Molecular logic of salt taste reception in special reference to transmembrane channel-like 4 (TMC4).

The taste is biologically of intrinsic importance. It almost momentarily perceives environmental stimuli for better survival. In the early 2000s, research into taste reception was greatly developed with discovery of the receptors. However, the mechanism of salt taste reception is not fully elucidated yet and many questions still remain. At present, next-generation sequencing and genome-editing technologies are available which would become pivotal tools to elucidate the remaining issues. Here we review current mechanisms of salt taste reception in particular and characterize the properties of transmembrane channel-like 4 as a novel salt taste-related molecule that we found using these sophisticated tools.

Spatial and Functional Crosstalk between the Mitochondrial Na+-Ca2+ Exchanger NCLX and the Sarcoplasmic Reticulum Ca2+ Pump SERCA in Cardiomyocytes.

The mitochondrial Na+-Ca2+ exchanger, NCLX, was reported to supply Ca2+ to sarcoplasmic reticulum (SR)/endoplasmic reticulum, thereby modulating various cellular functions such as the rhythmicity of cardiomyocytes, and cellular Ca2+ signaling upon antigen receptor stimulation and chemotaxis in B lymphocytes; however, there is little information on the spatial relationships of NCLX with SR Ca2+ handling proteins, and their physiological impact. Here we examined the issue, focusing on the interaction of NCLX with an SR Ca2+ pump SERCA in cardiomyocytes. A bimolecular fluorescence complementation assay using HEK293 cells revealed that the exogenously expressed NCLX was localized in close proximity to four exogenously expressed SERCA isoforms. Immunofluorescence analyses of isolated ventricular myocytes showed that the NCLX was localized to the edges of the mitochondria, forming a striped pattern. The co-localization coefficients in the super-resolution images were higher for NCLX-SERCA2, than for NCLX-ryanodine receptor and NCLX-Na+/K+ ATPase α-1 subunit, confirming the close localization of endogenous NCLX and SERCA2 in cardiomyocytes. The mathematical model implemented with the spatial and functional coupling of NCLX and SERCA well reproduced the NCLX inhibition-mediated modulations of SR Ca2+ reuptake in HL-1 cardiomyocytes. Taken together, these results indicated that NCLX and SERCA are spatially and functionally coupled in cardiomyocytes.

Physiological and Pathophysiological Roles of Mitochondrial Na+-Ca2+ Exchanger, NCLX, in Hearts.

It has been over 10 years since SLC24A6/SLC8B1, coding the Na+/Ca2+/Li+ exchanger (NCLX), was identified as the gene responsible for mitochondrial Na+-Ca2+ exchange, a major Ca2+ efflux system in cardiac mitochondria. This molecular identification enabled us to determine structure-function relationships, as well as physiological/pathophysiological contributions, and our understandings have dramatically increased. In this review, we provide an overview of the recent achievements in relation to NCLX, focusing especially on its heart-specific characteristics, biophysical properties, and spatial distribution in cardiomyocytes, as well as in cardiac mitochondria. In addition, we discuss the roles of NCLX in cardiac functions under physiological and pathophysiological conditions-the generation of rhythmicity, the energy metabolism, the production of reactive oxygen species, and the opening of mitochondrial permeability transition pores.

TMC4 is a novel chloride channel involved in high-concentration salt taste sensation.

"Salty taste" sensation is evoked when sodium and chloride ions are present together in the oral cavity. The presence of an epithelial cation channel that receives Na+ has previously been reported. However, no molecular entity involving Cl- receptors has been elucidated. We report the strong expression of transmembrane channel-like 4 (TMC4) in the circumvallate and foliate papillae projected to the glossopharyngeal nerve, mediating a high-concentration of NaCl. Electrophysiological analysis using HEK293T cells revealed that TMC4 was a voltage-dependent Cl- channel and the consequent currents were completely inhibited by NPPB, an anion channel blocker. TMC4 allowed permeation of organic anions including gluconate, but their current amplitudes at positive potentials were less than that of Cl-. Tmc4-deficient mice showed significantly weaker glossopharyngeal nerve response to high-concentration of NaCl than the wild-type littermates. These results indicated that TMC4 is a novel chloride channel that responds to high-concentration of NaCl.

