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Biomedical subjects

Yasuo Mori

Publications and source records attributed to Yasuo Mori.

At least 37 records · Page 2Linked to original sources
PubMed2006

A CaV2.1 calcium channel mutation rocker reduces the number of postsynaptic AMPA receptors in parallel fiber-Purkinje cell synapses.

The rocker mice are hereditary ataxic mutants that carry a point mutation in the gene encoding the CaV2.1 (P/Q-type) Ca2+ channel alpha1 subunit, and show the mildest symptoms among the reported CaV2.1 mutant mice. We studied the basic characteristics of the rocker mutant Ca2+ channel and their impacts on excitatory synaptic transmission in cerebellar Purkinje cells (PCs). In acutely dissociated PC somas, the rocker mutant channel showed a moderate reduction in Ca2+ channel current density, whereas its kinetics and voltage dependency of gating remained nearly normal. Despite the small changes in channel function, synaptic transmission in the parallel fiber (PF)-PC synapses was severely impaired. The climbing fiber inputs onto PCs showed a moderate impairment but could elicit normal complex spikes. Presynaptic function of the PF-PC synapses, however, was unexpectedly almost normal in terms of paired-pulse facilitation, sensitivity to extracellular Ca2+ concentration and glutamate concentration in synaptic clefts. Electron microscopic analyses including freeze-fracture replica labeling revealed that both the number and density of postsynaptic alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors substantially decreased without gross structural changes of the PF-PC synapses. We also observed an abnormal arborization of PC dendrites in young adult rocker mice (approximately 1 month old). These lines of evidence suggest that even a moderate dysfunction of CaV2.1 Ca2+ channel can cause substantial changes in postsynaptic molecular composition of the PF-PC synapses and dendritic structure of PCs.

Action Potentials↗
PubMed2005

Phoneutria nigriventer omega-Phonetoxin IIA: a new tool for anti-calcium channel autoantibody assays in Lambert-Eaton myasthenic syndrome.

Lambert-Eaton myasthenic syndrome (LEMS) is a neurological autoimmune disease in which downregulation of voltage-gated calcium channels (VGCCs) leads to reduced acetylcholine release from motoneuron terminals. 70% of cases are paraneoplastic and rapid diagnosis of LEMS can result in early detection of the underlying tumor. Serological assays based on the capacity of autoantibodies to precipitate VGCCs labeled with radioligands provide valuable data. We have established a novel assay using the spider venom peptide 125I-omega-Phonetoxin IIA (125I-omegaPtxIIA). 125I-omegaPtxIIA labeled recombinant Cav2.1 and Cav2.2 channels and endogenous VGCCs in rat brain membranes. Autoantibodies that immunoprecipitate a 125I-omegaPtxIIA/channel complex were detected in 26/31 (84%) LEMS patients. The patients that were seropositive in the 125I-omegaPtxIIA assay corresponded precisely to the population that was positive for Cav2.1 and/or Cav2.2 antibodies detected using two different omega-conotoxins. Thus, the 125I-omegaPtxIIA assay detects a broader spectrum of autoantibody specificities than current omega-conotoxin-based assays.

Adult↗
PubMed2005

Ca2+-calmodulin-dependent myosin light chain kinase is essential for activation of TRPC5 channels expressed in HEK293 cells.

