Novel Screening System for Biliary Excretion of Drugs Using Human Cholangiocyte Organoid Monolayers with Directional Drug Transport.

It has recently been reported that cholangiocyte organoids can be established from primary human hepatocytes. The purpose of this study was to culture the organoids in monolayers on inserts to investigate the biliary excretory capacity of drugs. Cholangiocyte organoids prepared from hepatocytes had significantly higher mRNA expression of CK19, a bile duct epithelial marker, compared to hepatocytes. The organoids also expressed mRNA for efflux transporters involved in biliary excretion of drugs, P-glycoprotein (P-gp), multidrug resistance-associated protein 2 (MRP2), and breast cancer resistance protein (BCRP). The subcellular localization of each protein was observed. These results suggest that the membrane-cultured cholangiocyte organoids are oriented with the upper side being the apical membrane side (A side, bile duct lumen side) and the lower side being the basolateral membrane side (B side, hepatocyte side), and that each efflux transporter is localized to the apical membrane side. Transport studies showed that the permeation rate from the B side to the A side was faster than from the A side to the B side for the substrates of each efflux transporter, but this directionality disappeared in the presence of inhibitor of each transporter. In conclusion, the cholangiocyte organoid monolayer system has the potential to quantitatively evaluate the biliary excretion of drugs. The results of the present study represent an unprecedented system using human cholangiocyte organoids, which may be useful as a screening model to directly quantify the contribution of biliary excretion to the clearance of drugs.

TRIC-A Channel Maintains Store Calcium Handling by Interacting With Type 2 Ryanodine Receptor in Cardiac Muscle.

RATIONALE: Trimeric intracellular cation (TRIC)-A and B are distributed to endoplasmic reticulum/sarcoplasmic reticulum intracellular Ca2+ stores. The crystal structure of TRIC has been determined, confirming the homotrimeric structure of a potassium channel. While the pore architectures of TRIC-A and TRIC-B are conserved, the carboxyl-terminal tail (CTT) domains of TRIC-A and TRIC-B are different from each other. Aside from its recognized role as a counterion channel that participates in excitation-contraction coupling of striated muscles, the physiological function of TRIC-A in heart physiology and disease has remained largely unexplored. OBJECTIVE: In cardiomyocytes, spontaneous Ca2+ waves, triggered by store overload-induced Ca2+ release mediated by the RyR2 (type 2 ryanodine receptor), develop extrasystolic contractions often associated with arrhythmic events. Here, we test the hypothesis that TRIC-A is a physiological component of RyR2-mediated Ca2+ release machinery that directly modulates store overload-induced Ca2+ release activity via CTT. METHODS AND RESULTS: We show that cardiomyocytes derived from the TRIC-A-/- (TRIC-A knockout) mice display dysregulated Ca2+ movement across sarcoplasmic reticulum. Biochemical studies demonstrate a direct interaction between CTT-A and RyR2. Modeling and docking studies reveal potential sites on RyR2 that show differential interactions with CTT-A and CTT-B. In HEK293 (human embryonic kidney) cells with stable expression of RyR2, transient expression of TRIC-A, but not TRIC-B, leads to apparent suppression of spontaneous Ca2+ oscillations. Ca2+ measurements using the cytosolic indicator Fura-2 and the endoplasmic reticulum luminal store indicator D1ER suggest that TRIC-A enhances Ca2+ leak across the endoplasmic reticulum by directly targeting RyR2 to modulate store overload-induced Ca2+ release. Moreover, synthetic CTT-A peptide facilitates RyR2 activity in lipid bilayer reconstitution system, enhances Ca2+ sparks in permeabilized TRIC-A-/- cardiomyocytes, and induces intracellular Ca2+ release after microinjection into isolated cardiomyocytes, whereas such effects were not observed with the CTT-B peptide. In response to isoproterenol stimulation, the TRIC-A-/- mice display irregular ECG and develop more fibrosis than the WT (wild type) littermates. CONCLUSIONS: In addition to the ion-conducting function, TRIC-A functions as an accessory protein of RyR2 to modulate sarcoplasmic reticulum Ca2+ handling in cardiac muscle.

[Electrophysiological Effect of Citreoviridin on Human InducedPluripotent Stem Cell-derived Cardiomyocytes].

