Critical amino acid residues regulating TRPA1 Zn2+ response: A comparative study across species.

Cellular zinc ions (Zn2+) are crucial for signal transduction in various cell types. The transient receptor potential (TRP) ankyrin 1 (TRPA1) channel, known for its sensitivity to intracellular Zn2+ ([Zn2+]i), has been a subject of limited understanding regarding its molecular mechanism. Here, we used metal ion-affinity prediction, three-dimensional structural modeling, and mutagenesis, utilizing data from the Protein Data Bank and AlphaFold database, to elucidate the [Zn2+]i binding domain (IZD) structure composed by specific AAs residues in human (hTRPA1) and chicken TRPA1 (gTRPA1). External Zn2+ induced activation in hTRPA1, while not in gTRPA1. Moreover, external Zn2+ elevated [Zn2+]i specifically in hTRPA1. Notably, both hTRPA1 and gTRPA1 exhibited inherent sensitivity to [Zn2+]i, as evidenced by their activation upon internal Zn2+ application. The critical AAs within IZDs, specifically histidine at 983/984, lysine at 711/717, tyrosine at 714/720, and glutamate at 987/988 in IZD1, and H983/H984, tryptophan at 710/716, E854/E855, and glutamine at 979/980 in IZD2, were identified in hTRPA1/gTRPA1. Furthermore, mutations, such as the substitution of arginine at 919 (R919) to H919, abrogated the response to external Zn2+ in hTRPA1. Among single-nucleotide polymorphisms (SNPs) at Y714 and a triple SNP at R919 in hTRPA1, we revealed that the Zn2+ responses were attenuated in mutants carrying the Y714 and R919 substitution to asparagine and proline, respectively. Overall, this study unveils the intrinsic sensitivity of hTRPA1 and gTRPA1 to [Zn2+]i mediated through IZDs. Furthermore, our findings suggest that specific SNP mutations can alter the responsiveness of hTRPA1 to extracellular and intracellular Zn2+.

Transient receptor potential melastatin 3 dysfunction in post COVID-19 condition and myalgic encephalomyelitis/chronic fatigue syndrome patients.

BACKGROUND: Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a severe multisystemic condition associated with post-infectious onset, impaired natural killer (NK) cell cytotoxicity and impaired ion channel function, namely Transient Receptor Potential Melastatin 3 (TRPM3). Long-term effects of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus has resulted in neurocognitive, immunological, gastrointestinal, and cardiovascular manifestations recently recognised as post coronavirus disease 2019 (COVID-19) condition. The symptomatology of ME/CFS overlaps significantly with post COVID-19; therefore, this research aimed to investigate TRPM3 ion channel function in post COVID-19 condition patients. METHODS: Whole-cell patch-clamp technique was used to measure TRPM3 ion channel activity in isolated NK cells of N = 5 ME/CFS patients, N = 5 post COVID-19 patients, and N = 5 healthy controls (HC). The TRPM3 agonist, pregnenolone sulfate (PregS) was used to activate TRPM3 function, while ononetin was used as a TRPM3 antagonist. RESULTS: As reported in previous research, PregS-induced TRPM3 currents were significantly reduced in ME/CFS patients compared with HC (p = 0.0048). PregS-induced TRPM3 amplitude was significantly reduced in post COVID-19 condition compared with HC (p = 0.0039). Importantly, no significant difference was reported in ME/CFS patients compared with post COVID-19 condition as PregS-induced TRPM3 currents of post COVID-19 condition patients were similar of ME/CFS patients currents (p > 0.9999). Isolated NK cells from post COVID-19 condition and ME/CFS patients were resistant to ononetin and differed significantly with HC (p < 0.0001). CONCLUSION: The results of this investigation suggest that post COVID-19 condition patients may have impaired TRPM3 ion channel function and provide further evidence regarding the similarities between post COVID-19 condition and ME/CFS. Impaired TRPM3 channel activity in post COVID-19 condition patients suggest impaired ion mobilisation which may consequently impede cell function resulting in chronic post-infectious symptoms. Further investigation into TRPM3 function may elucidate the pathomechanism, provide a diagnostic and therapeutic target for post COVID-19 condition patients and commonalities with ME/CFS patients.