Minor contribution of NCX to Na+-Ca2+ exchange activity in brain mitochondria.

NCLX was identified as a mitochondrial Na+-Ca2+ exchanger. However, contribution of NCLX to overall mitochondrial Na+-Ca2+ exchange activity remains unclear, especially in brain mitochondria where plasma membrane Na+-Ca2+ exchanger NCX also exists. We studied the issue using isolated mouse brain mitochondria. The Na+- as well as Li+-dependent Ca2+ efflux from mitochondria was significantly inhibited by a NCLX blocker, but was insensitive to NCX blockers, suggesting that NCLX comprises a major part in forward mode of mitochondrial Na+-Ca2+ exchange activity. On the other hand, the Na+-dependent Ca2+ influx into mitochondria, the reverse mode, was insensitive to all the blockers tested, suggesting unidentified Ca2+ transport systems.

Membrane current evoked by mitochondrial Na+-Ca2+ exchange in mouse heart.

The electrogenicity of mitochondrial Na+-Ca2+ exchange (NCXm) had been controversial and no membrane current through it had been reported. We succeeded for the first time in recording NCXm-mediated currents using mitoplasts derived from mouse ventricle. Under conditions that K+, Cl-, and Ca2+ uniporter currents were inhibited, extra-mitochondrial Na+ induced inward currents with 1 µM Ca2+ in the pipette. The half-maximum concentration of Na+ was 35.6 mM. The inward current was diminished without Ca2+ in the pipette, and was augmented with 10 µM Ca2+. The Na+-induced inward currents were largely inhibited by CGP-37157, an NCXm blocker. However, the reverse mode of NCXm, which should be detected as an outward current, was hardly induced by extra-mitochondrial application of Ca2+ with Na+ in the pipette. It was concluded that NCXm is electrogenic. This property may be advantageous for facilitating Ca2+ extrusion from mitochondria, which has large negative membrane potential.

Integration of mitochondrial energetics in heart with mathematical modelling.

It has been an unsolved question how cardiac mitochondrial energetics is regulated during working transition. Mathematical modelling is a powerful tool for exploring the complicated networks of mitochondrial metabolism. We summarize the recent progress and remaining questions about mitochondrial energetics in heart, especially focusing on approaches utilizing mathematical modelling. Feedback activation by ADP and/or inorganic phosphate is an old but still attractive hypothesis for explaining the regulation mechanisms of cardiac mitochondrial energetics. However, this hypothesis has not been fully validated by experiments because rises of ADP and/or inorganic phosphate concentrations during cardiac workload increase have not been detected in many experiments. The hypothesis of intracellular energetic units is an extended version of feedback activation, which has a similar problem. The each-step activation hypothesis beautifully reproduces metabolite constancy, although such master regulators have not been identified yet. Ca2+ has been the most plausible candidate because some of the mitochondrial dehydrogenases are activated by it. Recent experimental and simulation studies, however, throw doubt on its physiological relevance. Finally, we discuss issues to be solved to obtain a better view of cardiac mitochondrial energetics.

Physiological functions of mitochondrial Na+-Ca2+ exchanger, NCLX, in lymphocytes.

Roles of mitochondrial Na+-Ca2+ exchanger, NCLX, were studied in B lymphocytes such as heterozygous NCLX knockout DT40 cells, NCLX knockdown A20 cells, and native mouse spleen B lymphocytes treated with a NCLX blocker, CGP-37157. Cytosolic Ca2+ response to B cell receptor stimulation was impaired in these B lymphocytes, demonstrating importance of mitochondria-ER Ca2+ recycling via NCLX and sarco/endoplasmic reticulum Ca2+-ATPase SERCA, and interaction with store-operated Ca2+ entry. NCLX was also associated with motility and chemotaxis of B lymphocyte. Contrary to B lymphocytes, contribution of NCLX in mouse spleen T lymphocytes was minor.

