Biomedical subjects

Yasuo Mori

Publications and source records attributed to Yasuo Mori.

At least 19 recordsLinked to original sources

PubMed2006

Properties of human Cav2.1 channel with a spinocerebellar ataxia type 6 mutation expressed in Purkinje cells.

Spinocerebellar ataxia type 6 (SCA6) is caused by polyglutamine expansion in P/Q-type Ca2+ channels (Ca(v)2.1) and is characterized by predominant degeneration of cerebellar Purkinje cells. To characterize the Ca(v)2.1 channel with an SCA6 mutation in cerebellar Purkinje cells, we have generated knock-in mouse models that express human Ca(v)2.1 with 28 polyglutamine repeats (disease range) and with 13 polyglutamine repeats (normal range). Patch-clamp recordings of the Purkinje cells from homozygous control or SCA6 knock-in mice revealed a non-inactivating current that is highly sensitive to a spider toxin omega-Agatoxin IVA, indicating that the human Ca(v)2.1 expressed in Purkinje cells exhibits typical P-type properties in contrast to the previous data showing Q-type properties, when it was expressed in cultured cell lines. Furthermore, the voltage dependence of activation and inactivation and current density were not different between SCA6 and control, though these properties were altered in previous reports using non-neuronal cells as expression systems. Therefore, our results do not support the notion that the alteration of the channel properties may underlie the pathogenic mechanism of SCA6.

Age Factors↗

PubMed2006

Upregulation of TRPC1 in the development of cardiac hypertrophy.

The importance of Ca(2+) entry in the cardiac hypertrophic response is well documented, but the actual Ca(2+) entry channels remain unknown. Transient receptor potential (TRP) proteins are thought to form either homo- or heteromeric Ca(2+) entry channels that are involved in the proliferation and differentiation of various cells. The purpose of this study was to explore the potential involvement of TRP channels in the development of cardiac hypertrophy. The mRNA and protein expression of several TRP channel subunits were evaluated using hearts from abdominal aortic-banded (AAB) rats. Although TRPs C1, C3, C5, and C6 were constitutively expressed, only TRPC1 expression was significantly increased in the hearts of AAB rats compared to sham-operated rats. Using primary cultures of neonatal rat cardiomyocytes, we detected increases in the expression of TRPC1, brain natriuretic peptide (BNP), and atrial natriuretic factor (ANF), as well as increases in store-operated Ca(2+) entry (SOCE) and cell surface area, following endothelin-1 (ET-1) treatment. Silencing of the TRPC1 gene via small interfering RNA (siRNA) attenuated SOCE and prevented ET-1-, angiotensin-II (AT II)-, and phenylephrine (PE)-induced cardiac hypertrophy. In HEK 293T cells, overexpression of TRPC1 augmented SOCE, leading to an increase in nuclear factor of activated T cells (NFAT) promoter activity, while co-transfection with dominant-negative forms of TRPC1 suppressed it. In conclusion, TRPC1 functions in Ca(2+) influx, and its upregulation is involved in the development of cardiac hypertrophy; moreover, it plays an important role in the regulation of the signaling pathways that govern cardiac hypertrophy. These findings establish TRPC1 as a functionally important regulator of cardiac hypertrophy.

Animals↗

PubMed2006

Regulatory role of neuron-restrictive silencing factor in expression of TRPC1.

Neuron-restrictive silencer factor (NRSF) binds its consensus element to repress the transcription of various genes. The dominant-negative form (dnNRSF) has a hypertrophic effect on cardiogenesis through an unidentified mechanism. We examined the involvement of transient receptor potential (TRP) channel proteins, using transgenic mice overexpressing dnNRSF (dnNRSF mice). Electrophoretic mobility-shift assays revealed an interaction between NRSF and a neuron-restrictive silencer element-like sequence in intron 4 of TRPC1 genomic DNA. According to RT-PCR and Western analyses, TRPC1 was up-regulated in dnNRSF mouse heart. Transient overexpression of TRPC1 in HEK 293T cells increased the activity of the nuclear factor in activated T cells (NFAT) promoter and stimulated store-operated Ca(2+) channel (SOCC)-mediated Ca(2+) entry. Transfection of TRPC1 into primary cardiomyocytes increased NFAT activity, indicating a major role for TRPC1 in NFAT activation. Our findings strongly suggest that NRSF regulates TRP1 gene expression and causes changes in the levels of calcium entry through SOCCs.