Impaired TRPM3-dependent calcium influx and restoration using Naltrexone in natural killer cells of myalgic encephalomyelitis/chronic fatigue syndrome patients.

BACKGROUND: Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a serious disorder of unknown aetiology. While the pathomechanism of ME/CFS remains elusive, reduced natural killer (NK) cell cytotoxic function is a consistent immunological feature. NK cell effector functions rely on long-term sustained calcium (Ca2+) influx. In recent years evidence of transient receptor potential melastatin 3 (TRPM3) dysfunction supports the hypothesis that ME/CFS is potentially an ion channel disorder. Specifically, reports of single nucleotide polymorphisms, low surface expression and impaired function of TRPM3 have been reported in NK cells of ME/CFS patients. It has been reported that mu (µ)-opioid receptor (µOR) agonists, known collectively as opioids, inhibit TRPM3. Naltrexone hydrochloride (NTX), a µOR antagonist, negates the inhibitory action of µOR on TRPM3 function. Importantly, it has recently been reported that NTX restores impaired TRPM3 function in NK cells of ME/CFS patients. METHODS: Live cell immunofluorescent imaging was used to measure TRPM3-dependent Ca2+ influx in NK cells isolated from n = 10 ME/CFS patients and n = 10 age- and sex-matched healthy controls (HC) following modulation with TRPM3-agonist, pregnenolone sulfate (PregS) and TRPM3-antaognist, ononetin. The effect of overnight (24 h) NTX in vitro treatment on TRPM3-dependent Ca2+ influx was determined. RESULTS: The amplitude (p < 0.0001) and half-time of Ca2+ response (p < 0.0001) was significantly reduced at baseline in NK cells of ME/CFS patients compared with HC. Overnight treatment of NK cells with NTX significantly improved TRPM3-dependent Ca2+ influx in ME/CFS patients. Specifically, there was no significance between HC and ME/CFS patients for half-time response, and the amplitude of Ca2+ influx was significantly increased in ME/CFS patients (p < 0.0001). CONCLUSION: TRPM3-dependent Ca2+ influx was restored in ME/CFS patients following overnight treatment of isolated NK cells with NTX in vitro. Collectively, these findings validate that TRPM3 loss of function results in altered Ca2+ influx supporting the growing evidence that ME/CFS is a TRP ion channel disorder and that NTX provides a potential therapeutic intervention for ME/CFS.

Potent Activation of Human but Not Mouse TRPA1 by JT010.

Transient receptor potential (TRP) ankyrin repeat 1 (TRPA1), which is involved in inflammatory pain sensation, is activated by endogenous factors, such as intracellular Zn2+ and hydrogen peroxide, and by irritant chemical compounds. The synthetic compound JT010 potently and selectively activates human TRPA1 (hTRPA1) among the TRPs. Therefore, JT010 is a useful tool for analyzing TRPA1 functions in biological systems. Here, we show that JT010 is a potent activator of hTRPA1, but not mouse TRPA1 (mTRPA1) in human embryonic kidney (HEK) cells expressing hTRPA1 and mTRPA1. Application of 0.3-100 nM of JT010 to HEK cells with hTRPA1 induced large Ca2+ responses. However, in HEK cells with mTRPA1, the response was small. In contrast, both TRPA1s were effectively activated by allyl isothiocyanate (AITC) at 10-100 µM. Similar selective activation of hTRPA1 by JT010 was observed in electrophysiological experiments. Additionally, JT010 activated TRPA1 in human fibroblast-like synoviocytes with inflammation, but not TRPA1 in mouse dorsal root ganglion cells. As cysteine at 621 (C621) of hTRPA1, a critical cysteine for interaction with JT010, is conserved in mTRPA1, we applied JT010 to HEK cells with mutations in mTRPA1, where the different residue of mTRPA1 with tyrosine at 60 (Y60), with histidine at 1023 (H1023), and with asparagine at 1027 (N1027) were substituted with cysteine in hTRPA1. However, these mutants showed low sensitivity to JT010. In contrast, the mutation of hTRPA1 at position 669 from phenylalanine to methionine (F669M), comprising methionine at 670 in mTRPA1 (M670), significantly reduced the response to JT010. Moreover, the double mutant at S669 and M670 of mTRPA1 to S669E and M670F, respectively, induced slight but substantial sensitivity to 30 and 100 nM JT010. Taken together, our findings demonstrate that JT010 potently and selectively activates hTRPA1 but not mTRPA1.