Mammalian homologues of Drosophila transient receptor potential (TRP) proteins are responsible for receptor-activated Ca(2+) influx in vertebrate cells. We previously reported the involvement of intracellular Ca(2+) in the receptor-mediated activation of mammalian canonical transient receptor potential 5 (TRPC5) channels. Here we investigated the role of calmodulin, an important sensor of changes in intracellular Ca(2+), and its downstream cascades in the activation of recombinant TRPC5 channels in human embryonic kidney (HEK) 293 cells. Ca(2+) entry through TRPC5 channels, induced upon stimulation of the G-protein-coupled ATP receptor, was abolished by treatment with W-13, an inhibitor of calmodulin. ML-9 and wortmannin, inhibitors of Ca(2+)-calmodulin-dependent myosin light chain kinase (MLCK), and the expression of a dominant-negative mutant of MLCK inhibited the TRPC5 channel activity, revealing an essential role of MLCK in maintaining TRPC5 channel activity. It is important to note that ML-9 impaired the plasma membrane localization of TRPC5 channels. Furthermore, TRPC5 channel activity measured using the whole-cell patch-clamp technique was inhibited by ML-9, whereas TRPC5 channel activity observed in the cell-excised, inside-out patch was unaffected by ML-9. An antibody that recognizes phosphorylated myosin light chain (MLC) revealed that the basal level of phosphorylated MLC under unstimulated conditions was reduced by ML-9 in HEK293 cells. These findings strongly suggest that intracellular Ca(2+)-calmodulin constitutively activates MLCK, thereby maintaining TRPC5 channel activity through the promotion of plasma membrane TRPC5 channel distribution under the control of phosphorylation/dephosphorylation equilibrium of MLC.

Amino Acid Sequence↗
PubMed2005

The role of canonical transient receptor potential 7 in B-cell receptor-activated channels.

Phospholipase C signaling stimulates Ca2+ entry across the plasma membrane through multiple mechanisms. Ca2+ store depletion stimulates store-operated Ca2+-selective channels, or alternatively, other phospholipase C-dependent events activate Ca2+-permeable non-selective cation channels. Transient receptor potential 7 (TRPC7) is a non-selective cation channel that can be activated by both mechanisms when ectopically expressed, but the regulation of native TRPC7 channels is not known. We knocked out TRPC7 in DT40 B-cells, which expresses both forms of Ca2+ entry. No difference in the store-operated current I(crac) was detected between TRPC7-/- and wild-type cells. Wild-type cells demonstrated nonstore-operated cation entry and currents in response to activation of the B-cell receptor or protease-activated receptor 2, intracellular dialysis with GTPgammaS, or application of the synthetic diacylglycerol oleyl-acetyl-glycerol. These responses were absent in TRPC7-/- cells but could be restored by transfection with human TRPC7. In conclusion, in B-lymphocytes, TRPC7 appeared to participate in the formation of ion channels that could be activated by phospholipase C-linked receptors. This represents the first demonstration of a physiological function for endogenous TRPC7 channels.

Animals↗
PubMed2005

The non-selective cation-permeable channel TRPC3 is a tetrahedron with a cap on the large cytoplasmic end.

TRPC3 plays important roles in neuronal differentiation and immune cell maturation by mediating the cationic current in response to phospholipase C activation, Ca2+ depletion, and diacylglycerol stimulation. Here, we purified the TRPC3 channel using a glycosylated tetramer and observed the structure using electron microscopy. Negatively stained specimens demonstrate homogeneous protein particles containing an internal cavity-like structure. These particle images were picked up by automated pick-up programs, aligned, and classified by the growing neural gas network method. Similarly oriented projections were averaged to decrease the signal-to-noise ratio. The averaged images progress from the top view to the side views, which are representative of their raw images. The top view confirmed the hypothesis of a four-domain structure, and the side view demonstrates a large cytoplasmic domain with a capped structure at the bottom, which is near a predicted locus of ion release. The total image of the protein is a blunt-edged trapezoid of 200 x 200 x 235 A. This large dimension of TRPC3 is also supported by the Stokes radius (92 A) obtained from gel filtration chromatography.

Chromatography, Gel↗
PubMed2005

Comprehensive analysis of the ascidian genome reveals novel insights into the molecular evolution of ion channel genes.

