Translation reinitiation in c.453delC frameshift mutation of KCNH2 producing functional hERG K+ channels with mild dominant negative effect in the heterozygote patient-derived iPSC cardiomyocytes.

The c.453delC (p.Thr152Profs*14) frameshift mutation in KCNH2 is associated with an elevated risk of Long QT syndrome (LQTS) and fatal arrhythmia. Nevertheless, the loss-of-function mechanism underlying this mutation remains unexplored and necessitates an understanding of electrophysiology. To gain insight into the mechanism of the LQT phenotype, we conducted whole-cell patch-clamp and immunoblot assays, utilizing both a heterologous expression system and patient-derived induced pluripotent stem cell-cardiomyocytes (iPSC-CMs) with 453delC-KCNH2. We also explored the site of translational reinitiation by employing LC/MS mass spectrometry. Contrary to the previous assumption of early termination of translation, the findings of this study indicate that the 453delC-KCNH2 leads to an N-terminally truncated hERG channel, a potential from a non-canonical start codon, with diminished expression and reduced current (IhERG). The co-expression with wildtype KCNH2 produced heteromeric hERG channel with mild dominant-negative effect. Additionally, the heterozygote patient-derived iPSC-CMs exhibited prolonged action potential duration and reduced IhERG, which was ameliorated with the use of a hERG activator, PD-118057. The results of our study offer novel insights into the mechanisms involved in congenital LQTS associated with the 453delC mutation of KCNH2. The mutant results in the formation of less functional N-terminal-truncated channels with reduced amount of membrane expression. A hERG activator is capable of correcting abnormalities in both the heterologous expression system and patient-derived iPSC-CMs.

Identifying the Multitarget Pharmacological Mechanism of Action of Genistein on Lung Cancer by Integrating Network Pharmacology and Molecular Dynamic Simulation.

Food supplements have become beneficial as adjuvant therapies for many chronic disorders, including cancer. Genistein, a natural isoflavone enriched in soybeans, has gained potential interest as an anticancer agent for various cancers, primarily by modulating apoptosis, the cell cycle, and angiogenesis and inhibiting metastasis. However, in lung cancer, the exact impact and mechanism of action of genistein still require clarification. To provide more insight into the mechanism of action of genistein, network pharmacology was employed to identify the key targets and their roles in lung cancer pathogenesis. Based on the degree score, the hub genes AKT1, CASP3, EGFR, STAT3, ESR1, SRC, PTGS2, MMP9, PRAG, and AR were significantly correlated with genistein treatment. AKT1, EGFR, and STAT3 were enriched in the non-small cell lung cancer (NSCLC) pathway according to Kyoto Encyclopedia of Genes and Genomes analysis, indicating a significant connection to lung cancer development. Moreover, the binding affinity of genistein to NSCLC target proteins was further verified by molecular docking and molecular dynamics simulations. Genistein exhibited potential binding to AKT1, which is involved in apoptosis, cell migration, and metastasis, thus holding promise for modulating AKT1 function. Therefore, this study aimed to investigate the mechanism of action of genistein and its therapeutic potential for the treatment of NSCLC.

Requirement of ß subunit for the reduced voltage-gated Na+ current of a Brugada syndrome patient having novel double missense mutation (p.A385T/R504T) of SCN5A.