The inflammatory regulation of TRPA1 expression in human A549 lung epithelial cells.

Transient receptor potential ankyrin-1 (TRPA1) is an ion channel mediating pain and cough signals in sensory neurons. We and others have shown that TRPA1 is also expressed in some non-neuronal cells and supports inflammatory responses. To address the pathogenesis and to uncover potential targets for pharmacotherapy in inflammatory lung diseases, we set out to study the expression of TRPA1 in human A549 lung epithelial cells under inflammatory conditions. TRPA1 expression was determined by RT-qPCR and Western blotting at a mRNA and protein level, respectively and its function was studied by Fluo 3-AM intracellular Ca2+ measurement in A549 lung epithelial cells. TRPA1 promoter activity was assessed by reporter gene assay. TRPA1 expression was very low in A549 cells in the absence of inflammatory stimuli. Tumor necrosis factor-α (TNF-α) significantly increased TRPA1 expression and a synergy was found between TNF-α, interleukin-1ß (IL-1ß) and interferon-γ (IFN-γ). Reporter gene experiments indicate that the combination of TNF-α and IL-1ß increases TRPA1 promoter activity while the effect of IFN-γ seems to be non-transcriptional. Interestingly, the glucocorticoid dexamethasone downregulated TRPA1 expression in A549 cells by reducing TRPA1 mRNA stability in a transcription-dependent manner. Furthermore, pharmacological blockade of TRPA1 reduced the production of the pro-inflammatory cytokine IL-8. In conclusion, TRPA1 was found to be expressed and functional in human A549 lung epithelial cells under inflammatory conditions. The anti-inflammatory steroid dexamethasone reduced TRPA1 expression through post-transcriptional mechanisms. The results reveal TRPA1 as a potential mediator and drug target in inflammatory lung conditions.

HIF-1α Dependent Upregulation of ZIP8, ZIP14, and TRPA1 Modify Intracellular Zn2+ Accumulation in Inflammatory Synoviocytes.

Intracellular free zinc ([Zn2+]i) is mobilized in neuronal and non-neuronal cells under physiological and/or pathophysiological conditions; therefore, [Zn2+]i is a component of cellular signal transduction in biological systems. Although several transporters and ion channels that carry Zn2+ have been identified, proteins that are involved in Zn2+ supply into cells and their expression are poorly understood, particularly under inflammatory conditions. Here, we show that the expression of Zn2+ transporters ZIP8 and ZIP14 is increased via the activation of hypoxia-induced factor 1α (HIF-1α) in inflammation, leading to [Zn2+]i accumulation, which intrinsically activates transient receptor potential ankyrin 1 (TRPA1) channel and elevates basal [Zn2+]i. In human fibroblast-like synoviocytes (FLSs), treatment with inflammatory mediators, such as tumor necrosis factor-α (TNF-α) and interleukin-1α (IL-1α), evoked TRPA1-dependent intrinsic Ca2+ oscillations. Assays with fluorescent Zn2+ indicators revealed that the basal [Zn2+]i concentration was significantly higher in TRPA1-expressing HEK cells and inflammatory FLSs. Moreover, TRPA1 activation induced an elevation of [Zn2+]i level in the presence of 1 µM Zn2+ in inflammatory FLSs. Among the 17 out of 24 known Zn2+ transporters, FLSs that were treated with TNF-α and IL-1α exhibited a higher expression of ZIP8 and ZIP14. Their expression levels were augmented by transfection with an active component of nuclear factor-κB P65 and HIF-1α expression vectors, and they could be abolished by pretreatment with the HIF-1α inhibitor echinomycin (Echi). The functional expression of ZIP8 and ZIP14 in HEK cells significantly increased the basal [Zn2+]i level. Taken together, Zn2+ carrier proteins, TRPA1, ZIP8, and ZIP14, induced under HIF-1α mediated inflammation can synergistically change [Zn2+]i in inflammatory FLSs.