Animals↗

PubMed2006

TRPC3 and TRPC6 are essential for angiotensin II-induced cardiac hypertrophy.

Angiotensin (Ang) II participates in the pathogenesis of heart failure through induction of cardiac hypertrophy. Ang II-induced hypertrophic growth of cardiomyocytes is mediated by nuclear factor of activated T cells (NFAT), a Ca(2+)-responsive transcriptional factor. It is believed that phospholipase C (PLC)-mediated production of inositol-1,4,5-trisphosphate (IP(3)) is responsible for Ca(2+) increase that is necessary for NFAT activation. However, we demonstrate that PLC-mediated production of diacylglycerol (DAG) but not IP(3) is essential for Ang II-induced NFAT activation in rat cardiac myocytes. NFAT activation and hypertrophic responses by Ang II stimulation required the enhanced frequency of Ca(2+) oscillation triggered by membrane depolarization through activation of DAG-sensitive TRPC channels, which leads to activation of L-type Ca(2+) channel. Patch clamp recordings from single myocytes revealed that Ang II activated DAG-sensitive TRPC-like currents. Among DAG-activating TRPC channels (TRPC3, TRPC6, and TRPC7), the activities of TRPC3 and TRPC6 channels correlated with Ang II-induced NFAT activation and hypertrophic responses. These data suggest that DAG-induced Ca(2+) signaling pathway through TRPC3 and TRPC6 is essential for Ang II-induced NFAT activation and cardiac hypertrophy.

Angiotensin II↗

PubMed2006

The order of expression of transcription factors directs hierarchical specification of hematopoietic lineages.

The mechanism of lineage specification in multipotent stem cells has not been fully understood. We recently isolated progenitors with the eosinophil, basophil, or mast cell lineage potential, all of which originate from granulocyte/monocyte progenitors (GMPs). By using these prospectively purified progenitors, we show here that the expression timing of GATA-2 and CCAAT enhancer-binding protein alpha (C/EBPalpha) can differentially control their lineage commitment. The expression of GATA-2 instructed C/EBPalpha-expressing GMPs to commit exclusively into the eosinophil lineage, while it induced basophil and/or mast cell lineage commitment if C/EBPalpha was suppressed at the GMP stage. Furthermore, simply by switching the order of C/EBPalpha and GATA-2 transduction, even lymphoid-committed progenitors recaptured these developmental processes to be reprogrammed into each of these lineages. We propose that the order of expression of key transcription factors is critical for their interplay to selectively drive lineage specification programs, by which stem cells could generate multiple lineage cells in a hierarchical manner.

Animals↗

PubMed2006

Coupling of STIM1 to store-operated Ca2+ entry through its constitutive and inducible movement in the endoplasmic reticulum.

Depletion of intracellular calcium (Ca(2+)) stores induces store-operated Ca(2+) (SOC) entry across the plasma membrane (PM). STIM1, a putative Ca(2+) sensor in the endoplasmic reticulum (ER), has been recently shown to be necessary for SOC channel activation. Here we show that STIM1 dynamically moves in tubulovesicular shape on the ER and its subcompartment in resting living cells, whereas, upon Ca(2+) store depletion, it is rapidly redistributed into discrete puncta that are located underneath, but not inserted into the PM. Normal constitutive movement of STIM1 is mediated through the coiled-coil and Ser/Thr-rich C-terminal domains in the cytoplasmic region of STIM1, whereas subsequent inducible puncta formation further requires the sterile alpha motif domain protruding into the ER lumen. Each of these three domains (coiled-coil, Ser/Thr-rich, and sterile alpha motif) was essential for activating SOC channels. Hence, our findings based on structure-function experiments suggest that constitutive dynamic movement of STIM1 in the ER and its subcompartment is obligatory for subsequent depletion-dependent redistribution of STIM1 into puncta underneath the PM and activation of SOC channels.

Animals↗

PubMed2006

Nitric oxide activates TRP channels by cysteine S-nitrosylation.