Citreoviridin (CTV) is a mycotoxin produced by various fungi, including Penicillium citreonigrum. One of the toxicities reportedly associated with CTV is neurotoxicity. CTV is also suspected to be associated with acute cardiac beriberi (also known as "Shoshin-kakke") and Keshan disease, which can have adverse effects on the heart, so the in vivo and in vitro toxicity of CTV on the heart or cardiomyocytes in experimental animal models have been reported. However, the toxicity of CTV for the human heart, especially its electrophysiological effect, remains poorly understood. Therefore, to investigate the electrophysiological effect of CTV on the human cardiomyocytes, we conducted a multi-electrode array (MEA) using human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). The MEA revealed that 30 µmol/L of CTV stopped the beating of hiPSC-CMs, and the field potential duration and first peak amplitude were shortened at 10 µmol/L. Before the hiPSC-CMs stopped beating, the length of the inter-spike interval varied two- to four-fold. These results demonstrated that CTV induced an electrophysiological disturbance on human cardiomyocytes. This is first paper to elucidate the electrophysiological effect of CTV on human heart directly and may aid in analyzing the risk associated with CTV to ensure food safety.

An evaluation framework for new approach methodologies (NAMs) for human health safety assessment.

The need to develop new tools and increase capacity to test pharmaceuticals and other chemicals for potential adverse impacts on human health and the environment is an active area of development. Much of this activity was sparked by two reports from the US National Research Council (NRC) of the National Academies of Sciences, Toxicity Testing in the Twenty-first Century: A Vision and a Strategy (2007) and Science and Decisions: Advancing Risk Assessment (2009), both of which advocated for "science-informed decision-making" in the field of human health risk assessment. The response to these challenges for a "paradigm shift" toward using new approach methodologies (NAMS) for safety assessment has resulted in an explosion of initiatives by numerous organizations, but, for the most part, these have been carried out independently and are not coordinated in any meaningful way. To help remedy this situation, a framework that presents a consistent set of criteria, universal across initiatives, to evaluate a NAM's fit-for-purpose was developed by a multi-stakeholder group of industry, academic, and regulatory experts. The goal of this framework is to support greater consistency across existing and future initiatives by providing a structure to collect relevant information to build confidence that will accelerate, facilitate and encourage development of new NAMs that can ultimately be used within the appropriate regulatory contexts. In addition, this framework provides a systematic approach to evaluate the currently-available NAMs and determine their suitability for potential regulatory application. This 3-step evaluation framework along with the demonstrated application with case studies, will help build confidence in the scientific understanding of these methods and their value for chemical assessment and regulatory decision-making.

Development of torsadogenic risk assessment using human induced pluripotent stem cell-derived cardiomyocytes: Japan iPS Cardiac Safety Assessment (JiCSA) update.

Cardiac safety assessment is challenging because a better understanding of torsadogenic mechanisms beyond hERG blockade and QT interval prolongation is necessary for patient safety. Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) provide a new human cell-based platform to assess cardiac safety in non-clinical testing during drug development. The multi-electrode array (MEA) platform is a promising electrophysiological technology to assess QT interval prolongation and proarrhythmic potential of drug candidates using hiPSC-CMs. The Japan iPS Cardiac Safety Assessment (JiCSA) has established an MEA protocol to evaluate the applicability of hiPSC-CMs for assessing the torsadogenic potential of compounds and completed a large-scale validation study using 60 compounds. During our study, an international multi-site study of hiPSC-CMs was performed by the Comprehensive in Vitro Proarrhythmia Assay (CiPA) initiative using 28 compounds. We have comparatively analyzed our JiCSA datasets with those of CiPA using the CiPA logistical and ordinal linear regression model. Regardless of the protocol differences, the evaluation results of the 28 compounds were very similar and highly predictable for torsadogenic risks. Thus, an MEA-based approach using hiPSC-CMs would be a standard testing method to evaluate proarrhythmic potentials. This review paper would provide new insights into the hiPSC-CMs/MEA method required for its regulatory use.