Mutations within the SCN5A gene, which encodes the α-subunit 5 (NaV1.5) of the voltage-gated Na+ channel, have been linked to three distinct cardiac arrhythmia disorders: long QT syndrome type 3, Brugada syndrome (BrS), and cardiac conduction disorder. In this study, we have identified novel missense mutations (p.A385T/R504T) within SCN5A in a patient exhibiting overlap arrhythmia phenotypes. This study aims to elucidate the functional consequences of SCN5A mutants (p.A385T/R504T) to understand the clinical phenotypes. Whole-cell patch-clamp technique was used to analyze the NaV1.5 current (INa) in HEK293 cells transfected with the wild-type and mutant SCN5A with or without SCN1B co-expression. The amplitude of INa was not altered in mutant SCN5A (p.A385T/R504T) alone. Furthermore, a rightward shift of the voltage-dependent inactivation and faster recovery from inactivation was observed, suggesting a gain-of-function state. Intriguingly, the coexpression of SCN1B with p.A385T/R504T revealed significant reduction of INa and slower recovery from inactivation, consistent with the loss-of-function in Na+ channels. The SCN1B dependent reduction of INa was also observed in a single mutation p.R504T, but p.A385T co-expressed with SCN1B showed no reduction. In contrast, the slower recovery from inactivation with SCN1B was observed in A385T while not in R504T. The expression of SCN1B is indispensable for the electrophysiological phenotype of BrS with the novel double mutations; p.A385T and p.R504T contributed to the slower recovery from inactivation and reduced current density of NaV1.5, respectively.

Unveiling the Potentiality of Shikonin Derivatives Inhibiting SARS-CoV-2 Main Protease by Molecular Dynamic Simulation Studies.

Shikonin, a phytochemical present in the roots of Lithospermum erythrorhizon, is well-known for its broad-spectrum activity against cancer, oxidative stress, inflammation, viruses, and anti-COVID-19 agents. A recent report based on a crystallographic study revealed a distinct conformation of shikonin binding to the SARS-CoV-2 main protease (Mpro), suggesting the possibility of designing potential inhibitors based on shikonin derivatives. The present study aimed to identify potential shikonin derivatives targeting the Mpro of COVID-19 by using molecular docking and molecular dynamics simulations. A total of 20 shikonin derivatives were screened, of which few derivatives showed higher binding affinity than shikonin. Following the MM-GBSA binding energy calculations using the docked structures, four derivatives were retained with the highest binding energy and subjected to molecular dynamics simulation. Molecular dynamics simulation studies suggested that alpha-methyl-n-butyl shikonin, beta-hydroxyisovaleryl shikonin, and lithospermidin-B interacted with two conserved residues, His41 and Cys145, through multiple bonding in the catalytic sites. This suggests that these residues may effectively suppress SARS-CoV-2 progression by inhibiting Mpro. Taken together, the present in silico study concluded that shikonin derivatives may play an influential role in Mpro inhibition.

Higher expression of KCNK10 (TREK-2) K+ channels and their functional upregulation by lipopolysaccharide treatment in mouse peritoneal B1a cells.

Innate-like CD5+ B1a cells localized in serous cavities are activated by innate stimuli, such as lipopolysaccharide (LPS), leading to T cell-independent antibody responses. Although ion channels play crucial roles in the homeostasis and activation of immune cells, the electrophysiological properties of B1a cells have not been investigated to date. Previously, in the mouse B cell lymphoma cells, we found that the voltage-independent two-pore-domain potassium (K2P) channels generate a negative membrane potential and drive Ca2+ influx. Here, we newly compared the expression and activities of K2P channels in mouse splenic follicular B (FoB), marginal zone B (MZB), and peritoneal B1a cells. Next-generation sequencing analysis showed higher levels of transcripts for TREK-2 and TWIK-2 in B1a cells than those in FoB or MZB cells. Electrophysiological analysis, using patch clamp technique, revealed higher activity of TREK-2 with the characteristic large unitary conductance (~ 250 pS) in B1a than that in FoB or MZB cells. TREK-2 activity was further increased by LPS treatment (>2 h), which was more prominent in B1a than that in MZB or FoB cells. The cytosolic Ca2+ concentration of B cells was decreased by high-K+-induced depolarization (ΔRKCl (%)), suggesting the basal Ca2+ influx to be driven by negative membrane potential. The LPS treatment significantly increased the ΔRKCl (%) in B1a, though not in FoB and MZB cells. Our study was the first to compare the K2P channels in mouse primary B cell subsets, elucidating the functional upregulation of TREK-2 and augmentation of Ca2+ influx by the stimulation of Toll-like receptor 4 in B1a cells.

