First, NCS-1 activates InsP3R, releasing Ca 2+ from the ER [3,56,72], which could lead to direct binding of this ion to TRPA1 and thus increase its activity [10,16,75].Second, given the association between NCS-1 and TRPA1, EF-Hands may exert effects on the channel similar to those described with CaM [24].Studies with Ca 2+ and CaM regulating TRPA1 function suggest that the binding location of NCS-1 is probably close to the channel pore, where a slight conformational change in the effector sites could have a significant impact on Po and conductance [39,67].Other pathways might also be involved in the activation of the TRPA1 channel downstream of NCS-1 [32].
One of the potential regulatory pathways is the PI3K pathway, where NCS-1 is involved as an upregulating factor.In cortical neurons, NCS-1 mediates cell survival and neurite growth through the activation of PI3K [14,41,71].Another interesting result was obtained in the same type of breast cancer cells used in this study, where NCS-1 elicited enhanced cell survival, motility, and metastatic spread through the PI3K pathway [22,34].Here, we demonstrated that NCS-1 activates I TRA1 and that this effect is dependent on PI3K activation, highlighting a novel target of the PI3K pathway.Interestingly, PI3K modifies the channel activity of other members of the TRP family, such as TRPV1 in endothelial cells [11] and neurons [60] and TRPC1 in breast cancer cells [73].Furthermore, the relationship between TRPA1 and PI3K has been shown previously where decreased TRPA1 activity promotes the proliferation and migration of HUVECs, an effect that is abolished by administration of a PI3K inhibitor [32].
MDA-MB-231 cells are a recognized cancer model.In these cells, overexpression of NCS-1 increased migration dynamics, capacity to form distant metastases, and survival in vivo [1], predicting an unfavorable patient outcome [1,5,37].In clinical trials, expression of NCS-1 was also a predictive biomarker for the response to taxane-based neoadjuvant chemotherapy in breast cancer [38], and doxorubicin-induced breast cancer cell death [5].These NCS-1 dependent effects occur via control of Ca 2+ signaling, stress, and IP3K pathway activation [21].It is interesting that TRP channels have also been implicated in similar phenomena, such as the proliferation and migration of breast cancer cells.For example, TRPM7, TRPM8, and TRPV6, have been suggested as biomarkers [49,54].
NCS1 and TRPA1 have a role not only in cancer but also in the development of neuropathy, a reason to carry out tests in neuronal models such as SH-SY5Y cells.For example, paclitaxel induces the binding of NCS-1 to InsP3R, changing cytosolic Ca 2+ concentration and triggering intracellular signals that lead to paclitaxel-induced peripheral and central nervous system neuropathy [4,45].
In summary, our work shows the functional interaction between NCS-1 and TRPA1 in MDA-MB-231 cells, a breast cancer cell line, and SH-SY5Y cells, a neuronal model.We found that the levels of NCS-1 correlate with TRPA1 at both the mRNA and protein levels.Also, the functional properties of TRPA1 are modified by this NCS-1.We also observed that the PI3K pathway, but not PKA or PKC, is involved in this relationship, probably as an effector downstream of NCS-1 activation.These results are important to understanding the mechanisms underlying diverse conditions where Ca 2+ dynamics are compromised, inspiring further research and developing new treatments for breast cancer and chemotherapy induced neuropathy, among other related diseases.
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Fig. 1 A
Fig. 1 A. Comparative expression of TRPA1 mRNA in CTR, KD and OE MDA-MB231, measured by qRT-PCR.Human β-actin was amplified as a control.Note the different levels of expression in the three different MDA-MB231 cell lines.B. Representative Western blot showing the expression of TRPA1 protein in the same cell lines in two different batches of cells.Human β-actin was used as a control.Note the different protein levels in the three different MDA-MB231 cell lines.* denotes significance (p < 0.05) comparing with KD cells

Fig. 2 A
Fig. 2 A. Typical I-V recording of the current obtained in individual MDA-MB231 CTR cells, elicited by a ramp protocol from -100 mV to + 100 mV and activated by AITC (black trace), which is abolished when the TRPA1 inhibitor HC-030031 is present (gray trace).B. I-V relationship of the AITC-induced current in MDA-MB231 CTR cells in the absence (black trace) and presence (gray trace) of HC-030031.C. Typical I-V recording of the current obtained in individual SH-SY5Y cells, elicited by a ramp protocol from -100 mV to + 100 mV and activated by AITC (black trace), which is abolished when the TRPA1 inhibitor HC-030031 is present (gray trace).D. I-V relationship of the AITC-induced current in SH-SY5Y cells in the absence (black trace) and presence (gray trace) of HC-030031.E. Typical

Fig. 3 A
Fig. 3 A. Recordings of single channel activity (opening is downward) and corresponding amplitude histogram of single-channel currents through TRPA1, activated by AITC, in MDA-MB231 CTR cells, at -60 mV.B. Recordings of single channel activity (opening is downward) and corresponding amplitude histogram of singlechannel currents through TRPA1, activated by AITC, in the presence of HC030031, at -60 mV.C. Recordings of single channel activity (opening is downward) and corresponding amplitude histogram of

Fig. 4 A
Fig. 4 A. Recordings of single channel activity (opening is downward) and corresponding amplitude histogram of single-channel currents through TRPA1, activated by AITC, in SH-SY5Y cells, at -60 mV.B. C. Recordings of single channel activity (opening is downward) and corresponding amplitude histogram of single-channel currents through TRPA1, activated by AITC, in the presence of HC030031, at -60 mV.C. Recordings of single channel activity (opening is downward) and corresponding amplitude histogram

Fig. 5 A
Fig. 5 A. Comparison of the mean open probability (Po) in 8 different patches from MDA-MB231 CTR cells in the conditions described in A. B. Comparison of the mean open probability (Po) in 8 different patches from SH-SY5Y cells in the conditions described in B. C. Comparison of the mean inward (IG TRPA1 ) and outward (OG TRPA1 ) conductances in 6 different patches from MDA-MB231 CTR cells in the presence of NCS1 and under the effect of wortmannin (WOR), H89 and chelerythrine (CHEL), calculated from I-V linear regressions.D. Comparison of the mean inward (IG TRPA1 ) and outward (OG TRPA1 ) conductances in 6 different patches from SH-SY5Y cells in the presence of NCS1 and under the effect of wortmannin (WOR), H89 and chelerythrine (CHEL), calculated from I-V linear regressions