Proarrhythmia risk prediction using human induced pluripotent stem cell-derived cardiomyocytes.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are expected to become a useful tool for proarrhythmia risk prediction in the non-clinical drug development phase. Several features including electrophysiological properties, ion channel expression profile and drug responses were investigated using commercially available hiPSC-CMs, such as iCell-CMs and Cor.4U-CMs. Although drug-induced arrhythmia has been extensively examined by microelectrode array (MEA) assays in iCell-CMs, it has not been fully understood an availability of Cor.4U-CMs for proarrhythmia risk. Here, we evaluated the predictivity of proarrhythmia risk using Cor.4U-CMs. MEA assay revealed linear regression between inter-spike interval and field potential duration (FPD). The hERG inhibitor E-4031 induced reverse-use dependent FPD prolongation. We next evaluated the proarrhythmia risk prediction by a two-dimensional map, which we have previously proposed. We determined the relative torsade de pointes risk score, based on the extent of FPD with Fridericia's correction (FPDcF) change and early afterdepolarization occurrence, and calculated the margins normalized to free effective therapeutic plasma concentrations. The drugs were classified into three risk groups using the two-dimensional map. This risk-categorization system showed high concordance with the torsadogenic information obtained by a public database CredibleMeds. Taken together, these results indicate that Cor.4U-CMs can be used for drug-induced proarrhythmia risk prediction.

Trimeric intracellular cation channels and sarcoplasmic/endoplasmic reticulum calcium homeostasis.

Trimeric intracellular cation channels (TRIC) represents a novel class of trimeric intracellular cation channels. Two TRIC isoforms have been identified in both the human and the mouse genomes: TRIC-A, a subtype predominantly expressed in the sarcoplasmic reticulum (SR) of muscle cells, and TRIC-B, a ubiquitous subtype expressed in the endoplasmic reticulum (ER) of all tissues. Genetic ablation of either TRIC-A or TRIC-B leads to compromised K(+) permeation and Ca(2+) release across the SR/ER membrane, supporting the hypothesis that TRIC channels provide a counter balancing K(+) flux that reduces SR/ER membrane depolarization for maintenance of the electrochemical gradient that drives SR/ER Ca(2+) release. TRIC-A and TRIC-B seem to have differential functions in Ca(2+) signaling in excitable and nonexcitable cells. Tric-a(-/-) mice display defective Ca(2+) sparks and spontaneous transient outward currents in arterial smooth muscle and develop hypertension, in addition to skeletal muscle dysfunction. Knockout of TRIC-B results in abnormal IP3 receptor-mediated Ca(2+) release in airway epithelial cells, respiratory defects, and neonatal lethality. Double knockout mice lacking both TRIC-A and TRIC-B show embryonic lethality as a result of cardiac arrest. Such an aggravated lethality indicates that TRIC-A and TRIC-B share complementary physiological functions in Ca(2+) signaling in embryonic cardiomyocytes. Tric-a(-/-) and Tric-b(+/-) mice are viable and susceptible to stress-induced heart failure. Recent evidence suggests that TRIC-A directly modulates the function of the cardiac ryanodine receptor 2 Ca(2+) release channel, which in turn controls store-overload-induced Ca(2+) release from the SR. Thus, the TRIC channels, in addition to providing a countercurrent for SR/ER Ca(2+) release, may also function as accessory proteins that directly modulate the ryanodine receptor/IP3 receptor channel functions.

Contribution of calumin to embryogenesis through participation in the endoplasmic reticulum-associated degradation activity.

Calumin is an endoplasmic reticulum (ER)-transmembrane protein, and little is known about its physiological roles. Here we showed that calumin homozygous mutant embryos die at embryonic days (E) 10.5-11.5. At mid-gestation, calumin was expressed predominantly in the yolk sac. Apoptosis was enhanced in calumin homozygous mutant yolk sacs at E9.5, pointing to a possible link to the embryonic lethality. Calumin co-immunoprecipitated with ERAD components such as p97, BIP, derlin-1, derlin-2 and VIMP, suggesting its involvement in ERAD. Indeed, calumin knockdown in HEK 293 cells resulted in ERAD being less efficient, as demonstrated by attenuation in both degradations of a misfolded α1-antitrypsin variant and the ER-to-cytosol dislocation of cholera toxin A1 subunit. In calumin homozygous mutant yolk sac endoderm cells, ER stress-associated alterations were observed, including lipid droplet accumulation, fragmentation of the ER and dissociation of ribosomes from the ER. In this context, the ER-overload response, assumed to be cytoprotective, was also triggered in the mutant endoderm cells, but seemed to fully counteract the excessive ER stress generated due to defective ERAD. Taken together, our findings suggested that calumin serves to maintain the yolk sac integrity through participation in the ERAD activity, contributing to embryonic development.

Facilitated hyperpolarization signaling in vascular smooth muscle-overexpressing TRIC-A channels.