Thermosensitivity of the voltage-dependent activation of calcium homeostasis modulator 1 (calhm1) ion channel.

Calcium homeostasis modulator 1 (calhm1) proteins form an outwardly rectifying nonselective ion channel having exceedingly slow kinetics and low sensitivity to voltage that is shifted by lowering extracellular Ca2+ ([Ca2+]e). Here we found that physiological temperature dramatically facilitates the voltage-dependent activation of the calhm1 current (Icalhm1); increased amplitude (Q10, 7-15) and fastened speed of activation. Also, the leftward shift of the half-activation voltage (V1/2) was similary observed in the normal and lower [Ca2+]e. Since calhm1 is highly expressed in the brain and taste cells, the thermosensitivity should be considered in their electrophysiology.

Teaching cardiac excitation-contraction coupling using a mathematical computer simulation model of human ventricular myocytes.

To understand the excitation-contraction (E-C) coupling of cardiomyocytes, including the electrophysiological mechanism of their characteristically long action potential duration, is one of the major learning goals in medical physiology. However, the integrative interpretation of the responses occurring during the contraction-relaxation cycle is challenging due to the dynamic interaction of underlying factors. Starting in 2017, we adopted the mathematical computer simulation model of human ventricular myocyte (Cardiac E-C_Sim), hypothesizing that this educational technology may facilitate students' learning of cardiac physiology. Here, we describe the overall process for the educational application of Cardiac E-C_Sim in the human physiology practicum of Seoul National University College of Medicine. We also report the results from questionnaires covering detailed assessment of the practicum class. The analysis of results and feedback opinions enabled us to understand how the students had approached the problem-solving process. As a whole, the students could better accomplish the learning goals using Cardiac E-C_Sim, followed by constructive discussions on the complex and dynamic mechanisms of cardiac E-C coupling. We suggest that the combined approach of lecture-based teaching and computer simulations guided by a manual containing clinical context would be broadly applicable in physiology education.

Dual regulatory effects of PI(4,5)P2 on TREK-2 K+ channel through antagonizing interaction between the alkaline residues (K330 and R355-357) in the cytosolic C-terminal helix.

TWIK-related two-pore domain K+ channel-2 (TREK-2) has voltageindependent activity and shows additional activation by acidic intracellular pH (pHi) via neutralizing the E332 in the cytoplasmic C terminal (Ct). We reported opposite regulations of TREK-2 by phosphatidylinositol 4,5-bisphosphate (PIP2) via the alkaline K330 and triple Arg residues (R355-357); inhibition and activation, respectively. The G334 between them appeared critical because its mutation (G334A) endowed hTREK-2 with tonic activity, similar to the mutation of the inhibitory K330 (K330A). To further elucidate the role of putative bent conformation at G334, we compared the dual mutation forms, K330A/G334A and G334A/R355-7A, showing higher and lower basal activity, respectively. The results suggested that the tonic activity of G334A owes to a dominant influence from R355-7. Since there are additional triple Arg residues (R377-9) distal to R355-7, we also examined the triple mutant (G334A/R355-7A/R377-9A) that showed tonic inhibition same with G334A/R355-7A. Despite the state of tonic inhibition, the activation by acidic pHi was preserved in both G334A/R355-7A and G334A/R355-7A/R377-9A, similar to the R355-7A. Also, the inhibitory effect of ATP could be commonly demonstrated under the activation by acidic pHi in R355-7A, G334A/R355-7A, and G334A/R355-7A/R377-9A. These results suggest that the putative bent conformation at G334 is important to set the tug-of-war between K330 and R355-7 in the PIP2-dependent regulation of TREK-2.

Gardenia jasminoides extract and its constituent, genipin, inhibit activation of CD3/CD28 co-stimulated CD4+ T cells via ORAI1 channel.