Mechanically activated Piezo1 channels of cardiac fibroblasts stimulate p38 mitogen-activated protein kinase activity and interleukin-6 secretion.

Piezo1 is a mechanosensitive cation channel with widespread physiological importance; however, its role in the heart is poorly understood. Cardiac fibroblasts help preserve myocardial integrity and play a key role in regulating its repair and remodeling following stress or injury. Here we investigated Piezo1 expression and function in cultured human and mouse cardiac fibroblasts. RT-PCR experiments confirmed that Piezo1 mRNA in cardiac fibroblasts is expressed at levels similar to those in endothelial cells. The results of a Fura-2 intracellular Ca2+ assay validated Piezo1 as a functional ion channel that is activated by its agonist, Yoda1. Yoda1-induced Ca2+ entry was inhibited by Piezo1 blockers (gadolinium and ruthenium red) and was reduced proportionally by siRNA-mediated Piezo1 knockdown or in murine Piezo1+/- cells. Results from cell-attached patch clamp recordings on human cardiac fibroblasts established that they contain mechanically activated ion channels and that their pressure responses are reduced by Piezo1 knockdown. Investigation of Yoda1 effects on selected remodeling genes indicated that Piezo1 activation increases both mRNA levels and protein secretion of IL-6, a pro-hypertrophic and profibrotic cytokine, in a Piezo1-dependent manner. Moreover, Piezo1 knockdown reduced basal IL-6 expression from cells cultured on softer collagen-coated substrates. Multiplex kinase activity profiling combined with kinase inhibitor experiments and phosphospecific immunoblotting established that Piezo1 activation stimulates IL-6 secretion via the p38 mitogen-activated protein kinase downstream of Ca2+ entry. In summary, cardiac fibroblasts express mechanically activated Piezo1 channels coupled to secretion of the paracrine signaling molecule IL-6. Piezo1 may therefore be important in regulating cardiac remodeling.

Piezo1 integration of vascular architecture with physiological force.

The mechanisms by which physical forces regulate endothelial cells to determine the complexities of vascular structure and function are enigmatic. Studies of sensory neurons have suggested Piezo proteins as subunits of Ca(2+)-permeable non-selective cationic channels for detection of noxious mechanical impact. Here we show Piezo1 (Fam38a) channels as sensors of frictional force (shear stress) and determinants of vascular structure in both development and adult physiology. Global or endothelial-specific disruption of mouse Piezo1 profoundly disturbed the developing vasculature and was embryonic lethal within days of the heart beating. Haploinsufficiency was not lethal but endothelial abnormality was detected in mature vessels. The importance of Piezo1 channels as sensors of blood flow was shown by Piezo1 dependence of shear-stress-evoked ionic current and calcium influx in endothelial cells and the ability of exogenous Piezo1 to confer sensitivity to shear stress on otherwise resistant cells. Downstream of this calcium influx there was protease activation and spatial reorganization of endothelial cells to the polarity of the applied force. The data suggest that Piezo1 channels function as pivotal integrators in vascular biology.

Regulation of NRF2, AP-1 and NF-κB by cigarette smoke exposure in three-dimensional human bronchial epithelial cells.