The TRIC channel subtypes, namely TRIC-A and TRIC-B, are intracellular monovalent cation-specific channels and likely mediate counterion movements to support efficient Ca(2+) release from the sarco/endoplasmic reticulum. Vascular smooth muscle cells (VSMCs) contain both TRIC subtypes and two Ca(2+) release mechanisms; incidental opening of ryanodine receptors (RyRs) generates local Ca(2+) sparks to induce hyperpolarization and relaxation, whereas agonist-induced activation of inositol trisphosphate receptors produces global Ca(2+) transients causing contraction. Tric-a knock-out mice develop hypertension due to insufficient RyR-mediated Ca(2+) sparks in VSMCs. Here we describe transgenic mice overexpressing TRIC-A channels under the control of a smooth muscle cell-specific promoter. The transgenic mice developed congenital hypotension. In Tric-a-overexpressing VSMCs from the transgenic mice, the resting membrane potential decreased because RyR-mediated Ca(2+) sparks were facilitated and cell surface Ca(2+)-dependent K(+) channels were hyperactivated. Under such hyperpolarized conditions, L-type Ca(2+) channels were inactivated, and thus, the resting intracellular Ca(2+) levels were reduced in Tric-a-overexpressing VSMCs. Moreover, Tric-a overexpression impaired inositol trisphosphate-sensitive stores to diminish agonist-induced Ca(2+) signaling in VSMCs. These altered features likely reduced vascular tonus leading to the hypotensive phenotype. Our Tric-a-transgenic mice together with Tric-a knock-out mice indicate that TRIC-A channel density in VSMCs is responsible for controlling basal blood pressure at the whole-animal level.

Secondary Publication: Proposal for Points of Consideration for Pluripotent Stem Cell Culture.

Human pluripotent stem cells, such as human embryonic stem cells and human induced pluripotent stem cells, are used in basic research and various applied fields, including drug discovery and regenerative medicine. Stem cell technologies have developed rapidly in recent years, and the supply of culture materials has improved. This has facilitated the culture of human pluripotent stem cells and has enabled an increasing number of researchers and bioengineers to access this technology. At the same time, it is a challenge to share the basic concepts and techniques of this technology among researchers and technicians to ensure the reproducibility of research results. Human pluripotent stem cells differ from conventional somatic cells in many aspects, and many points need to be considered in their handling, even for those experienced in cell culture. Therefore, we have prepared this proposal, "Points of Consideration for Pluripotent Stem Cell Culture," to promote the effective use of human pluripotent stem cells. This proposal includes seven items to be considered and practices to be confirmed before using human pluripotent stem cells. These are laws/guidelines and consent/material transfer agreements, diversity of pluripotent stem cells, culture materials, thawing procedure, media exchange and cell passaging, freezing procedure, and culture management. We aim for the concept of these points of consideration to be shared by researchers and technicians involved in the cell culture of pluripotent stem cells. In this way, we hope the reliability of research using pluripotent stem cells can be improved, and cell culture technology will advance.

TRIC channels supporting efficient Ca(2+) release from intracellular stores.

Trimeric intracellular cation-selective (TRIC) channel subtypes, namely TRIC-A and TRIC-B, are derived from distinct genes and distributed throughout the sarco/endoplasmic reticulum (SR/ER) and nuclear membranes. TRIC-A is preferentially expressed at high levels in excitable tissues, while TRIC-B is ubiquitously detected at relatively low levels in various tissues. TRIC channels are composed of ~300 amino acid residues and contain three putative membrane-spanning segments to form a bullet-shaped homo-trimeric assembly. Both native and purified recombinant TRIC subtypes form functional monovalent cation-selective channels in a lipid bilayer reconstitution system. The electrophysiological data indicate that TRIC channels behave as K(+) channels under intracellular conditions, although the detailed channel characteristics remain to be investigated. The pathophysiological defects detected in knockout mice suggest that TRIC channels support SR/ER Ca(2+) release mediated by ryanodine (RyR) and inositol trisphosphate receptor (IP(3)R) channels. For example, Tric-a-knockout mice develop hypertension resulting from vascular hypertonicity, and the mutant vascular smooth muscle cells exhibit insufficient RyR-mediated Ca(2+) release for inducing hyperpolarization. Tric-b-knockout mice show respiratory failure at birth, and IP(3)R-mediated Ca(2+) release essential for surfactant handling is impaired in the mutant alveolar epithelial cells. Moreover, double-knockout mice lacking both TRIC subtypes show embryonic heart failure, and SR Ca(2+) handling is deranged in the mutant cardiomyocytes. Current evidence strongly suggests that TRIC channels mediate counter-K(+) movements, in part, to facilitate physiological Ca(2+) release from intracellular stores.