Gardenia jasminoides (GJ) is a widely used herbal medicine with antiinflammatory properties, but its effects on the ORAI1 channel, which is important in generating intracellular calcium signaling for T cell activation, remain unknown. In this study, we investigated whether 70% ethanolic GJ extract (GJEtOH) and its subsequent fractions inhibit ORAI1 and determined which constituents contributed to this effect. Whole-cell patch clamp analysis revealed that GJEtOH (64.7% ± 3.83% inhibition at 0.1 mg/ml) and all its fractions showed inhibitory effects on the ORAI1 channel. Among the GJ fractions, the hexane fraction (GJHEX, 66.8% ± 9.95% at 0.1 mg/ml) had the most potent inhibitory effects in hORAI1-hSTIM1 co-transfected HEK293T cells. Chemical constituent analysis revealed that the strong ORAI1 inhibitory effect of GJHEX was due to linoleic acid, and in other fractions, we found that genipin inhibited ORAI1. Genipin significantly inhibited IORAI1 and interleukin-2 production in CD3/ CD28-stimulated Jurkat T lymphocytes by 35.9% ± 3.02% and 54.7% ± 1.32% at 30 µM, respectively. Furthermore, the same genipin concentration inhibited the proliferation of human primary CD4+ T lymphocytes stimulated with CD3/CD28 antibodies by 54.9% ± 8.22%, as evaluated by carboxyfluorescein succinimidyl ester assay. Our findings suggest that genipin may be one of the active components of GJ responsible for T cell suppression, which is partially mediated by activation of the ORAI1 channel. This study helps us understand the mechanisms of GJ in the treatment of inflammatory diseases.

Luteolin reduces fluid hypersecretion by inhibiting TMEM16A in interleukin-4 treated Calu-3 airway epithelial cells.

Rhinorrhea in allergic rhinitis (AR) is characterized by the secretion of electrolytes in the nasal discharge. The secretion of Cl- and HCO3- is mainly regulated by cystic fibrosis transmembrane conductance regulator (CFTR) or via the calciumactivated Cl- channel anoctamin-1 (ANO1) in nasal gland serous cells. Interleukin-4 (IL-4), which is crucial in the development of allergic inflammation, increases the expression and activity of ANO1 by stimulating histamine receptors. In this study, we investigated ANO1 as a potential therapeutic target for rhinorrhea in AR using an ANO1 inhibitor derived from a natural herb. Ethanolic extracts (30%) of Spirodela polyrhiza (SPEtOH) and its five major flavonoids constituents were prepared. To elucidate whether the activity of human ANO1 (hANO1) was modulated by SPEtOH and its chemical constituents, a patch clamp experiment was performed in hANO1-HEK293T cells. Luteolin, one of the major chemical constituents in SPEtOH, significantly inhibited hANO1 activity in hANO1-HEK293T cells. Further, SPEtOH and luteolin specifically inhibited the calcium-activated chloride current, but not CFTR current in human airway epithelial Calu-3 cells. Calu-3 cells were cultured to confluency on transwell inserts in the presence of IL-4 to measure the electrolyte transport by Ussing chamber. Luteolin also significantly inhibited the ATP-induced increase in electrolyte transport, which was increased in IL-4 sensitized Calu-3 cells. Our findings indicate that SPEtOH- and luteolin may be suitable candidates for the prevention and treatment of allergic rhinitis. SPEtOH- and luteolin-mediated ANO1 regulation provides a basis for the development of novel approaches for the treatment of allergic rhinitis-induced rhinorrhea.

The agonistic action of URO-K10 on Kv7.4 and 7.5 channels is attenuated by co-expression of KCNE4 ancillary subunit.