Cigarette smoke (CS) is a complex mixture of chemicals and interacts with various physiological processes. We previously reported that nuclear factor erythroid 2-related factor 2 (NRF2) was the most sensitive transcription factor to aqueous CS extract (AqCSE) exposure in monolayer cultured human bronchial epithelial cell lines. Recently, in vitro three-dimensional (3D) culture models have been used to supplement pharmacological and toxicological assessments. Bronchial epithelium models in particular are useful for the evaluation of substances that directly contact the respiratory tract, such as CS. In the present study, we used 3D-cultured human bronchial epithelial cells (HBECs) to assess activation of transcription factors and relevant gene expression in response to AqCSE, primarily focusing on NRF2 and nuclear factor-kappa B (NF-κB) pathways. The 3D-cultured HBECs exposed to AqCSE showed expression of NRF2 and its nuclear translocation in addition to upregulation of genes related to oxidative stress. Our results suggest that the NRF2 pathway was the dominant pathway when 3D-cultured HBECs were exposed to AqCSE at a low dose, supporting our previous findings that NRF2 was the most sensitive transcription factor in response to AqCSE. Expression and nuclear translocation of NF-κB were not increased, although proinflammatory genes were upregulated. However, another inflammation-related transcription factor, activation protein 1, was induced by AqCSE. Gene classification analysis suggested that induction of the inflammatory response by AqCSE was dependent on NRF2 and activation protein 1 rather than NF-κB.

Castration Induces Down-Regulation of A-Type K+ Channel in Rat Vas Deferens Smooth Muscle.

A-type K+ channels contribute to regulating the propagation and frequency of action potentials in smooth muscle cells (SMCs). The present study (i) identified the molecular components of A-type K+ channels in rat vas deferens SMs (VDSMs) and (ii) showed the long-term, genomic effects of testosterone on their expression in VDSMs. Transcripts of the A-type K+ channel α subunit, Kv4.3L and its regulatory ß subunits, KChIP3, NCS1, and DPP6-S were predominantly expressed in rat VDSMs over the other related subtypes (Kv4.2, KChIP1, KChIP2, KChIP4, and DPP10). A-type K+ current (IA) density in VDSM cells (VDSMCs) was decreased by castration without changes in IA kinetics, and decreased IA density was compensated for by an oral treatment with 17α-methyltestosterone (MET). Correspondingly, in the VDSMs of castrated rats, Kv4.3L and KChIP3 were down-regulated at both the transcript and protein expression levels. Changes in Kv4.3L and KChIP3 expression levels were compensated for by the treatment with MET. These results suggest that testosterone level changes in testosterone disorders and growth processes control the functional expression of A-type K+ channels in VDSMCs.

PIEZO1 and TRPV4, which Are Distinct Mechano-Sensors in the Osteoblastic MC3T3-E1 Cells, Modify Cell-Proliferation.

Mechanical-loading and unloading can modify osteoblast functioning. Ca2+ signaling is one of the earliest events in osteoblasts to induce a mechanical stimulus, thereby demonstrating the importance of the underlying mechanical sensors for the sensation. Here, we examined the mechano-sensitive channels PIEZO1 and TRPV4 were involved in the process of mechano-sensation in the osteoblastic MC3T3-E1 cells. The analysis of mRNA expression revealed a high expression of Piezo1 and Trpv4 in these cells. We also found that a PIEZO1 agonist, Yoda1, induced Ca2+ response and activated cationic currents in these cells. Ca2+ response was elicited when mechanical stimulation (MS), with shear stress, was induced by fluid flow in the MC3T3-E1 cells. Gene knockdown of Piezo1 in the MC3T3-E1 cells, by transfection with siPiezo1, inhibited the Yoda1-induced response, but failed to inhibit the MS-induced response. When MC3T3-E1 cells were transfected with siTrpv4, the MS-induced response was abolished and Yoda1 response was attenuated. Moreover, the MS-induced response was inhibited by a TRPV4 antagonist HC-067047 (HC). Yoda1 response was also inhibited by HC in MC3T3-E1 cells and HEK cells, expressing both PIEZO1 and TRPV4. Meanwhile, the activation of PIEZO1 and TRPV4 reduced the proliferation of MC3T3-E1, which was reversed by knockdown of PIEZO1, and TRPV4, respectively. In conclusion, TRPV4 and PIEZO1 are distinct mechano-sensors in the MC3T3-E1 cells. However, PIEZO1 and TRPV4 modify the proliferation of these cells, implying that PIEZO1 and TRPV4 may be functional in the osteoblastic mechano-transduction. Notably, it is also found that Yoda1 can induce TRPV4-dependent Ca2+ response, when both PIEZO1 and TRPV4 are highly expressed.