TRIC-B channels display labile gating: evidence from the TRIC-A knockout mouse model.

Sarcoplasmic/endoplasmic reticulum (SR) and nuclear membranes contain two related cation channels named TRIC-A and TRIC-B. In many tissues, both subtypes are co-expressed, making it impossible to distinguish the distinct single-channel properties of each subtype. We therefore incorporated skeletal muscle SR vesicles derived from Tric-a-knockout mice into bilayers in order to characterise the biophysical properties of native TRIC-B without possible misclassification of the channels as TRIC-A, and without potential distortion of functional properties by detergent purification protocols. The native TRIC-B channels were ideally selective for cations. In symmetrical 210 mM K(+), the maximum (full) open channel level (199 pS) was equivalent to that observed when wild-type SR vesicles were incorporated into bilayers. Analysis of TRIC-B gating revealed complex and variable behaviour. Four main sub-conductance levels were observed at approximately 80 % (161 pS), 60 % (123 pS), 46 % (93 pS), and 30 % (60 pS) of the full open state. Seventy-five percent of the channels were voltage sensitive with Po being markedly reduced at negative holding potentials. The frequent, rapid transitions between TRIC-B sub-conductance states prevented development of reliable gating models using conventional single-channel analysis. Instead, we used mean-variance plots to highlight key features of TRIC-B gating in a more accurate and visually useful manner. Our study provides the first biophysical characterisation of native TRIC-B channels and indicates that this channel would be suited to provide counter current in response to Ca(2+) release from the SR. Further experiments are required to distinguish the distinct functional properties of TRIC-A and TRIC-B and understand their individual but complementary physiological roles.

[TRIC channel and hypertension].

TRIC channel subtypes form bullet-shaped homo-trimeric assemblies and behave as K (+) channels in intracellular membrane systems. The pathophysiological defects observed in knockout mice suggest that TRIC channels mediate counter-K (+) movements to facilitate Ca(2 +) release from intracellular stores in various cell types. In vascular smooth muscle cells (VSMCs) , Ca(2 +) release mediated by ryanodine receptors (RyRs) generates local Ca(2 +) sparks, which activate cell-surface Ca(2 +) -dependent K (+) channels and induce hyperpolarization. Tric-a-knockout mice develop hypertension due to elevated resting tonus in the mutant VSMCs. In Tric-a-knockout VSMCs, RyR-mediated Ca(2 +) sparks are compromised and the hyperpolarization signaling is thus impaired. Under such depolarized conditions, voltage-dependent L-type Ca(2 +) channels are hyper-activated to enhance resting tonus in Tric-a-knockout VSMCs. Therefore, the expression level of TRIC-A channels in VSMCs seems to set resting blood pressure at whole animal level. Moreover, our association study identified several single nucleotide polymorphisms (SNPs) around the TRIC-A gene that increase a hypertension risk and restrict the efficiency of antihypertensive drugs. The observations suggest that the TRIC-A SNPs can provide biomarkers for the diagnosis and personalized medical treatment of essential hypertension.

[Toward Regulatory Acceptance of MPS-Cardiac Safety Assessment as an Example].

Microphysiological system (MPS) are "Cell/tissue culture systems that reproduce in vivo organ functions in vitro by placing organ compartments that mimic the physiological environment of various organs such as the liver, small intestine, and lungs in micro-spaces." The MPS are attracting attention around the world as tools to improve human predictability in drug discovery research. In the U.S., in 2012, the NIH (National Institutes of Health) allocated a large budget to academia for research development of MPS. In Japan, the National Institute of Advanced Industrial Science and Technology and the NIHS (National Institute of Health Sciences) have been playing a central role in commercialization, performance evaluation, and standardization of MPS devices developed by academia for the liver, small intestine, kidney, and BBB as target organs/tissues in the AMED-MPS project that started in 2017. Pharmaceutical companies are beginning to utilize MPS in drug discovery research. However, MPS have only just been raised as a topic of discussion between regulatory authorities and pharmaceutical companies, and it will be necessary to overcome many barriers before data obtained by MPS can be included in drug approval documents and be widely accepted administratively. In this review, I would like to introduce cardiac safety evaluation as a concrete example to show what paths MPS should take to gain regulatory approval. In addition, I would like also to introduce human 3D heart tissue, which was developed in NIHS, as a cardiac MPS.