KCNQ family constitutes slowly-activating potassium channels among voltage-gated potassium channel superfamily. Recent studies suggested that KCNQ4 and 5 channels are abundantly expressed in smooth muscle cells, especially in lower urinary tract including corpus cavernosum and that both channels can exert membrane stabilizing effect in the tissues. In this article, we examined the electrophysiological characteristics of overexpressed KCNQ4, 5 channels in HEK293 cells with recently developed KCNQ-specific agonist. With submicromolar EC50, the drug not only increased the open probability of KCNQ4 channel but also increased slope conductance of the channel. The overall effect of the drug in whole-cell configuration was to increase maximal whole-cell conductance, to prolongate the activation process, and left-shift of the activation curve. The agonistic action of the drug, however, was highly attenuated by the co-expression of one of the ß ancillary subunits of KCNQ family, KCNE4. Strong in vitro interactions between KCNQ4, 5 and KCNE4 were found through Foster Resonance Energy Transfer and co-immunoprecipitation. Although the expression levels of both KCNQ4 and KCNE4 are high in mesenteric arterial smooth muscle cells, we found that 1 µM of the agonist was sufficient to almost completely relax phenylephrine-induced contraction of the muscle strip. Significant expression of KCNQ4 and KCNE4 in corpus cavernosum together with high tonic contractility of the tissue grants highly promising relaxational effect of the KCNQspecific agonist in the tissue.

Triple arginine residues in the proximal C-terminus of TREK K+ channels are critical for biphasic regulation by phosphatidylinositol 4,5-bisphosphate.

TWIK-related two-pore domain K+ channels (TREKs) are activated by acidic intracellular pH (pHi), membrane stretch, temperature, and arachidonic acid (AA). Phosphatidylinositol 4,5-bisphosphate (PIP2) exerts concentration-dependent biphasic regulations, which have been observed: inhibition by high PIP2, activation by partial decrease of PIP2, and inhibition by depletion of PIP2. Consistently, the stimulation of voltage-sensitive PIP2 phosphatase (Dr-VSP) induces initial activation and subsequent inhibition of TREKs. Lys in the proximal C-terminus (pCt) is responsible for the inhibition by high PIP2, which is generated by phosphatidylinositol kinases with ATP; its neutralizing mutation [K330A of human TREK-2 (hTREK-2)] induces tonic high activity, irrespective of ATP. Here we focus on triple successive Arg in pCt (R3-pCt) as a candidate region for the stimulatory regulation by lower PIP2. Their neutralized mutant (R3A-pCt; RRR340-2A and RRR355-7A in hTREK-1 and -2, respectively) showed negligible basal current and was not affected by ATP removal or by Dr-VSP activation. Phosphatidic acid, a phospholipid agonist of TREKs, did not activate R3A-pCt. In contrast, acidic pHi, AA, and high temperature activated R3A-pCt normally, whereas activation by membrane stretch was attenuated. In hTREK-2, combined neutralizations of the inhibitory K330 and R3-pCt (K330A/RRR355-7A) did not recover the suppressed current. In contrast, combined neutralization of pHi-sensing Glu (E332A/R355-7A) induced tonic high current and no further activation by pHi. Interestingly, when the Gly between K330/E332 and R3-pCt was mutated (G334A), hTREK-2 was tonic activated with reversed responses to ATP and acidic pHi. Therefore, we propose that the PIP2-dependent converse regulation of TREKs by Lys and R3-pCt with Gly implies structural flexibility.

Identification of critical amino acids in the proximal C-terminal of TREK-2 K+ channel for activation by acidic pHi and ATP-dependent inhibition.

TWIK-related two-pore domain K+ channels (TREKs) are regulated by intracellular pH (pHi) and Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2). Previously, Glu306 in proximal C-terminal (pCt) of mouse TREK-1 was identified as the pHi-sensing residue. The direction of PI(4,5)P2 sensitivity is controversial, and we have recently shown that TREKs are inhibited by intracellular ATP via endogenous PI(4,5)P2 formation. Here we investigate the anionic and cationic residues of pCt for the pHi and ATP-sensitivity in human TREK-2 (hTREK-2). In inside-out patch clamp recordings (ITREK-2,i-o), acidic pHi-induced activation was absent in E332A and was partly attenuated in E335A. Neutralization of cationic Lys (K330A) also eliminated the acidic pHi sensitivity of ITREK-2,i-o. Unlike the inhibition of wild-type (WT) ITREK-2,i-o by intracellular ATP, neither E332A nor K330A was sensitive to ATP. Nevertheless, exogenous PI(4,5)P2 (10 µM) abolished ITREK-2 i-o in all the above mutants as well as in WT, indicating unspecific inhibition by exogenous PI(4,5)P2. In whole-cell recordings of TREK-2 (ITREK-2,w-c), K330A and E332A showed higher or fully active basal activity, showing attenuated or insignificant activation by 2-APB, arachidonic acid, or acidic pHe 6.9. ITREK-1,w-c of WT is largely suppressed by pHe 6.9, and the inhibition is slightly attenuated in K312A and E315A. The results show concerted roles of the oppositely charged Lys and Glu in pCt for the ATP-dependent low basal activity and pHi sensitivity.