Transient receptor potential (TRP) proteins form plasma-membrane cation channels that act as sensors for diverse cellular stimuli. Here, we report a novel activation mechanism mediated by cysteine S-nitrosylation in TRP channels. Recombinant TRPC1, TRPC4, TRPC5, TRPV1, TRPV3 and TRPV4 of the TRPC and TRPV families, which are commonly classified as receptor-activated channels and thermosensor channels, induce entry of Ca(2+) into cells in response to nitric oxide (NO). Labeling and functional assays using cysteine mutants, together with membrane sidedness in activating reactive disulfides, show that cytoplasmically accessible Cys553 and nearby Cys558 are nitrosylation sites mediating NO sensitivity in TRPC5. The responsive TRP proteins have conserved cysteines on the same N-terminal side of the pore region. Notably, nitrosylation of native TRPC5 upon G protein-coupled ATP receptor stimulation elicits entry of Ca(2+) into endothelial cells. These findings reveal the structural motif for the NO-sensitive activation gate in TRP channels and indicate that NO sensors are a new functional category of cellular receptors extending over different TRP families.

Animals↗

PubMed2006

Oligo-Asp tag/Zn(II) complex probe as a new pair for labeling and fluorescence imaging of proteins.

To accomplish the selective labeling of a specific protein in complicated biological systems, a peptide tag incorporated into the protein and a complementary small molecular probe are required. Although a variety of peptide tag/probe pairs have been developed as molecular tools for protein analyses, the availability of pairs suitable for real-time imaging of proteins is still limited. We now report a new peptide tag/artificial probe pair composed of a genetically encodable oligo-aspartate sequence (D4 tag, (D4)n, n = 1-3) and the corresponding multinuclear Zn(II) complexes (Zn(II)-DpaTyrs). The strong binding affinity of the Zn(II)-DpaTyr probes with the D4 tag was a result of the multiple coordination bonds and the multivalent effect. It was measured quantitatively by isothermal titration calorimetry. The high affinity between the tag and the probe, indispensable for the selective protein labeling, enabled the pair to be used for the labeling and fluorescence imaging of a membrane-bound receptor protein tethering a triply repeated D4 tag ((D4)3) in an intact cell configuration without significantly affecting the receptor signal transduction.

Amino Acid Sequence↗

PubMed2006

Arachidonic acid can function as a signaling modulator by activating the TRPM5 cation channel in taste receptor cells.

Vertebrate sensory cells such as vomeronasal neurons and Drosophila photoreceptor cells use TRP channels to respond to exogenous stimuli. In mammalian taste cells, bitter and sweet substances as well as some amino acids are received by G protein-coupled receptors (T2Rs or T1Rs). As a result of activation of G protein and phospholipase Cbeta2, the TRPM5 channel is activated. Intracellular Ca(2+) is known to be a TRPM5 activator, but the participation of lipid activators remains unreported. To clarify the effect of arachidonic acid on TRPM5 in taste cells, we investigated the expression profile of a series of enzymes involved in controlling the intracellular free arachidonic acid level, with the result that in a subset of taste bud cells, monoglyceride lipase (MGL) and cyclooxygenase-2 (COX-2) are expressed as well as the previously reported group IIA phospholipase A(2) (PLA(2)-IIA). Double-labeling analysis revealed that MGL, COX-2 and PLA(2)-IIA are co-expressed in some cells that express TRPM5. We then investigated whether arachidonic acid activates TRPM5 via a heterologous expression system in HEK293 cells, and found that its activation occurred at 10 microM arachidonic acid. These results strongly suggest the possibility that arachidonic acid acts as a modulator of TRPM5 in taste signaling pathways.

Animals↗

PubMed2006

Transient receptor potential (TRP) protein 7 acts as a G protein-activated Ca2+ channel mediating angiotensin II-induced myocardial apoptosis.