(-)-Englerin A-evoked Cytotoxicity Is Mediated by Na+ Influx and Counteracted by Na+/K+-ATPase.

(-)-Englerin A ((-)-EA) has a rapid and potent cytotoxic effect on several types of cancer cell that is mediated by plasma membrane ion channels containing transient receptor potential canonical 4 (TRPC4) protein. Because these channels are Ca2+-permeable, it was initially thought that the cytotoxicity arose as a consequence of Ca2+ overload. Here we show that this is not the case and that the effect of (-)-EA is mediated by a heteromer of TRPC4 and TRPC1 proteins. Both TRPC4 and TRPC1 were required for (-)-EA cytotoxicity; however, although TRPC4 was necessary for the (-)-EA-evoked Ca2+ elevation, TRPC1 was not. TRPC1 either had no role or was a negative regulator of Ca2+ entry. By contrast, both TRPC4 and TRPC1 were necessary for monovalent cation entry evoked by (-)-EA, and (-)-EA-evoked cell death was dependent upon entry of the monovalent cation Na+ We therefore hypothesized that Na+/K+-ATPase might act protectively by counteracting the Na+ load resulting from sustained Na+ entry. Indeed, inhibition of Na+/K+-ATPase by ouabain potently and strongly increased (-)-EA-evoked cytotoxicity. The data suggest that (-)-EA achieves cancer cell cytotoxicity by inducing sustained Na+ entry through heteromeric TRPC1/TRPC4 channels and that the cytotoxic effect of (-)-EA can be potentiated by Na+/K+-ATPase inhibition.

Picomolar, selective, and subtype-specific small-molecule inhibition of TRPC1/4/5 channels.

The concentration of free cytosolic Ca2+ and the voltage across the plasma membrane are major determinants of cell function. Ca2+-permeable non-selective cationic channels are known to regulate these parameters, but understanding of these channels remains inadequate. Here we focus on transient receptor potential canonical 4 and 5 proteins (TRPC4 and TRPC5), which assemble as homomers or heteromerize with TRPC1 to form Ca2+-permeable non-selective cationic channels in many mammalian cell types. Multiple roles have been suggested, including in epilepsy, innate fear, pain, and cardiac remodeling, but limitations in tools to probe these channels have restricted progress. A key question is whether we can overcome these limitations and develop tools that are high-quality, reliable, easy to use, and readily accessible for all investigators. Here, through chemical synthesis and studies of native and overexpressed channels by Ca2+ and patch-clamp assays, we describe compound 31, a remarkable small-molecule inhibitor of TRPC1/4/5 channels. Its potency ranged from 9 to 1300 pm, depending on the TRPC1/4/5 subtype and activation mechanism. Other channel types investigated were unaffected, including TRPC3, TRPC6, TRPV1, TRPV4, TRPA1, TRPM2, TRPM8, and store-operated Ca2+ entry mediated by Orai1. These findings suggest identification of an important experimental tool compound, which has much higher potency for inhibiting TRPC1/4/5 channels than previously reported agents, impressive specificity, and graded subtype selectivity within the TRPC1/4/5 channel family. The compound should greatly facilitate future studies of these ion channels. We suggest naming this TRPC1/4/5-inhibitory compound Pico145.

Loss of Transient Receptor Potential Melastatin 3 ion channel function in natural killer cells from Chronic Fatigue Syndrome/Myalgic Encephalomyelitis patients.

