Thermal stress involved in TRPV2 promotes tumorigenesis through the pathways of HSP70/27 and PI3K/Akt/mTOR in esophageal squamous cell carcinoma.

BACKGROUND: The transient receptor potential vanilloid receptor 2 (TRPV2) has been found to participate in the pathogenesis of various types of cancers, however, its role(s) in the tumorigenesis of ESCC remain poorly understood. METHODS: Western blotting and immunohistochemistry were performed to determine the expression profiles of TRPV2 in the ESCC patient tissues. A series of in vitro and in vivo experiments were conducted to reveal the role of TRPV2 in the tumorigenesis of ESCC. RESULTS: Our study first uncovered that the activation of TRPV2 by recurrent acute thermal stress (54 °C) or O1821 (20 µM) promoted cancerous behaviours in ESCC cells. The pro-angiogenic capacity of the ESCC cells was found to be enhanced profoundly and both tumour formation and metastasis that originated from the cells were substantially promoted in nude mouse models upon the activation of TRPV2. These effects were inhibited significantly by tranilast (120 µM) and abolished by TRPV2 knockout. Conversely, overexpression of TRPV2 could switch the cells to tumorigenesis upon activation of TRPV2. Mechanistically, the driving role of TRPV2 in the progression of ESCC is mainly regulated by the HSP70/27 and PI3K/Akt/mTOR signalling pathways. CONCLUSIONS: We revealed that TRPV2-PI3K/Akt/mTOR is a novel and promising target for the prevention and treatment of ESCC.

Ameliorating Effect of Umbilical Cord Mesenchymal Stem Cells in a Human Induced Pluripotent Stem Cell Model of Dravet Syndrome.

Dravet syndrome (DS) is a form of severe childhood-onset refractory epilepsy typically caused by a heterozygous loss-of-function mutation. DS patient-derived induced pluripotent stem cells (iPSCs) are appropriate human cells for exploring disease mechanisms and testing new therapeutic strategies in vitro. Repeated spontaneous seizures can cause neuroinflammatory reactions and oxidative stress, resulting in neuronal toxicity, neuronal dysfunction, blood-brain barrier disruption, and hippocampal inflammation. Antiepileptic drug therapy does not delay the development of chronic epilepsy. The application of mesenchymal stem cells (MSCs) is one therapeutic strategy for thwarting epilepsy development. This study evaluated the effects of human umbilical cord mesenchymal stem cell-conditioned medium (HUMSC-CM) in a new in vitro model of neurons differentiated from DS patient-derived iPSCs. In the presence of HUMSC-CM, increases in superoxide dismutase 1 (SOD1), superoxide dismutase 2 (SOD2), glutathione peroxidase (GPX), and glutathione (GSH) levels were found to contribute to a reduction in reactive oxygen species (ROS) levels. In parallel, inflammation was rescued in DS patient-derived neuronal cells via increased expression of anti-inflammatory cytokines (TGF-ß, IL-6, and IL-10) and significant downregulation of tumor necrosis factor-α and interleukin-1ß expression. The intracellular calcium concentration ([Ca2+]i) and malondialdehyde (MDA) and ROS levels were decreased in DS patient-derived cells. In addition, action potential (AP) firing ability was enhanced by HUMSC-CM. In conclusion, HUMSC-CM can effectively eliminate ROS, affect migration and neurogenesis, and promote neurons to enter a highly functional state. Therefore, HUMSC-CM is a promising therapeutic strategy for the clinical treatment of refractory epilepsy such as DS.

Hair follicle-derived mesenchymal stem cells decrease alopecia areata mouse hair loss and reduce inflammation around the hair follicle.

BACKGROUND: Alopecia areata (AA) is a common autoimmune hair loss disease with increasing incidence. Corticosteroids are the most widely used for hair loss treatment; however, long-term usage of hormonal drugs is associated with various side effects. Mesenchymal stem cells (MSCs) therapy has been studied extensively to curb autoimmune diseases without affecting immunity against diseases. METHODS: Hair follicle-derived MSCs (HF-MSCs) were harvested from the waste material of hair transplants, isolated and expanded. The therapeutic effect of HF-MSCs for AA treatment was investigated in vitro AA-like hair follicle organ model and in vivo C3H/HeJ AA mice model. RESULTS: AA-like hair follicle organ in vitro model was successfully established by pre-treatment of mouse vibrissa follicles by interferon-γ (IFN-γ). The AA-like symptoms were relieved when IFN-γ induced AA in vitro model was co-cultured with HF-MSC for 2 days. In addition, when skin grafted C3H/HeJ AA mice models were injected with 106 HF-MSCs once a week for 3 weeks, the transcription profiling and immunofluorescence analysis depicted that HF-MSCs treatment significantly decreased mouse hair loss and reduced inflammation around HF both in vitro and in vivo. CONCLUSIONS: This study provides a new therapeutic approach for alopecia areata based on HF-MSCs toward its future clinical application.