Uncovering the arrhythmogenic potential of TRPM4 activation in atrial-derived HL-1 cells using novel recording and numerical approaches.

AIMS: Transient receptor potential cation channel subfamily melastatin member 4 (TRPM4), a Ca2+-activated nonselective cation channel abundantly expressed in the heart, has been implicated in conduction block and other arrhythmic propensities associated with cardiac remodelling and injury. The present study aimed to quantitatively evaluate the arrhythmogenic potential of TRPM4. METHODS AND RESULTS: Patch clamp and biochemical analyses were performed using expression system and an immortalized atrial cardiomyocyte cell line (HL-1), and numerical model simulation was employed. After rapid desensitization, robust reactivation of TRPM4 channels required high micromolar concentrations of Ca2+. However, upon evaluation with a newly devised, ionomycin-permeabilized cell-attached (Iono-C/A) recording technique, submicromolar concentrations of Ca2+ (apparent Kd = ∼500 nM) were enough to activate this channel. Similar submicromolar Ca2+ dependency was also observed with sharp electrode whole-cell recording and in experiments coexpressing TRPM4 and L-type voltage-dependent Ca2+ channels. Numerical simulations using a number of action potential (AP) models (HL-1, Nygren, Luo-Rudy) incorporating the Ca2+- and voltage-dependent gating parameters of TRPM4, as assessed by Iono-C/A recording, indicated that a few-fold increase in TRPM4 activity is sufficient to delay late AP repolarization and further increases (≥ six-fold) evoke early afterdepolarization. These model predictions are consistent with electrophysiological data from angiotensin II-treated HL-1 cells in which TRPM4 expression and activity were enhanced. CONCLUSIONS: These results collectively indicate that the TRPM4 channel is activated by a physiological range of Ca2+ concentrations and its excessive activity can cause arrhythmic changes. Moreover, these results demonstrate potential utility of the first AP models incorporating TRPM4 gating for in silico assessment of arrhythmogenicity in remodelling cardiac tissue.

Patient-Specific Human Induced Pluripotent Stem Cell Model Assessed with Electrical Pacing Validates S107 as a Potential Therapeutic Agent for Catecholaminergic Polymorphic Ventricular Tachycardia.

INTRODUCTION: Human induced pluripotent stem cells (hiPSCs) offer a unique opportunity for disease modeling. However, it is not invariably successful to recapitulate the disease phenotype because of the immaturity of hiPSC-derived cardiomyocytes (hiPSC-CMs). The purpose of this study was to establish and analyze iPSC-based model of catecholaminergic polymorphic ventricular tachycardia (CPVT), which is characterized by adrenergically mediated lethal arrhythmias, more precisely using electrical pacing that could promote the development of new pharmacotherapies. METHOD AND RESULTS: We generated hiPSCs from a 37-year-old CPVT patient and differentiated them into cardiomyocytes. Under spontaneous beating conditions, no significant difference was found in the timing irregularity of spontaneous Ca2+ transients between control- and CPVT-hiPSC-CMs. Using Ca2+ imaging at 1 Hz electrical field stimulation, isoproterenol induced an abnormal diastolic Ca2+ increase more frequently in CPVT- than in control-hiPSC-CMs (control 12% vs. CPVT 43%, p<0.05). Action potential recordings of spontaneous beating hiPSC-CMs revealed no significant difference in the frequency of delayed afterdepolarizations (DADs) between control and CPVT cells. After isoproterenol application with pacing at 1 Hz, 87.5% of CPVT-hiPSC-CMs developed DADs, compared to 30% of control-hiPSC-CMs (p<0.05). Pre-incubation with 10 µM S107, which stabilizes the closed state of the ryanodine receptor 2, significantly decreased the percentage of CPVT-hiPSC-CMs presenting DADs to 25% (p<0.05). CONCLUSIONS: We recapitulated the electrophysiological features of CPVT-derived hiPSC-CMs using electrical pacing. The development of DADs in the presence of isoproterenol was significantly suppressed by S107. Our model provides a promising platform to study disease mechanisms and screen drugs.