BACKGROUND: Chronic Fatigue Syndrome (CFS)/ Myalgic Encephalomyelitis (ME) is a debilitating disorder that is accompanied by reduced cytotoxic activity in natural killer (NK) cells. NK cells are an essential innate immune cell, responsible for recognising and inducing apoptosis of tumour and virus infected cells. Calcium is an essential component in mediating this cellular function. Transient Receptor Potential Melastatin 3 (TRPM3) cation channels have an important regulatory role in mediating calcium influx to help maintain cellular homeostasis. Several single nucleotide polymorphisms have been reported in TRPM3 genes from isolated peripheral blood mononuclear cells, NK and B cells in patients with CFS/ME and have been proposed to correlate with illness presentation. Moreover, a significant reduction in both TRPM3 surface expression and intracellular calcium mobilisation in NK cells has been found in CFS/ME patients compared with healthy controls. Despite the functional importance of TRPM3, little is known about the ion channel function in NK cells and the epiphenomenon of CFS/ME. The objective of the present study was to characterise the TRPM3 ion channel function in NK cells from CFS/ME patients in comparison with healthy controls using whole cell patch-clamp techniques. METHODS: NK cells were isolated from 12 age- and sex-matched healthy controls and CFS patients. Whole cell electrophysiology recording has been used to assess TRPM3 ion channel activity after modulation with pregnenolone sulfate and ononetin. RESULTS: We report a significant reduction in amplitude of TRPM3 current after pregnenolone sulfate stimulation in isolated NK cells from CFS/ME patients compared with healthy controls. In addition, we found pregnenolone sulfate-evoked ionic currents through TRPM3 channels were significantly modulated by ononetin in isolated NK cells from healthy controls compared with CFS/ME patients. CONCLUSIONS: TRPM3 activity is impaired in CFS/ME patients suggesting changes in intracellular Ca2+ concentration, which may impact NK cellular functions. This investigation further helps to understand the intracellular-mediated roles in NK cells and confirm the potential role of TRPM3 ion channels in the aetiology and pathomechanism of CFS/ME.

PIEZO1 Channel Is a Potential Regulator of Synovial Sarcoma Cell-Viability.

Detection of mechanical stress is essential for diverse biological functions including touch, audition, and maintenance of vascular myogenic tone. PIEZO1, a mechano-sensing cation channel, is widely expressed in neuronal and non-neuronal cells and is expected to be involved in important biological functions. Here, we examined the possibility that PIEZO1 is involved in the regulation of synovial sarcoma cell-viability. Application of a PIEZO1 agonist Yoda1 effectively induced Ca2+ response and cation channel currents in PIEZO1-expressing HEK (HEK-Piezo1) cells and synovial sarcoma SW982 (SW982) cells. Mechanical stress, as well as Yoda1, induced the activity of an identical channel of conductance with 21.6 pS in HEK-Piezo1 cells. In contrast, Yoda1 up to 10 μM had no effects on membrane currents in HEK cells without transfecting PIEZO1. A knockdown of PIEZO1 with siRNA in SW982 cells abolished Yoda1-induced Ca2+ response and significantly reduced cell cell-viability. Because PIEZO1 is highly expressed in SW982 cells and its knockdown affects cell-viability, this gene is a potential target against synovial sarcoma.

Transcriptional repression of HER2 by ANO1 Cl- channel inhibition in human breast cancer cells with resistance to trastuzumab.

The Ca2+-activated Cl- channel ANO1 contributes to tumorigenesis and metastasis in several carcinomas including breast cancer (BCA). Cl- channels have recently been attracting attention as 'transcriptional modulators'. Human epidermal growth factor receptor 2 (HER2) is overexpressed in approximately 30% of patients with BCA, and anti-HER2 monoclonal antibodies such as trastuzumab have emerged as a treatment for metastatic BCA. Among the seven human BCA cell lines examined in the present study, MDA-MB-453 and YMB-1 cells were HER2-positive; however, YMB-1 cell viability showed resistance to trastuzumab. Whole-cell patch-clamp configurations indicated that ANO1 was the main Cl- conductance in YMB-1 cells, and the pharmacological and siRNA-mediated inhibition of ANO1 significantly prevented HER2 transcription in YMB-1 cells. The expression levels of insulin-like growth factor-binding protein 5 (IGFBP5), which is a risk factor for BCA recurrence and metastasis, was not affected by the inhibition of ANO1 in YMB-1 cells. These results suggest that ANO1 Cl- channels may function as a transcriptional regulator of HER2, and ANO1 inhibitors have potential in the treatment of BCA patients with resistance to HER2-targeted therapy.