Proarrhythmic effects induced by benzethonium chloride and domiphen bromide in vitro and in vivo.

Benzethonium chloride (BZT) and domiphen bromide (DMP) are widely used as antimicrobials in drugs, vaccines and industry. However, no cardiac safety data has been developed on both compounds. Previously we reported BZT and DMP as high-affinity human ether-a-go-go related gene (HERG) channel inhibitors with unknown proarrhythmic risk. Here, we investigate the cardiotoxicity of BZT and DMP in vitro and in vivo, aiming to improve the safety-in-use of both antimicrobials. In the present study, human iPSC derived cardiomyocytes (hiPSC-CMs) were generated and rabbit models were used to examine the proarrhythmic potential of BZT and DMP. Our results found that BZT and DMP induced time- and dose-dependent decrease in the contractile parameters of hiPSC-CMs, prolonged FPDc (≥ 0.1 µM), caused tachycardia/fibrillation-like oscillation (0.3-1 µM), ultimately progressing to irreversible arrest of beating (≥ 1 µM). The IC50 values of BZT and DMP derived from normalized beat rate were 0.13 µM and 0.10 µM on hiPSC-CMs at 76 days. Moreover, in vivo rabbit ECG data demonstrated that 12.85 mg/kg BZT and 3.85 mg/kg DMP evoked QTc prolongation, noncomplex arrhythmias and ventricular tachycardias. Our findings support the cardiac safety of 0.01 µM BZT/DMP in vitro and the intravenous infusion of 3.85 mg/kg BZT and 1.28 mg/kg DMP in vivo, whereas higher concentrations of both compounds cause mild to moderate cardiotoxicity that should not be neglected during medical and industrial applications.

Generation of UCiPSC-derived neurospheres for cell therapy and its application.

BACKGROUND: Neural stem cell (NSC) therapy remains one of the most potential approaches for the treatment of neurological disorders. The discovery of human induced pluripotent stem cells (hiPSCs) and the establishment of hiPSC-derived human neural stem cells (hiNSCs) have revolutionized the technique of cell therapy. Meanwhile, it is often required that NSCs are stored and transported to a long distance for research or treatment purposes. Although high survival rates could be maintained, conventional methods for cell transportation (dry ice or liquid nitrogen) are inconvenient and expensive. Therefore, the establishment of a safe, affordable, and low-cost strategy to store and transport easily accessible hiPSCs and hiNSCs, with characteristics that match fetal hNSCs, is incredibly urgent. METHODS: We reprogrammed human urinary cells to iPSCs using a non-integrating, virus-free technique and differentiated the iPSCs toward iNSCs/neurospheres and neurons, under Good Manufacturing Practice (GMP)-compatible conditions. The pluripotency of iPSCs and iNSCs was characterized by a series of classical methods (surface markers, karyotype analysis, and in vitro as well as in vivo differentiation capabilities, etc.). RESULTS: Here, our results showed that we successfully generated hiNSCs/neurospheres from more available, non-invasive, and more acceptable urinary cells by a virus-free technique. Next, we demonstrated that the iNSCs differentiated into mature cerebral cortical neurons and neural networks. Interestingly, hiNSCs survived longer as neurospheres at ambient temperature (AT) than those cultured in a monolayer. Within 7 days approximately, the neural viability remained at > 80%, while hiNSCs cultured in a monolayer died almost immediately. Neurospheres exposed to AT that were placed under standard culture conditions (37 °C, 5% CO2) recovered their typical morphology, and retained their proliferation and differentiation abilities. CONCLUSIONS: In this study, we provided a simple method for the storage of NSCs as neurospheres at AT as an alternative method to more costly and inconvenient traditional methods of cryopreservation. This will enable hiNSCs to be transported over long distances at AT and facilitate the therapeutic application of NSCs as neurospheres without any further treatment.