Mitochondrial dysfunction reduces the activity of KIR2.1 K+ channel in myoblasts via impaired oxidative phosphorylation.

Myoblast fusion depends on mitochondrial integrity and intracellular Ca2+ signaling regulated by various ion channels. In this study, we investigated the ionic currents associated with [Ca2+]i regulation in normal and mitochondrial DNA-depleted (ρ0) L6 myoblasts. The ρ0 myoblasts showed impaired myotube formation. The inwardly rectifying K+ current (IKir) was largely decreased with reduced expression of KIR2.1, whereas the voltage-operated Ca2+ channel and Ca2+-activated K+ channel currents were intact. Sustained inhibition of mitochondrial electron transport by antimycin A treatment (24 h) also decreased the IKir. The ρ0 myoblasts showed depolarized resting membrane potential and higher basal [Ca2+]i. Our results demonstrated the specific downregulation of IKir by dysfunctional mitochondria. The resultant depolarization and altered Ca2+ signaling might be associated with impaired myoblast fusion in ρ0 myoblasts.

Inhibition of TREK-2 K(+) channels by PI(4,5)P2: an intrinsic mode of regulation by intracellular ATP via phosphatidylinositol kinase.

TWIK-related two-pore domain K(+) channels 1 and 2 (TREKs) are activated under various physicochemical conditions. However, the directions in which they are regulated by PI(4,5)P2 and intracellular ATP are not clearly presented yet. In this study, we investigated the effects of ATP and PI(4,5)P2 on overexpressed TREKs (HEK293T and COS-7) and endogenously expressed TREK-2 (mouse astrocytes and WEHI-231 B cells). In all of these cells, both TREK-1 and TREK-2 currents were spontaneously increased by dialysis with ATP-free pipette solution for whole-cell recording (ITREK-1,w-c and ITREK-2w-c) or by membrane excision for inside-out patch clamping without ATP (ITREK-1,i-o and ITREK-2,i-o). Steady state ITREK-2,i-o was reversibly decreased by 3 mM ATP applied to the cytoplasmic side, and this reduction was prevented by wortmannin, a PI-kinase inhibitor. An exogenous application of PI(4,5)P2 inhibited the spontaneously increased ITREKs,i-o, suggesting that intrinsic PI(4,5)P2 maintained by intracellular ATP and PI kinase may set the basal activity of TREKs in the intact cells. The inhibition of intrinsic TREK-2 by ATP was more prominent in WEHI-231 cells than astrocytes. Interestingly, unspecific screening of negative charges by poly-L-lysine also inhibited ITREK-2,i-o. Application of PI(4,5)P2 after the poly-L-lysine treatment showed dose-dependent dual effects, initial activation and subsequent inhibition of ITREK-2,i-o at low and high concentrations, respectively. In HEK293T cells coexpressing TREK-2 and a voltage-sensitive PI(4,5)P2 phosphatase, sustained depolarization increased ITREK-2,w-c initially (<5 s) but then decreased the current below the control level. In HEK293T cells coexpressing TREK-2 and type 3 muscarinic receptor, application of carbachol induced transient activation and sustained suppression of ITREK-2,w-c and cell-attached ITREK-2. The inhibition of TREK-2 by unspecific electrostatic quenching, extensive dephosphorylation, or sustained hydrolysis of PI(4,5)P2 suggests the existence of dual regulatory modes that depend on PI(4,5)P2 concentration.