Ion fluxes through membrane ion channels play crucial roles both in neuronal signaling and the homeostatic control of body electrolytes. Despite our knowledge about the respective ion channels, just how diversification of ion channel genes underlies adaptation of animals to the physical environment remains unknown. Here we systematically survey up to 160 putative ion channel genes in the genome of Ciona intestinalis and compare them with corresponding gene sets from the genomes of the nematode Chaenorhabditis elegans, the fruit fly Drosophila melanogaster, and the more closely related genomes of vertebrates. Ciona has a set of so-called "prototype" genes for ion channels regulating neuronal excitability, or for neurotransmitter receptors, suggesting that genes responsible for neuronal signaling in mammals appear to have diversified mainly via gene duplications of the more restricted members of ancestral genomes before the ascidian/vertebrate divergence. Most genes responsible for modulation of neuronal excitability and pain sensation are absent from the ascidian genome, suggesting that these genes arose after the divergence of urochordates. In contrast, the divergent genes encoding connexins, transient receptor potential-related channels and chloride channels, channels involved rather in homeostatic control, indicate gene duplication events unique to the ascidian lineage. Because several invertebrate-unique channel genes exist in Ciona genome, the crown group of extant vertebrates not only acquired novel channel genes via gene/genome duplications but also discarded some ancient genes that have persisted in invertebrates. Such genome-wide information of ion channel genes in basal chordates enables us to begin correlating the innovation and remodeling of genes with the adaptation of more recent chordates to their physical environment.

Animals↗
PubMed2005

Effects of calmodulin and Ca2+ channel blockers on omega-conotoxin GVIA binding to crude membranes from alpha1B subunit (Cav2.2) expressed BHK cells and mice brain lacking the alpha1B subunits.

Characteristics for the specific binding of 125I-omega-CTX GVIA and 125I-omega-CTX MVIIC to crude membranes from BHKN101 cells expressing the alpha1B subunits of Cav2.2 channels and from mice brain lacking the alpha1B subunits of Cav2.2 channels, particularly, the effects of CaM and various Ca2+ channel blockers on these specific bindings were investigated. Specific binding of 125I-omega-CTX GVIA to the crude membranes from BHKN101 cells was observed, but not from control BHK6 cells. omega-CTX GVIA, omega-CTX MVIIC and omega-CTX SVIB inhibited the specific binding of 125I-omega-CTX GVIA to crude membranes from BHKN101 cells, and the IC50 values for omega-CTXGVIA, omega-CTX MVIIC and omega-CTX SVIB were 0.07, 8.5 and 1.7 nM, respectively. However, omega-agatoxin IVA and calciseptine at concentrations of 10(-9)-10(-6) M did not inhibit specific binding. Specific binding was also about 80% inhibited by 20 microg protein/ml CaM. The amount of 125I-omega-CTX GVIA (30 pM) specifically bound to membranes from brain of knockout mice lacking alpha1B subunits of Cav2.2 channels was about 30% of that to the crude membranes from brain of wild-type. On the other hand, specific binding of 125I-omega-CTX MVIIC (200 pM) was observed on the crude membranes of both BHKN101 and control BHK6 cells. The specific binding of 125I-omega-CTX MVIIC (200 pM) was not inhibited by omega-CTX GVIA and omega-CTX SVIB, and also omega-Aga IVA and calciseptine at concentrations of 10(-9)-10(-7) M, although specific binding was almost completely dose dependently inhibited by non-radiolabeled omega-CTX MVIIC (IC50 value was about 0.1 nM). 20 microg protein/ml CaM did not inhibit specific binding. Therefore, these results suggest that BHKN101 cells have a typical Cav2.2 channels which are also inhibited by CaM and have not specific binding sites for omega-CTX MVIIC, although omega-CTX MVIIC is a blocker for both Cav2.1 (alpha1A; P/Q-type) and Cav2.2 channels.

Animals↗
PubMed2004

Identification of R(-)-isomer of efonidipine as a selective blocker of T-type Ca2+ channels.