Rational Programming of Cas12a for Early-Stage Detection of COVID-19 by Lateral Flow Assay and Portable Real-Time Fluorescence Readout Facilities.

Coronavirus disease 2019 (COVID-19) caused by the SARS-CoV-2 virus has led to a global pandemic with a high spread rate and pathogenicity. Thus, with limited testing solutions, it is imperative to develop early-stage diagnostics for rapid and accurate detection of SARS-CoV-2 to contain the rapid transmission of the ongoing COVID-19 pandemic. In this regard, there remains little knowledge about the integration of the CRISPR collateral cleavage mechanism in the lateral flow assay and fluorophotometer. In the current study, we demonstrate a CRISPR/Cas12a-based collateral cleavage method for COVID-19 diagnosis using the Cas12a/crRNA complex for target recognition, reverse transcription loop-mediated isothermal amplification (RT-LAMP) for sensitivity enhancement, and a novel DNA capture probe-based lateral flow strip (LFS) or real-time fluorescence detector as the parallel system readout facility, termed CRICOLAP. Our novel approach uses a customized reporter that hybridizes an optimized complementary capture probe fixed at the test line for naked-eye result readout. The CRICOLAP system achieved ultra-sensitivity of 1 copy/µL in ~32 min by portable real-time fluorescence detection and ~60 min by LFS. Furthermore, CRICOLAP validation using 60 clinical nasopharyngeal samples previously verified with a commercial RT-PCR kit showed 97.5% and 100% sensitivity for S and N genes, respectively, and 100% specificity for both genes of SARS-CoV-2. CRICOLAP advances the CRISPR/Cas12a collateral cleavage result readout in the lateral flow assay and fluorophotometer, and it can be an alternative method for the decentralized field-deployable diagnosis of COVID-19 in remote and limited-resource locations.

A heterozygous SCN1A (c.A5768G/+) mutant human induced pluripotent stem cell line (USTCi002-A) generated using TALEN-mediated precise gene editing.

Severe mycological epilepsy of infancy is a catastrophic disease with preferential dysfunction of interneurons, frequentepisoderate, cognitive and sudden death. The disease is mainly caused by heterozygous loss-of-function mutation of SCN1A gene encoding α subunit of the sodium channel Nav1.1. To generate mutations in normal iPSC, Transcription activator-like effector nucleases was used to introduce the epilepsy-causing mutation A5768G into the endogenous locus of SCN1A gene. The gene editing induced pluripotent stem cell line and normal iPSC were obtained from the same donor to eliminate significantly the genetic background noise.

Generation of a tdTomato-GAD67 reporter human epilepsia mutation induced pluripotent stem cell line, USTCi001-A-2, using CRISPR/Cas9 editing.

Dravet syndrome is an epileptic encephalopathy largely due to haploinsufficiency of the voltage-gated sodium channel Nav1.1 that is expressed primarily in GABAergic neurons. In order to distinguish the different subtypes, we used gene editing to introduce tdTomato gene into the genome of iPSCs to label the GABAergic neurons in the differentiated neuronal networks. The gene-edited cell line demonstrates normal karyotype, expresses the main pluripotency markers, and shows the presence of differentiation into the three embryonic germ layers in teratomas.

Transient receptor potential ankyrin 1 contributes to the ATP-elicited oxidative stress and inflammation in THP-1-derived macrophage.

P2X7 receptor (P2X7R) is an ATP-gated non-selective cation channel which mediates ATP-induced inflammation in macrophages. Transient receptor potential (TRP) receptors are nociceptors in cellular membrane which can perceive the stimuli of environmental irritant. The interaction between TRP channels and P2X7R has been found while the details about inflammation are still unclear. In this study, we suggested that transient receptor potential ankyrin 1 (TRPA1), a member of TRP superfamily, participates in ATP-induced oxidative stress and inflammation in human acute monocytic leukemia cell line (THP-1)-derived macrophage. The co-localization between TRPA1 and P2X7R was detected using immunofluorescence in THP-1-derived macrophage and transfected human embryonic kidney cell line (HEK293T). The mechanism by which ATP or 3'-O-(4-Benzoylbenzoyl)-ATP (BzATP) induces the activation of macrophages was verified by calcium imaging, mitochondrial reactive oxygen species (mtROS) detection, mitochondrial membrane potential (∆Ψm) measurement, flow cytometry, enzyme-linked immunosorbent assay (ELISA), western blotting, CCK-8 assay, and the lactate dehydrogenase (LDH) release cytotoxic assay. The BzATP and ATP induced calcium overload, mitochondria injury, interleukin-1ß (IL-1ß) secretion, and cytotoxicity can be inhibited by TRPA1 antagonists. These results indicated that TRPA1 can co-localize with P2X7R and mediate ATP-induced oxidative stress and inflammation. Therefore, the inhibition of TRPA1 may provide a potential therapy for ATP-elicited inflammatory diseases, including atherosclerosis.