A simulation study on the constancy of cardiac energy metabolites during workload transition.

KEY POINTS: The cardiac energy metabolites such as ATP, phosphocreatine, ADP and NADH are kept relatively constant during physiological cardiac workload transition. How this is accomplished is not yet clarified, though Ca2+ has been suggested to be one of the possible mechanisms. We constructed a detailed mathematical model of cardiac mitochondria based on experimental data and studied whether known Ca2+ -dependent regulation mechanisms play roles in the metabolite constancy. Model simulations revealed that the Ca2+ -dependent regulation mechanisms have important roles under the in vitro condition of isolated mitochondria where malate and glutamate were mitochondrial substrates, while they have only a minor role and the composition of substrates has marked influence on the metabolite constancy during workload transition under the simulated in vivo condition where many substrates exist. These results help us understand the regulation mechanisms of cardiac energy metabolism during physiological cardiac workload transition. ABSTRACT: The cardiac energy metabolites such as ATP, phosphocreatine, ADP and NADH are kept relatively constant over a wide range of cardiac workload, though the mechanisms are not yet clarified. One possible regulator of mitochondrial metabolism is Ca2+ , because it activates several mitochondrial enzymes and transporters. Here we constructed a mathematical model of cardiac mitochondria, including oxidative phosphorylation, substrate metabolism and ion/substrate transporters, based on experimental data, and studied whether the Ca2+ -dependent activation mechanisms play roles in metabolite constancy. Under the in vitro condition of isolated mitochondria, where malate and glutamate were used as mitochondrial substrates, the model well reproduced the Ca2+ and inorganic phosphate (Pi ) dependences of oxygen consumption, NADH level and mitochondrial membrane potential. The Ca2+ -dependent activations of the aspartate/glutamate carrier and the F1 Fo -ATPase, and the Pi -dependent activation of Complex III were key factors in reproducing the experimental data. When the mitochondrial model was implemented in a simple cardiac cell model, simulation of workload transition revealed that cytoplasmic Ca2+ concentration ([Ca2+ ]cyt ) within the physiological range markedly increased NADH level. However, the addition of pyruvate or citrate attenuated the Ca2+ dependence of NADH during the workload transition. Under the simulated in vivo condition where malate, glutamate, pyruvate, citrate and 2-oxoglutarate were used as mitochondrial substrates, the energy metabolites were more stable during the workload transition and NADH level was almost insensitive to [Ca2+ ]cyt . It was revealed that mitochondrial substrates have a significant influence on metabolite constancy during cardiac workload transition, and Ca2+ has only a minor role under physiological conditions.

Dysregulation of a potassium channel, THIK-1, targeted by caspase-8 accelerates cell shrinkage.

Activation of caspases is crucial for the execution of apoptosis. Although the caspase cascade associated with activation of the initiator caspase-8 (CASP8) has been investigated in molecular and biochemical detail, the physiological role of CASP8 is not fully understood. Here, we identified a two-pore domain potassium channel, tandem-pore domain halothane-inhibited K+ channel 1 (THIK-1), as a novel CASP8 substrate. The intracellular region of THIK-1 was cleaved by CASP8 in apoptotic cells. Overexpression of THIK-1, but not its mutant lacking the CASP8-target sequence in the intracellular portion, accelerated cell shrinkage in response to apoptotic stimuli. In contrast, knockdown of endogenous THIK-1 by RNA interference resulted in delayed shrinkage and potassium efflux. Furthermore, a truncated THIK-1 mutant lacking the intracellular region, which mimics the form cleaved by CASP8, led to a decrease of cell volume of cultured cells without apoptotic stimulation and excessively promoted irregular development of Xenopus embryos. Taken together, these results indicate that THIK-1 is involved in the acceleration of cell shrinkage. Thus, we have demonstrated a novel physiological role of CASP8: creating a cascade that advances the cell to the next stage in the apoptotic process.

Roles of the mitochondrial Na(+)-Ca(2+) exchanger, NCLX, in B lymphocyte chemotaxis.