Atractylodes macrocephala Koidz Alleviates Symptoms in Zymosan-Induced Irritable Bowel Syndrome Mouse Model through TRPV1, NaV1.5, and NaV1.7 Channel Modulation.

(1) Background: Irritable bowel syndrome (IBS) is a common disease in the gastrointestinal (GI) tract. Atractylodes macrocephala Koidz (AMK) is known as one of the traditional medicines that shows a good efficacy in the GI tract. (2) Methods: We investigated the effect of AMK in a network pharmacology and zymosan-induced IBS animal model. In addition, we performed electrophysiological experiments to confirm the regulatory mechanisms related to IBS. (3) Results: Various characteristics of AMK were investigated using TCMSP data and various analysis systems. AMK restored the macroscopic changes and weight to normal. Colonic mucosa and inflammatory factors were reduced. These effects were similar to those of amitriptyline and sulfasalazine. In addition, transient receptor potential (TRP) V1, voltage-gated Na+ (NaV) 1.5, and NaV1.7 channels were inhibited. (4) Conclusion: These results suggest that AMK may be a promising therapeutic candidate for IBS management through the regulation of ion channels.

Discovery of a novel natural compound, vitekwangin B, with ANO1 protein reduction properties and anticancer potential.

Background: Prostate cancer and non-small cell lung cancer (NSCLC) present significant challenges in the development of effective therapeutic strategies. Hormone therapies for prostate cancer target androgen receptors and prostate-specific antigen markers. However, treatment options for prostatic small-cell neuroendocrine carcinoma are limited. NSCLC, on the other hand, is primarily treated with epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors but exhibits resistance. This study explored a novel therapeutic approach by investigating the potential anticancer properties of vitekwangin B, a natural compound derived from Vitex trifolia. Methods: Vitekwangin B was chromatographically isolated from the fruits of V. trifolia. ANO1 protein levels in prostate cancer and NSCLC cells were verified and evaluated again after vitekwangin B treatment. Results: Vitekwangin B did not inhibit anoctamin1 (ANO1) channel function but significantly reduced ANO1 protein levels. These results demonstrate that vitekwangin B effectively inhibited cancer cell viability and induced apoptosis in prostate cancer and NSCLC cells. Moreover, it exhibited minimal toxicity to liver cells and did not affect hERG channel activity, making it a promising candidate for further development as an anticancer drug. Conclusion: Vitekwangin B may offer a new direction for cancer therapy by targeting ANO1 protein, potentially improving treatment outcomes in patients with prostate cancer and NSCLC. Further research is needed to explore its full potential and overcome existing drug resistance challenges.

ANO1-downregulation induced by schisandrathera D: a novel therapeutic target for the treatment of prostate and oral cancers.

Anoctamin 1 (ANO1), a drug target for various cancers, including prostate and oral cancers, is an intracellular calcium-activated chloride ion channel that plays various physiopathological roles, especially in the induction of cancer growth and metastasis. In this study, we tested a novel compound isolated from Schisandra sphenanthera, known as schisandrathera D, for its inhibitory effect on ANO1. Schisandrathera D dose-dependently suppressed the ANO1 activation-mediated decrease in fluorescence of yellow fluorescent protein; however, it did not affect the adenosine triphosphate-induced increase in the intracellular calcium concentration or forskolin-induced cystic fibrosis transmembrane conductance regulator activity. Specifically, schisandrathera D gradually decreased the levels of ANO1 protein and significantly reduced the cell viability in ANO1-expressing cells when compared to those in ANO1-knockout cells. These effects could be attributed to the fact that schisandrathera D displayed better binding capacity to ANO1 protein than the previously known ANO1 inhibitor, Ani9. Finally, schisandrathera D increased the levels of caspase-3 and cleaved poly (ADP-ribose) polymerase 1, thereby indicating that its anticancer effect is mediated through apoptosis. Thus, this study highlights that schisandrathera D, which reduces ANO1 protein levels, has apoptosis-mediated anticancer effects in prostate and oral cancers, and thus, can be further developed into an anticancer agent.