Transient receptor potential ankyrin 1 contributes to the lysophosphatidylcholine-induced oxidative stress and cytotoxicity in OLN-93 oligodendrocyte.

Transient receptor potential ankyrin 1 (TRPA1), the non-selective cation channel, was found that can mediate the generation of multiple sclerosis, while the mechanism is still controversial. Lysophosphatidylcholine (LPC) is a critical trigger of multiple sclerosis which results from the syndrome of neuronal inflammation and demyelination. In this work, we suggested that TRPA1 can mediate the LPC-induced oxidative stress and cytotoxicity in OLN-93 oligodendrocyte. The expression of TRPA1 in OLN-93 was detected by using quantitative real-time PCR (qRT-PCR) and immunofluorescence. The calcium overload induced by LPC via TRPA1 was detected by calcium imaging. The mechanism of LPC-induced mitochondrial reactive oxygen species (mtROS) generation, mitochondria membrane depolarization, nitric oxide (NO) increase, and development of superoxide production via TRPA1 was verified by using confocal imaging. The cell injury elicited by LPC via TRPA1 was confirmed by both CCK-8 and LDH cytotoxicity detection. These results indicate that TRPA1 plays an important role of the LPC-induced oxidative stress and cell damage in OLN-93 oligodendrocyte. Therefore, inhibition of TRPA1 may protect the LPC-induced demyelination.

Generation of corrected-hiPSC (USTCi001-A-1) from epilepsy patient iPSCs using TALEN-mediated editing of the SCN1A gene.

Dravet syndrome is a neurological disorder characterized by treatment-resistant polymorphic seizures, primarily caused by loss-of-function in the SCN1A gene. To develop an in vitro model of this disease, in a previously study we generated an induced pluripotent stem cell line from a 10-year-old boy carrying the NM_001165963.1:c.5768A to G (Q1923R) mutation in SCN1A. Using TALEN-mediated genome editing, we have now generated an isogenic control line in which the disease-causing mutation found in the epilepsy patient iPSCs was corrected, in order to eliminate the interference of different genetic backgrounds in future analyses.

Generation of induced pluripotent stem cell line (CSUXHi002-A) from a patient with spinocerebellar ataxia type 1.

Spinocerebellar ataxia type 1 (SCA1) is a hereditary neurodegenerative disease caused by CAG repeated expansion in ATXN1 gene. We generated induced pluripotent stem cells (iPSCs) from the urine exfoliated epithelial cells of SCA1 patient by using the integration-free methods. The patient derived iPSCs retained the mutation (the 65 CAG expansion tracts in ATXN1 gene), displayed normal karyotypes, expressed pluripotency markers and had the potential to differentiate towards three germ layers in vivo. This type of stem cell model will be valuable for elucidating the pathological mechanism and screening potential drugs of SCA1.

Generation of iPSC line (USTCi001-A) from human skin fibroblasts of a patient with epilepsy.

Epilepsy is a neurological disorder, characterized by recurrent (two or more) epileptic seizures resulting from excessive and abnormal cortical neural activity.Fibroblasts were collected from a 10-year-old male with antecedent febrile seizures (PEFS+) and carrying a heterozygous A > G mutation of Nav1.1 α subunit gene. The induced USTCi001-A retained the mutation, expressed pluripotent markers, showed normal karyotype, and displayed in vitro differentiation potential toward cells of the three embryonic germ layers.

Transient Receptor Potential Ankyrin 1 Contributes to Lysophosphatidylcholine-Induced Intracellular Calcium Regulation and THP-1-Derived Macrophage Activation.