Transient receptor potential (TRP) proteins have been identified as cation channels that are activated by agonist-receptor coupling and mediate various cellular functions. TRPC7, a homologue of TRP channels, has been shown to act as a Ca2+ channel activated by G protein-coupled stimulation and to be abundantly expressed in the heart with an as-yet-unknown function. We studied the role of TRPC7 in G protein-activated signaling in HEK293 cells and cultured cardiomyocytes in vitro transfected with FLAG-tagged TRPC7 cDNA and in Dahl salt-sensitive rats with heart failure in vivo. TRPC7-transfected HEK293 cells showed an augmentation of carbachol-induced intracellular Ca2+ transient, which was attenuated under a Ca2+-free condition or in the presence of SK&F96365 (a Ca2+-permeable channel blocker). Upon stimulation with angiotensin II (Ang II), cultured neonatal rat cardiomyocytes transfected with TRPC7 exhibited a significant increase in apoptosis detected by TUNEL staining, accompanied with a decrease in the expression of atrial natriuretic factor and destruction of actin fibers, as compared with non-transfected cardiomyocytes. Ang II-induced apoptosis was inhibited by CV-11974 (Candesartan; Ang II type 1 [AT1] receptor blocker), SK&F96365, and FK506 (calcineurin inhibitor). In Dahl salt-sensitive rats, apoptosis and TRPC7 expression were increased in the failing myocardium, and a long-term treatment with temocapril, an angiotensin-converting enzyme inhibitor, suppressed both. Our findings suggest that TRPC7 could act as a Ca2+ channel activated by AT1 receptors, leading to myocardial apoptosis possibly via a calcineurin-dependent pathway. TRPC7 might be a key initiator linking AT1-activation to myocardial apoptosis, and thereby contributing to the process of heart failure.

Angiotensin II↗

PubMed2006

Blocker-resistant presynaptic voltage-dependent Ca2+ channels underlying glutamate release in mice nucleus tractus solitarii.

The visceral sensory information from the internal organs is conveyed via the vagus and glossopharyngeal primary afferent fibers and transmitted to the second-order neurons in the nucleus of the solitary tract (NTS). The glutamate release from the solitary tract (TS) axons to the second-order NTS neurons remains even in the presence of toxins that block N- and P/Q-type voltage-dependent Ca(2+) channels (VDCCs). The presynaptic VDCC playing the major role at this synapse remains unidentified. To address this issue, we examined two hypotheses in this study. First, we examined whether the remaining large component occurs through activation of a omega-conotoxin GVIA (omega-CgTX)-insensitive variant of N-type VDCC by using the mice genetically lacking its pore-forming subunit alpha(1B). Second, we examined whether R-type VDCCs are involved in transmitter release at the TS-NTS synapse. The EPSCs evoked by stimulation of the TS were recorded in medullary slices from young mice. omega-Agatoxin IVA (omega-AgaIVA; 200 nM) did not significantly affect the EPSC amplitude in the mice genetically lacking N-type VDCC. SNX-482 (500 nM) and Ni(2+) (100 microM) did not significantly reduce EPSC amplitude in ICR mice. These results indicate that, unlike in most of the brain synapses identified to date, the largest part of the glutamate release at the TS-NTS synapse in mice occurs through activation of non-L, non-P/Q, non-R, non-T and non-N (including its posttranslational variants) VDCCs at least according to their pharmacological properties identified to date.

Analysis of Variance↗

PubMed2006

Native TRPC7 channel activation by an inositol trisphosphate receptor-dependent mechanism.

In DT40 B lymphocytes, Canonical Transient Receptor Potential 7 (TRPC7) functions as a diacylglycerol-activated non-selective cation channel. However, previous work indicated that the non-store-operated Ca2+ entry in this cell type depends upon inositol trisphosphate receptors (IP3R). With the cell-attached configuration oleyl-acetyl-glycerol (OAG) induced single channel activity (75 pS) that was not observed in TRPC7-/- cells but was rescued by expression of TRPC7 under conditions expected to produce relatively low levels of expression ((LowT7)TRPC7-/-). A DT40 cell line lacking IP3R(IP3R-/- cells) showed no OAG-induced single channel activity, but this activity was rescued by transient expression of an IP3R((IP3R)IP3R-/-). Single channel properties in (LowT7)TRPC7-/- or (IP3R)IP3R-/- DT40 cells were indistinguishable from one another and from wild-type cells. Thus, TRPC7 forms, or is part of, the channel underlying endogenous diacylglycerol-activated currents in DT40 B lymphocytes, and this activity of native TRPC7 requires IP3R. However, with conditions expected to produce greater expression levels, TRPC7 functioned independently of the presence of IP3R. This finding may serve to resolve previously conflicting reports from expression studies of TRPC channels.