Molecular mechanisms underlying pimaric acid-induced modulation of voltage-gated K+ channels.

Voltage-gated K+ (KV) channels, which control firing and shape of action potentials in excitable cells, are supposed to be potential therapeutic targets in many types of diseases. Pimaric acid (PiMA) is a unique opener of large conductance Ca2+-activated K+ channel. Here, we report that PiMA modulates recombinant rodent KV channel activity. The enhancement was significant at low potentials (<0 mV) but not at more positive potentials. Application of PiMA significantly shifted the voltage-activation relationships (V1/2) of rodent KV1.1, 1.2, 1.3, 1.4, 1.6 and 2.1 channels (KV1.1-KV2.1) but KV4.3 to lower potentials and prolonged their half-decay times of the deactivation (T1/2D). The amino acid sequence which is responsible for the difference in response to PiMA was examined between KV1.1-KV2.1 and KV4.3 by site-directed mutagenesis of residues in S5 and S6 segments of Kv1.1. The point mutation of Phe332 into Tyr mimics the effects of PiMA on V1/2 and T1/2D and also abolished the further change by addition of PiMA. The results indicate that PiMA enhances voltage sensitivity of KV1.1-KV2.1 channels and suggest that the lipophilic residues including Phe332 in S5 of KV1.1-KV2.1 channels may be critical for the effects of PiMA, providing beneficial information for drug development of KV channel openers.

(-)-Englerin†A is a potent and selective activator of TRPC4 and TRPC5 calcium channels.

Current therapies for common types of cancer such as renal cell cancer are often ineffective and unspecific, and novel pharmacological targets and approaches are in high demand. Here we show the unexpected possibility for the rapid and selective killing of renal cancer cells through activation of calcium-permeable nonselective transient receptor potential canonical (TRPC) calcium channels by the sesquiterpene (-)-englerin A. This compound was found to be a highly efficient, fast-acting, potent, selective, and direct stimulator of TRPC4 and TRPC5 channels. TRPC4/5 activation through a high-affinity extracellular (-)-englerin A binding site may open up novel opportunities for drug discovery aimed at renal cancer.

The NADPH oxidase inhibitor diphenyleneiodonium activates the human TRPA1 nociceptor.

Transient receptor potential ankyrin 1 (TRPA1) is a Ca(2+)-permeable nonselective cation channel expressed in neuronal and nonneuronal cells and plays an important role in acute and inflammatory pain. Here, we show that an NADPH oxidase (NOX) inhibitor, diphenyleneiodonium (DPI), functions as a TRPA1 activator in human embryonic kidney cells expressing human TRPA1 (HEK-TRPA1) and in human fibroblast-like synoviocytes. Application of DPI at 0.03-10 µM induced a Ca(2+) response in HEK-TRPA1 cells in a concentration-dependent manner. The Ca(2+) response was effectively blocked by a selective TRPA1 antagonist, HC-030031 (HC). In contrast, DPI had no effect on HEK cells expressing TRPV1-V4 or TRPM8. Four other NOX inhibitors, apocynin (APO), VAS2870 (VAS), plumbagin, and 2-acetylphenothiazine, also induced a Ca(2+) response in HEK-TRPA1 cells, which was inhibited by pretreatment with HC. In the presence of 5 mM glutathione, the Ca(2+) response to DPI was effectively reduced. Moreover, mutation of cysteine 621 in TRPA1 substantially inhibited the DPI-induced Ca(2+) response, while it did not inhibit the APO- and VAS-induced responses. The channel activity was induced by DPI in excised membrane patches with both outside-out and inside-out configurations. Internal application of neomycin significantly inhibited the DPI-induced inward currents. In inflammatory synoviocytes with TRPA1, DPI evoked a Ca(2+) response that was sensitive to HC. In mice, intraplantar injection of DPI caused a pain-related response which was inhibited by preadministration with HC. Taken together, our findings demonstrate that DPI and other NOX inhibitors activate human TRPA1 without mediating NOX.