Lysophosphatidylcholine (LPC) is a major atherogenic lipid that stimulates an increase in mitochondrial reactive oxygen species (mtROS) and the release of cytokines under inflammasome activation. However, the potential receptors of LPC in macrophages are poorly understood. Members of the transient receptor potential (TRP) channel superfamily, which is crucially involved in transducing environmental irritant stimuli into nociceptor activity, are potential receptors of LPC. In this study, we investigated whether LPC can induce the activation of transient receptor potential ankyrin 1 (TRPA1), a member of the TRP superfamily. The functional expression of TRPA1 was first detected by quantitative real-time polymerase chain reaction (qRT-PCR), western blotting and calcium imaging in human acute monocytic leukemia cell line (THP-1)-derived macrophages. The mechanism by which LPC induces the activation of macrophages through TRPA1 was verified by cytoplasmic and mitochondrial calcium imaging, mtROS detection, a JC-1 assay, enzyme-linked immunosorbent assay, the CCK-8 assay and the lactate dehydrogenase (LDH) cytotoxic assay. LPC induced the activation of THP-1-derived macrophages via calcium influx, and this activation was suppressed by potent and selective inhibitors of TRPA1. These results indicated that TRPA1 can mediate mtROS generation, mitochondrial membrane depolarization, the secretion of IL-1ß and cytotoxicity through cellular and mitochondrial Ca2+ influx in LPC-treated THP-1-derived macrophages. Therefore, the inhibition of TRPA1 may protect THP-1-derived macrophages against LPC-induced injury.

Direct Conversion of Human Urine Cells to Neurons by Small Molecules.

Transdifferentiation of other cell type into human neuronal cells (hNCs) provides a platform for neural disease modeling, drug screening and potential cell-based therapies. Among all of the cell donor sources, human urine cells (hUCs) are convenient to obtain without invasive harvest procedure. Here, we report a novel approach for the transdifferentiation of hUCs into hNCs. Our study demonstrated that a combination of seven small molecules (CAYTFVB) cocktail induced transdifferentiation of hUCs into hNCs. These chemical-induced neuronal cells (CiNCs) exhibited typical neuron-like morphology and expressed mature neuronal markers. The neuronal-like morphology revealed in day 1, and the Tuj1-positive CiNCs reached to about 58% in day 5 and 38.36% Tuj1+/MAP2+ double positive cells in day 12. Partial electrophysiological properties of CiNCs was obtained using patch clamp. Most of the CiNCs generated using our protocol were glutamatergic neuron populations, whereas motor neurons, GABAergic or dopaminergic neurons were merely detected. hUCs derived from different donors were converted into CiNCs in this work. This method may provide a feasible and noninvasive approach for reprogramming hNCs from hUCs for disease models and drug screening.

Generation of iPSC line (GIBHi001-A) from a patient with autism spectrum disorder.

Autism spectrum disorder (ASD) is a neurological disorder with complex etiologies. In this study, urine cells were collected from a 16-year-old male with ASD and reprogrammed with the human SKOM transcription factors. The patient has a heterozygous C > T mutation of FCGR1B gene that was confirmed by sequencing analysis. The pluripotency was verified by gene expression and capacity of differentiation towards the three germ layers. This kind of iPSC will be valuable for further understanding the pathogenesis of ASD and help to develop drugs for treating ASD.

Promoting Osteogenesis of Human Umbilical Cord Mesenchymal Stem Cells Using 1α, 25-(OH)2D3 to Change Intracellular Calcium Concentration.

Vitamin D plays a major role in the regulation of calcium homeostasis and affects bone metabolism. There is currently limited detailed knowledge about the vitamin D endocrine system in human bone cells. Here, we investigated the direct effects of 1α, 25-dihydroxyvitamin D3 (1α, 25-(OH)2D3 or 'VD3') on osteogenesis of human umbilical cord mesenchymal stem cells (HUCMSCs). We also studied the impact of VD3 on intracellular Ca2+ concentrations in osteogenic cells. The results of alizarin red staining and alkaline phosphatase activity tests showed that VD3 could not induce osteogenic differentiation in HUCMSCs. However, addition of VD3 to the osteogenic differentiationinducing medium could promote HUCMSC to differentiate into osteoblasts. Calcium imaging showed that the addition of VD3 increased intracellular Ca2+ concentrations in osteogenic HUCMSCs. Thus, we concluded that adding VD3 increased intracellular Ca2+ concentrations in osteogenic HUCMSCs and promoted their osteogenesis.