[In vitro in vivo extrapolation (IVIVE) in safety pharmacology to improve human predictability -new evaluation systems for new drug modalities].

In the current drug development process, most safety pharmacological tests are animal experiments optimized for low molecular weight compounds. However, development trends have shifted to new modality drugs such as human specific mRNA, antisense oligonucleotides, and siRNA, etc., indicating that now the importance of the human predictability in safety pharmacology is more important than ever. In parallel with that, in vitro use of human cells, such as human iPS cell-derived tissue cells and chimeric mice with human hepatocytes, has been studied strenuously. In this review, we will discuss about IVIVE in safety pharmacology to improve human predictability in this trend of drug development.

Contribution of K(ir)2 potassium channels to ATP-induced cell death in brain capillary endothelial cells and reconstructed HEK293 cell model.

Cellular turnover of brain capillary endothelial cells (BCECs) by the balance of cell proliferation and death is essential for maintaining the homeostasis of the blood-brain barrier. Stimulation of metabotropic ATP receptors (P2Y) transiently increased intracellular Ca²(+) concentration ([Ca²(+)](i)) in t-BBEC 117, a cell line derived from bovine BCECs. The [Ca²(+)](i) rise induced membrane hyperpolarization via the activation of apamin-sensitive small-conductance Ca²(+)-activated K(+) channels (SK2) and enhanced cell proliferation in t-BBEC 117. Here, we found anomalous membrane hyperpolarization lasting for over 10 min in response to ATP in ∼15% of t-BBEC 117, in which inward rectifier K(+) channel (K(ir)2.1) was extensively expressed. Once anomalous hyperpolarization was triggered by ATP, it was removed by Ba²(+) but not by apamin. Prolonged exposure to ATPγS increased the relative population of t-BBEC 117, in which the expression of K(ir)2.1 mRNAs was significantly higher and Ba²(+)-sensitive anomalous hyperpolarization was observed. The cultivation of t-BBEC 117 in serum-free medium also increased this population and reduced the cell number. The reduction of cell number was enhanced by the addition of ATPγS and the enhancement was antagonized by Ba²(+). In the human embryonic kidney 293 cell model, where SK2 and K(ir)2.1 were heterologously coexpressed, [Ca²(+)](i) rise by P2Y stimulation triggered anomalous hyperpolarization and cell death. In conclusion, P2Y stimulation in BCECs enhances cell proliferation by SK2 activation in the majority of cells but also triggers cell death in a certain population showing a substantial expression of K(ir)2.1. This dual action of P2Y stimulation may effectively facilitate BCEC turnover.

Ca2+ overload and sarcoplasmic reticulum instability in tric-a null skeletal muscle.

The sarcoplasmic reticulum (SR) of skeletal muscle contains K(+), Cl(-), and H(+) channels may facilitate charge neutralization during Ca(2+) release. Our recent studies have identified trimeric intracellular cation (TRIC) channels on SR as an essential counter-ion permeability pathway associated with rapid Ca(2+) release from intracellular stores. Skeletal muscle contains TRIC-A and TRIC-B isoforms as predominant and minor components, respectively. Here we test the physiological function of TRIC-A in skeletal muscle. Biochemical assay revealed abundant expression of TRIC-A relative to the skeletal muscle ryanodine receptor with a molar ratio of TRIC-A/ryanodine receptor ∼5:1. Electron microscopy with the tric-a(-/-) skeletal muscle showed Ca(2+) overload inside the SR with frequent formation of Ca(2+) deposits compared with the wild type muscle. This elevated SR Ca(2+) pool in the tric-a(-/-) muscle could be released by caffeine, whereas the elemental Ca(2+) release events, e.g. osmotic stress-induced Ca(2+) spark activities, were significantly reduced likely reflecting compromised counter-ion movement across the SR. Ex vivo physiological test identified the appearance of "alternan" behavior with isolated tric-a(-/-) skeletal muscle, i.e. transient and drastic increase in contractile force appeared within the decreasing force profile during repetitive fatigue stimulation. Inhibition of SR/endoplasmic reticulum Ca(2+ ATPase) function could lead to aggravation of the stress-induced alternans in the tric-a(-/-) muscle. Our data suggests that absence of TRIC-A may lead to Ca(2+) overload in SR, which in combination with the reduced counter-ion movement may lead to instability of Ca(2+) movement across the SR membrane. The observed alternan behavior with the tric-a(-/-) muscle may reflect a skeletal muscle version of store overload-induced Ca(2+) release that has been reported in the cardiac muscle under stress conditions.