Lymphocyte chemotaxis plays important roles in immunological reactions, although the mechanism of its regulation is still unclear. We found that the cytosolic Na(+)-dependent mitochondrial Ca(2+) efflux transporter, NCLX, regulates B lymphocyte chemotaxis. Inhibiting or silencing NCLX in A20 and DT40 B lymphocytes markedly increased random migration and suppressed the chemotactic response to CXCL12. In contrast to control cells, cytosolic Ca(2+) was higher and was not increased further by CXCL12 in NCLX-knockdown A20 B lymphocytes. Chelating intracellular Ca(2+) with BAPTA-AM disturbed CXCL12-induced chemotaxis, suggesting that modulation of cytosolic Ca(2+) via NCLX, and thereby Rac1 activation and F-actin polymerization, is essential for B lymphocyte motility and chemotaxis. Mitochondrial polarization, which is necessary for directional movement, was unaltered in NCLX-knockdown cells, although CXCL12 application failed to induce enhancement of mitochondrial polarization, in contrast to control cells. Mouse spleen B lymphocytes were similar to the cell lines, in that pharmacological inhibition of NCLX by CGP-37157 diminished CXCL12-induced chemotaxis. Unexpectedly, spleen T lymphocyte chemotaxis was unaffected by CGP-37157 treatment, indicating that NCLX-mediated regulation of chemotaxis is B lymphocyte-specific, and mitochondria-endoplasmic reticulum Ca(2+) dynamics are more important in B lymphocytes than in T lymphocytes. We conclude that NCLX is pivotal for B lymphocyte motility and chemotaxis.

The destiny of Ca(2+) released by mitochondria.

Mitochondrial Ca(2+) is known to regulate diverse cellular functions, for example energy production and cell death, by modulating mitochondrial dehydrogenases, inducing production of reactive oxygen species, and opening mitochondrial permeability transition pores. In addition to the action of Ca(2+) within mitochondria, Ca(2+) released from mitochondria is also important in a variety of cellular functions. In the last 5 years, the molecules responsible for mitochondrial Ca(2+) dynamics have been identified: a mitochondrial Ca(2+) uniporter (MCU), a mitochondrial Na(+)-Ca(2+) exchanger (NCLX), and a candidate for a mitochondrial H(+)-Ca(2+) exchanger (Letm1). In this review, we focus on the mitochondrial Ca(2+) release system, and discuss its physiological and pathophysiological significance. Accumulating evidence suggests that the mitochondrial Ca(2+) release system is not only crucial in maintaining mitochondrial Ca(2+) homeostasis but also participates in the Ca(2+) crosstalk between mitochondria and the plasma membrane and between mitochondria and the endoplasmic/sarcoplasmic reticulum.

Identification of chemicals inducing cardiomyocyte proliferation in developmental stage-specific manner with pluripotent stem cells.

BACKGROUND: The proliferation of cardiomyocytes is highly restricted after postnatal maturation, limiting heart regeneration. Elucidation of the regulatory machineries for the proliferation and growth arrest of cardiomyocytes is imperative. Chemical biology is efficient to dissect molecular mechanisms of various cellular events and often provides therapeutic potentials. We have been investigating cardiovascular differentiation with pluripotent stem cells. The combination of stem cell and chemical biology can provide novel approaches to investigate the molecular mechanisms and manipulation of cardiomyocyte proliferation. METHODS AND RESULTS: To identify chemicals that regulate cardiomyocyte proliferation, we performed a screening of a defined chemical library based on proliferation of mouse pluripotent stem cell-derived cardiomyocytes and identified 4 chemical compound groups: inhibitors of glycogen synthase kinase-3, p38 mitogen-activated protein kinase, and Ca(2+)/calmodulin-dependent protein kinase II, and activators of extracellular signal-regulated kinase. Several appropriate combinations of chemicals synergistically enhanced proliferation of cardiomyocytes derived from both mouse and human pluripotent stem cells, notably up to a 14-fold increase in mouse cardiomyocytes. We also examined the effects of identified chemicals on cardiomyocytes in various developmental stages and species. Whereas extracellular signal-regulated kinase activators and Ca(2+)/calmodulin-dependent protein kinase II inhibitors showed proliferative effects only on cardiomyocytes in early developmental stages, glycogen synthase kinase-3 and p38 mitogen-activated protein kinase inhibitors substantially and synergistically induced re-entry and progression of cell cycle in neonatal but also as well as adult cardiomyocytes. CONCLUSIONS: Our approach successfully uncovered novel molecular targets and mechanisms controlling cardiomyocyte proliferation in distinct developmental stages and offered pluripotent stem cell-derived cardiomyocytes as a potent tool to explore chemical-based cardiac regenerative strategies.