Recurrent activations of transient receptor potential vanilloid-1 and vanilloid-4 promote cellular proliferation and migration in esophageal squamous cell carcinoma cells.

Some members of the transient receptor potential vanilloid (TRPV) subfamily of cation channels are thermosensitive. Earlier studies have revealed the distribution and functions of these thermo-TRPVs (TRPV1-4) in various organs, but their expression and function in the human esophagus are not fully understood. Here, we probed for the expression of the thermo-TRPVs in one nontumor human esophageal squamous cell line and two esophageal squamous cell carcinoma (ESCC) cell lines. TRPV1, TRPV2, and TRPV4 proteins were found to be upregulated in ESCC cells, while TRPV3 was not detectable in any of these cell lines. Subsequently, channel function was evaluated via monitoring of Ca2+ transients by Ca2+ imaging and nonselective cation channel currents were recorded by whole-cell patch clamp. We found that TRPV4 was activated by heat at 28 °C-35 °C, whereas TRPV1 and TRPV2 were activated by higher, noxious temperatures (44 °C and 53 °C, respectively). Furthermore, TRPV1 was activated by capsaicin (EC 50 = 20.32 µm), and this effect was antagonized by AMG9810; TRPV2 was activated by a newly developed cannabinoid compound, O1821, and inhibited by tranilast. In addition, TRPV4 was activated by hypotonic solutions (220 m Osm), and this effect was abolished by ruthenium red. The effects of TRPV1 and TRPV4 on ESCC were also explored. Our data, for the first time, showed that the overactivation of TRPV1 and TRPV4 promoted the proliferation and/or migration of ESCC cells. In summary, TRPV1, TRPV2, and TRPV4 were functionally expressed in human esophageal squamous cells, and thermo-TRPVs might play an important role in the development of ESCC.

Novel role of TRPV2 in promoting the cytotoxicity of H2O2-mediated oxidative stress in human hepatoma cells.

Oxidative stress is important for the initiation and progression of cancers, which confers the cells with a survival advantage by inducing oxidative adaption and drug resistance. Therefore, developing strategies to promote oxidative stress-induced cytotoxicity could be important for cancer therapy. Herein, we found that H2O2-mediated oxidative stress increases TRPV2 expression in human hepatoma (HepG2 and Huh-7) cells. This occurred at the mRNA and protein levels in a dose-dependent manner. The significance of TRPV2 in promoting H2O2-induced cell death was demonstrated in gain and loss of function studies with overexpression and knockdown of TRPV2, respectively. Mechanistically, H2O2-induced cell death involves inhibition of pro-survival signaling proteins (Akt, Nrf2) and activation of pro-death signaling proteins (p38, JNK1). Overexpression of TRPV2 in H2O2-treated hepatoma cells aggravates the inhibition of Akt and Nrf2, while it enhances the activation of p38 and JNK1 at the early stage of cell death. Interestingly, increased expression of TRPV2 in HepG2 cells improved the efficacy of stress-associated chemicals to induce cell death. Our findings suggest that TRPV2 acts as an important enhancer for H2O2-induced cytotoxicity. This process occurred by the inhibition of Akt and Nrf2 as well as the early activation of p38 and JNK1. These findings have important implications for inhibition of oxidative adaption and drug resistance.

Inhibition of HERG potassium channels by domiphen bromide and didecyl dimethylammonium bromide.

Domiphen bromide and didecyl dimethylammonium bromide were widely used environmental chemicals with potent activity on blockade of human ether-a-go-go related gene (HERG) channels. But the mechanism of their action is not clear. The kinetics of block of HERG channels by domiphen bromide and didecyl dimethylammonium bromide was studied in order to characterize the inhibition of HERG currents by these quaternary ammonium compounds (QACs). Domiphen bromide and didecyl dimethylammonium bromide inhibited HERG channel currents in a dose-dependent manner with IC50 values of 9nM and 5nM, respectively. Block of HERG channel by domiphen bromide and didecyl dimethylammonium bromide was voltage-dependent and use-dependent. Domiphen bromide and didecyl dimethylammonium bromide caused substantial negative shift of the activation curves, accelerated activated process, but had no effects on the deactivation and reactivation processes. The docking models implied that these two compounds bound to PAS domain of HERG channels and inhibited its function. Our data demonstrated that domiphen bromide and didecyl dimethylammonium bromide blocked the HERG channel with a preference for the activated channel state.