Animals↗

PubMed2006

Extracellular-added ADP-ribose increases intracellular free Ca2+ concentration through Ca2+ release from stores, but not through TRPM2-mediated Ca2+ entry, in rat beta-cell line RIN-5F.

Intracellular ADP-ribose is an activator of TRPM2, which is a Ca2+-permeable channel and mediates H2O2-induced cell death, in the TRPM2-expressing rat beta-cell line RIN-5F. We examined the effect of extracellular-added ADP-ribose on intracellular Ca2+ concentration in RIN-5F cells. ADP-ribose induced Ca2+ release from the thapsigargin-sensitive Ca2+ store, but not Ca2+ entry across the plasma membrane. A phospholipase C (PLC) inhibitor and a non-specific IP3 receptor inhibitor blocked its Ca2+ release. H2O2-induced Ca2+ entry through TRPM2 was not affected by extracellular ADP-ribose. These findings suggest that extracellular-added ADP-ribose induces Ca2+ release through the PLC-IP3 pathway and does not act as a TRPM2 activator.

Adenosine Diphosphate Ribose↗

PubMed2006

A critical role of TRPM2 in neuronal cell death by hydrogen peroxide.

A brief exposure to hydrogen peroxide (H2O2) induces severe deterioration of primary cultured neurons in vitro. We have investigated a link between the H2O2-induced neuronal death and Ca2+-permeable TRPM2 channels regulated by ADP-ribose (ADPR). In cultured cerebral cortical neurons from fetal rat, TRPM2 proteins were detected at cell bodies and neurite extensions. Application of H2O2 to the cultured neurons elicited an increase in intracellular Ca2+ concentration ([Ca2+]i) caused by Ca2+ influx and the Ca2+-dependent neuronal death in a similar concentration range. Molecular cloning of TRPM2 cDNA from rat brain revealed several differences in amino acid sequences within the Nudix box region as compared with those of human and mouse TRPM2. ADPR-induced current responses, H2O2-induced Ca2+ influx, and H2O2-induced cell death were induced in human embryonic kidney cells heterologously expressing rat TRPM2. Treatment of cultured neurons with small interfering RNA against rat TRPM2,which efficiently suppressed immunoreactive TRPM2 content and the H2O2-induced Ca2+ influx,significantly inhibited H2O2-induced neuronal death. These results suggest that TRPM2 plays a pivotal role in H2O2-induced neuronal death as redox-sensitive Ca2+-permeable channels expressed in neurons.

Animals↗

PubMed2006

TRPM2 activation by cyclic ADP-ribose at body temperature is involved in insulin secretion.

There are eight thermosensitive TRP (transient receptor potential) channels in mammals, and there might be other TRP channels sensitive to temperature stimuli. Here, we demonstrate that TRPM2 can be activated by exposure to warm temperatures (>35 degrees C) apparently via direct heat-evoked channel gating. beta-NAD(+)- or ADP-ribose-evoked TRPM2 activity is robustly potentiated at elevated temperatures. We also show that, even though cyclic ADP-ribose (cADPR) does not activate TRPM2 at 25 degrees C, co-application of heat and intracellular cADPR dramatically potentiates TRPM2 activity. Heat and cADPR evoke similar responses in rat insulinoma RIN-5F cells, which express TRPM2 endogenously. In pancreatic islets, TRPM2 is coexpressed with insulin, and mild heating of these cells evokes increases in both cytosolic Ca(2+) and insulin release, which is K(ATP) channel-independent and protein kinase A-mediated. Heat-evoked responses in both RIN-5F cells and pancreatic islets are significantly diminished by treatment with TRPM2-specific siRNA. These results identify TRPM2 as a potential molecular target for cADPR, and suggest that TRPM2 regulates Ca(2+) entry into pancreatic beta-cells at body temperature depending on the production of cADPR-related molecules, thereby regulating insulin secretion.