The mitochondrial Na+-Ca2+ exchanger, NCLX, regulates automaticity of HL-1 cardiomyocytes.

Mitochondrial Ca(2+) is known to change dynamically, regulating mitochondrial as well as cellular functions such as energy metabolism and apoptosis. The NCLX gene encodes the mitochondrial Na(+)-Ca(2+) exchanger (NCXmit), a Ca(2+) extrusion system in mitochondria. Here we report that the NCLX regulates automaticity of the HL-1 cardiomyocytes. NCLX knockdown using siRNA resulted in the marked prolongation of the cycle length of spontaneous Ca(2+) oscillation and action potential generation. The upstrokes of action potential and Ca(2+) transient were markedly slower, and sarcoplasmic reticulum (SR) Ca(2+) handling were compromised in the NCLX knockdown cells. Analyses using a mathematical model of HL-1 cardiomyocytes demonstrated that blocking NCXmit reduced the SR Ca(2+) content to slow spontaneous SR Ca(2+) leak, which is a trigger of automaticity. We propose that NCLX is a novel molecule to regulate automaticity of cardiomyocytes via modulating SR Ca(2+) handling.

Mitochondria Na(+)-Ca (2+) exchange in cardiomyocytes and lymphocytes.

Mitochondria Na(+)-Ca(2+) exchange (NCX(mit)) was first discovered by Carafoli et al. in 1974. Thereafter, the mechanisms and roles of NCX(mit) have been extensively studied. We review NCX(mit) in cardiomyocytes and lymphocytes by presenting our recent studies on it. Studies of NCX(mit) in rat ventricular cells demonstrated that NCX(mit) is voltage dependent and electrogenic. A targeted knockdown and knockout of NCLX in HL-1 cardiomyocytes and B lymphocytes, respectively, significantly reduced the NCX(mit) activity, indicating that NCLX is a major component of NCX(mit) in these cells. The store-operated Ca(2+) entry was greatly attenuated in NCLX knockout lymphocytes, suggesting that substantial amount of Ca(2+) enters into mitochondria and is released to cytosol via NCX(mit). NCX(mit) or NCLX has pivotal roles in Ca(2+) handling in mitochondria and cytoplasm.

Structure and function of ∆1-tetrahydrocannabinolic acid (THCA) synthase, the enzyme controlling the psychoactivity of Cannabis sativa.

∆1-Tetrahydrocannabinolic acid (THCA) synthase catalyzes the oxidative cyclization of cannabigerolic acid (CBGA) into THCA, the precursor of the primary psychoactive agent ∆1-tetrahydrocannabinol in Cannabis sativa. The enzyme was overproduced in insect cells, purified, and crystallized in order to investigate the structure-function relationship of THCA synthase, and the tertiary structure was determined to 2.75Å resolution by X-ray crystallography (R(cryst)=19.9%). The THCA synthase enzyme is a member of the p-cresol methyl-hydroxylase superfamily, and the tertiary structure is divided into two domains (domains I and II), with a flavin adenine dinucleotide coenzyme positioned between each domain and covalently bound to His114 and Cys176 (located in domain I). The catalysis of THCA synthesis involves a hydride transfer from C3 of CBGA to N5 of flavin adenine dinucleotide and the deprotonation of O6' of CBGA. The ionized residues in the active site of THCA synthase were investigated by mutational analysis and X-ray structure. Mutational analysis indicates that the reaction does not involve the carboxyl group of Glu442 that was identified as the catalytic base in the related berberine bridge enzyme but instead involves the hydroxyl group of Tyr484. Mutations at the active-site residues His292 and Tyr417 resulted in a decrease in, but not elimination of, the enzymatic activity of THCA synthase, suggesting a key role for these residues in substrate binding and not direct catalysis.