Up-regulation of K(ir)2.1 by ER stress facilitates cell death of brain capillary endothelial cells.

Brain capillary endothelial cells (BCECs) form blood brain barrier (BBB) to maintain brain homeostasis. Cell turnover of BCECs by the balance of cell proliferation and cell death is critical for maintaining the integrity of BBB. Here we found that stimuli with tunicamycin, endoplasmic reticulum (ER) stress inducer, up-regulated inward rectifier K(+) channel (K(ir)2.1) and facilitated cell death in t-BBEC117, a cell line derived from bovine BCECs. The activation of K(ir) channels contributed to the establishment of deeply negative resting membrane potential in t-BBEC117. The deep resting membrane potential increased the resting intracellular Ca(2+) concentration due to Ca(2+) influx through non-selective cation channels and thereby partly but significantly regulated cell death in t-BBEC117. The present results suggest that the up-regulation of K(ir)2.1 is, at least in part, responsible for cell death/cell turnover of BCECs induced by a variety of cellular stresses, particularly ER stress, under pathological conditions.

New molecular components supporting ryanodine receptor-mediated Ca(2+) release: roles of junctophilin and TRIC channel in embryonic cardiomyocytes.

Ca(2+) mobilization from intracellular stores is mediated by Ca(2+) release channels, designated ryanodine and IP(3) receptors, and directly regulates important cellular reactions including muscle contraction, endo/exocrine secretion, and neural excitability. In order to function as an intracellular store, the endo/sarcoplasmic reticulum is equipped with cooperative Ca(2+) uptake, storage and release machineries, comprising synergic collaborations among integral-membrane, cytoplasmic and luminal proteins. Our recent studies have demonstrated that junctophilins form junctional membrane complexes between the plasma membrane and the endo/sarcoplasmic reticulum in excitable cells, and that TRIC (trimeric intracellular cation) channels act as novel monovalent cation-specific channels on intracellular membrane systems. Knockout mice have provided evidence that both junctophilins and TRIC channels support efficient ryanodine receptor-mediated Ca(2+) release in muscle cells. This review focuses on cardiac Ca(2+) release by discussing pathological defects of mutant cardiomyocytes lacking ryanodine receptors, junctophilins, or TRIC channels.

F-phenibut (ß-(4-Fluorophenyl)-GABA), a potent GABAB receptor agonist, activates an outward-rectifying K+ current and suppresses the generation of action potentials in mouse cerebellar Purkinje cells.

The GABA analog phenibut (ß-Phenyl-GABA) is a GABAB receptor agonist that has been licensed for various uses in Russia. Phenibut is also available as a dietary supplement from online vendors worldwide, and previous studies have indicated that phenibut overdose results in intoxication, withdrawal symptoms, and addiction. F-phenibut (ß-(4-Fluorophenyl)-GABA), a derivative of phenibut, has not been approved for clinical use. However, it is also available as a nootropic supplement from online suppliers. F-phenibut binds to GABAB with a higher affinity than phenibut; therefore, F-phenibut may lead to more serious intoxication than phenibut. However, the mechanisms by which F-phenibut acts on GABAB receptors and influences neuronal function remain unknown. In the present study, we compared the potency of F-phenibut, phenibut, and the GABAB agonist (±)-baclofen (baclofen) using in vitro patch-clamp recordings obtained from mouse cerebellar Purkinje cells slice preparations Our findings indicate that F-phenibut acted as a potent GABAB agonist. EC50 of outward current density evoked by the three GABAB agonists decreased in the following order: phenibut (1362 µM) > F-phenibut (23.3 µM) > baclofen (6.0 µM). The outward current induced by GABAB agonists was an outward-rectifying K+ current, in contrast to the previous finding that GABAB agonists activates an inward-rectifying K+ current. The K+ current recorded in the present study was insensitive to extracellular Ba2+, intra- or extracellular Cs+, and intra- or extracellular tetraethylammonium-Cl. Moreover, F-phenibut suppressed action potential generation in Purkinje cells. Thus, abuse of F-phenibut may lead to severe damage by inhibiting the excitability of GABAB-expressing neurons.