Animals↗

PubMed2006

A functional AMPA receptor-calcium channel complex in the postsynaptic membrane.

Ca(2+) channels play critical roles in the regulation of synaptic activity. In contrast to the well established function of voltage-activated Ca(2+) channels in the presynaptic membrane for neurotransmitter release, some studies are just beginning to elucidate the functions of the Ca(2+) channels in the postsynaptic membrane. In this study, we demonstrated the functional association of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors with the neuronal Ca(2+) channels. A series of biochemical studies showed the specific association of Ca(v)2.1 (alpha(1A)-class) and Ca(v)2.2 (alpha(1B)-class) with AMPA receptors in the postsynaptic membrane. Our electrophysiological and Ca(2+) imaging analyses of recombinant Ca(v)2.1 and AMPA receptors also showed functional coupling of the two channels. Considering the critical roles of postsynaptic intracellular concentration of Ca(2+) ([Ca(2+)](i)) increase and AMPA receptor trafficking for long-term potentiation (LTP) and long-term depression (LTD), the functional association of Ca(2+) channels with the AMPA receptors may provide new insights into the mechanism of synaptic plasticity.

Animals↗

PubMed2006

Intracellular-produced hydroxyl radical mediates H2O2-induced Ca2+ influx and cell death in rat beta-cell line RIN-5F.

The melastatin-related transient receptor potential channel TRPM2 is a Ca(2+)-permeable channel that is activated by H(2)O(2), and the Ca(2+) influx through TRPM2 mediates cell death. However, the responsible oxidants for TRPM2 activation remain to be identified. In the present study, we investigated the involvement of hydroxyl radical on TRPM2 activation in TRPM2-expressing HEK293 cells and the rat beta-cell line RIN-5F. In both cell types, H(2)O(2) induced Ca(2+) influx in a concentration-dependent manner. However, the addition of hydroxyl radical, which was produced by mixing FeSO(4) and H(2)O(2), to the cells, did not increase intracellular Ca(2+) concentration. Interestingly, when H(2)O(2) was added to the cells under intracellular Fe(2+)-accumulated conditions, Ca(2+) influx was markedly enhanced compared to H(2)O(2) alone. In addition, the H(2)O(2)-induced Ca(2+) influx was reduced by hydroxyl radical scavengers and an iron chelator. Under intracellular Fe(2+)-accumulated conditions, H(2)O(2)-induced RIN-5F cell death through TRPM2 activation was also markedly enhanced. Hydroxyl radical scavengers and an iron chelator suppressed the RIN-5F cell death by H(2)O(2). These results strongly suggest that the intracellular hydroxyl radical plays a key role in the activation of TRPM2 during H(2)O(2) treatment, and TRPM2 activation mediated by hydroxyl radical is implicated in H(2)O(2)-induced cell death in the beta-cell line RIN-5F.

Adenosine Triphosphate↗

PubMed2006

Cyclosporin and Timothy syndrome increase mode 2 gating of CaV1.2 calcium channels through aberrant phosphorylation of S6 helices.

Calcium channels in the plasma membrane rarely remain open for much more than a millisecond at any one time, which avoids raising intracellular calcium to toxic levels. However, the dihydropyridine-sensitive calcium channels of the CaV1 family, which selectively couple electrical excitation to endocrine secretion, cardiovascular contractility, and neuronal transcription, have a unique second mode of gating, "mode 2," that involves frequent openings of much longer duration. Here we report that two human conditions, cyclosporin neurotoxicity and Timothy syndrome, increase mode 2 gating of the recombinant rabbit CaV1.2 channel. In each case, mode 2 gating depends on a Ser residue at the cytoplasmic end of the S6 helix in domain I (Ser-439, Timothy syndrome) or domain IV (Ser-1517, cyclosporin). Both Ser reside in consensus sequences for type II calmodulin-dependent protein kinase. Pharmacologically inhibiting type II calmodulin-dependent protein kinase or mutating the Ser residues to Ala prevents the increase in mode 2 gating. We propose that aberrant phosphorylation, or "phosphorylopathy," of the CaV1.2 channel protein contributes to the excitotoxicity associated with Timothy syndrome and with chronic cyclosporin treatment of transplant patients.

Amino Acid Substitution↗