NHE-1 blockade reversed changes in calcium transient in myocardial slices from isoproterenol-induced hypertrophied rat left ventricle.

We previously reported that left ventricular (LV) slices from isoproterenol (ISO)-induced hypertrophied rat hearts showed an increase of energy expenditure due to remodeling of Ca(2+) handling in excitation-contraction coupling, i.e., suppressed SERCA2a activity and enhanced Na(+)/Ca(2+)exchanger-1 (NCX-1) activity. Na(+)/H(+) exchanger-1 (NHE-1) inhibitor (NHEI) has been demonstrated to exert beneficial effects in the development of cardiac remodeling. We hypothesized that a novel NHE-1 selective inhibitor, BIIB723 prevents remodeling of Ca(2+) handling in LV slices of ISO-induced hypertrophied rat hearts mediated by inhibiting NCX-1 activity. The significant shortening in duration of multi-cellular Ca(2+) transient in ISO group was normalized in ISO+BIIB723 group. The significant increase in amplitude of multi-cellular Ca(2+) waves (CaW) generated at high [Ca(2+)](o) of LV slices in ISO group was also normalized in ISO+BIIB723 group. However, the enhanced NCX-1 activity was not antagonized by BIIB723. We recently reported that ISO-induced down-regulation of a Ca(2+) handling protein, SERCA2a, was normalized by BIIB723. Therefore, it seems likely that BIIB723 normalized shortened multi-cellular Ca(2+) transient duration and increased CaW amplitude in LV slices mediated via normalization of SERCA2a activity. Furthermore, the results presented here suggest the multi-cellular Ca(2+) transient duration and CaW amplitude in LV slices might be better indices reflecting SERCA2a activity than SERCA2a protein expression level.

Pivotal role of mitochondrial Na⁺₋Ca²âº exchange in antigen receptor mediated Ca²âº signalling in DT40 and A20 B lymphocytes.

Cytoplasmic Ca(2+) concentration ([Ca(2+)](i)) increases upon activation of antigen-receptor in lymphocytes. Mitochondria have been suggested to regulate the [Ca(2+)](i) response, but the molecular mechanisms and the roles are poorly understood. To clarify them, we carried out a combination study of mathematical simulations and knockout or knockdown of NCLX, a gene candidate for the mitochondrial Na(+)-Ca(2+) exchanger (NCX(mit)), in B lymphocytes. A mathematical model of Ca(2+) dynamics in B lymphocytes demonstrated that NCX(mit) inhibition reduces basal Ca(2+) content of endoplasmic reticulum (ER) and suppresses B-cell antigen receptor (BCR)-mediated [Ca(2+)](i) rise. The predictions were validated in DT40 B lymphocytes of heterozygous NCLX knockout (NCLX(+/-)). In NCLX(+/-) cells, mitochondrial Ca(2+) efflux via NCX(mit) was strongly decelerated, suggesting NCLX is a gene responsible for NCX(mit) in B lymphocytes. Consistent with the predictions, ER Ca(2+) content declined and [Ca(2+)](i) hardly rose upon BCR activation in NCLX(+/-) cells. ER Ca(2+) uptake was reduced to ∼58% of the wild-type (WT), while it was comparable to WT when mitochondrial respiration was disturbed. Essentially the same results were obtained by a pharmacological inhibition or knockdown of NCLX by siRNA in A20 B lymphocytes. Unexpectedly, ER Ca(2+) leak was augmented and co-localization of mitochondria with ER was lower in NCLX(+/-) and NCLX silenced cells. Taken together, we concluded that NCLX is a key Ca(2+) provider to ER, and that NCLX-mediated Ca(2+) recycling between mitochondria and ER is pivotal in B cell responses to antigen.