Differential pathways for calcium influx activated by concanavalin A and CD3 stimulation in Jurkat T cells.

Sustained increase in [Ca(2+)](c) (Δ[Ca(2+)](c)) is a critical early signal from T-cell receptor (TCR/CD3). In general, Ca(2+)-release activated Ca(2+) channels (CRAC) are responsible for the Ca(2+) influx and Δ[Ca(2+)](c) after TCR/CD3 stimulation. However, T cells also express Ca(2+)-permeable nonselective cation channels such as TRPM2 and TRPC. Gd(3+) is a relatively selective blocker for CRAC at micromolar concentrations. Here, Jurkat T cells were used to investigate the Gd(3+)-resistant Ca(2+) influx (Δ[Ca(2+)](c,Gd)) induced by concanavalin A (ConA, 1 µg/ml), a widely used mitogenic agent for T cells, or by anti-CD3 Ab (αCD3). αCD3-induced Δ[Ca(2+)](c) was partly (~60%) inhibited by 1 µM Gd(3+) while thapsigargin-induced Δ[Ca(2+)] was almost completely abolished. ConA-induced Δ[Ca(2+)] was mostly inhibited by 1 µM Gd(3+) during the early phase (<30 s of ConA application) and became resistant during the late phase (>2 min). Induction of Δ[Ca(2+)](c,Gd) by αCD3 and ConA was inhibited by 2-aminoethoxydiphenyl borate (2-APB) and by N-(p-amylcinnamoyl) anthranilic acid, indicating that TRPM2 and TRPC are involved in this process. Treatment with Pyr-3, a TRPC3-specific inhibitor, potently suppressed Δ[Ca(2+)](c,Gd) by αCD3 (IC(50), 0.16 µM). Patch clamp experiments demonstrated that the TRPM2 channels were activated by ConA, and the TRPC-like channels were activated by αCD3. Our present study suggests that TRPM2 and TRPC3 are activated by ConA and TCR/CD3, respectively, in Jurkat T cells and are responsible for the induction of Δ[Ca(2+)](c,Gd).

Lower troponin expression in the right ventricle of rats explains interventricular differences in E-C coupling.

Despite distinctive functional and anatomic differences, a precise understanding of the cardiac interventricular differences in excitation-contraction (E-C) coupling mechanisms is still lacking. Here, we directly compared rat right and left cardiomyocytes (RVCM and LVCM). Whole-cell patch clamp, the IonOptix system, and fura-2 fluorimetry were used to measure electrical properties (action potential and ionic currents), single-cell contractility, and cytosolic Ca2+ ([Ca2+]i), respectively. Myofilament proteins were analyzed by immunoblotting. RVCM showed significantly shorter action potential duration (APD) and higher density of transient outward K+ current (Ito). However, the triggered [Ca2+]i change (Ca2+ transient) was not different, while the decay rate of the Ca2+ transient was slower in RVCM. Although the relaxation speed was also slower, the sarcomere shortening amplitude (ΔSL) was smaller in RVCM. SERCA activity was ∼60% lower in RVCM, which is partly responsible for the slower decay of the Ca2+ transient. Immunoblot analysis revealed lower expression of the cardiac troponin complex (cTn) in RVCM, implying a smaller Ca2+ buffering capacity (κS), which was proved by in situ analysis. The introduction of these new levels of cTn, Ito, and SERCA into a mathematical model of rat LVCM reproduced the similar Ca2+ transient, slower Ca2+ decay, shorter APD, and smaller ΔSL of RVCM. Taken together, these data show reduced expression of cTn proteins in the RVCM, which provides an explanation for the interventricular difference in the E-C coupling kinetics.