Efonidipine, a derivative of dihydropyridine Ca(2+) antagonist, is known to block both L- and T-type Ca(2+) channels. It remains to be clarified, however, whether efonidipine affects other voltage-dependent Ca(2+) channel subtypes such as N-, P/Q- and R-types, and whether the optical isomers of efonidipine have different selectivities in blocking these Ca(2+) channels, including L- and T-types. To address these issues, the effects of efonidipine and its R(-)- and S(+)-isomers on these Ca(2+) channel subtypes were examined electrophysiologically in the expression systems using Xenopus oocytes and baby hamster kidney cells (BHK tk-ts13). Efonidipine, a mixture of R(-)- and S(+)-isomers, exerted blocking actions on L- and T-types, but no effects on N-, P/Q- and R-type Ca(2+) channels. The selective blocking actions on L- and T-type channels were reproduced by the S(+)-efonidipine isomer. By contrast, the R(-)-efonidipine isomer preferentially blocked T-type channels. The blocking actions of efonidipine and its enantiomers were dependent on holding potentials. These findings indicate that the R(-)-isomer of efonidipine is a specific blocker of the T-type Ca(2+) channel.

Animals↗
PubMed2004

Activation of RasGRP3 by phosphorylation of Thr-133 is required for B cell receptor-mediated Ras activation.

The Ras signaling pathway plays a critical role in B lymphocyte development and activation, but its activation mechanism has not been well understood. At least one mode of Ras regulation in B cells involves a Ras-guanyl nucleotide exchange factor, RasGRP3. We demonstrate here that RasGRP3 undergoes phosphorylation at Thr-133 upon B cell receptor cross-linking, thereby resulting in its activation. Deletion of phospholipase C-gamma2 or pharmacological interference with conventional PKCs resulted in marked reduction in both Thr-133 phosphorylation and Ras activation. Moreover, mutation of Thr-133 in RasGRP3 alone severely impaired its ability to activate Ras in B cell receptor signaling. Hence, our data suggest that PKC, after being activated by diacylglycerol, phosphorylates RasGRP3, thereby contributing to its full activation.

Amino Acid Sequence↗
PubMed2004

FPL 64176 modification of Ca(V)1.2 L-type calcium channels: dissociation of effects on ionic current and gating current.

FPL 64176 (FPL) is a nondihydropyridine compound that dramatically increases macroscopic inward current through L-type calcium channels and slows activation and deactivation. To understand the mechanism by which channel behavior is altered, we compared the effects of the drug on the kinetics and voltage dependence of ionic currents and gating currents. Currents from a homogeneous population of channels were obtained using cloned rabbit Ca(V)1.2 (alpha1C, cardiac L-type) channels stably expressed in baby hamster kidney cells together with beta1a and alpha2delta1 subunits. We found a striking dissociation between effects of FPL on ionic currents, which were modified strongly, and on gating currents, which were not detectably altered. Inward ionic currents were enhanced approximately 5-fold for a voltage step from -90 mV to +10 mV. Kinetics of activation and deactivation were slowed dramatically at most voltages. Curiously, however, at very hyperpolarized voltages (< -250 mV), deactivation was actually faster in FPL than in control. Gating currents were measured using a variety of inorganic ions to block ionic current and also without blockers, by recording gating current at the reversal potential for ionic current (+50 mV). Despite the slowed kinetics of ionic currents, FPL had no discernible effect on the fundamental movements of gating charge that drive channel gating. Instead, FPL somehow affects the coupling of charge movement to opening and closing of the pore. An intriguing possibility is that the drug causes an inactivated state to become conducting without otherwise affecting gating transitions.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed2004

Multiple regulation by calcium of murine homologues of transient receptor potential proteins TRPC6 and TRPC7 expressed in HEK293 cells.

We investigated, by using the patch clamp technique, Ca2+-mediated regulation of heterologously expressed TRPC6 and TRPC7 proteins in HEK293 cells, two closely related homologues of the transient receptor potential (TRP) family and molecular candidates for native receptor-operated Ca2+ entry channels. With nystatin-perforated recording, the magnitude and time courses of activation and inactivation of carbachol (CCh; 100 microM)-activated TRPC6 currents (I(TRPC6)) were enhanced and accelerated, respectively, by extracellular Ca2+ (Ca2+(o)) whether it was continuously present or applied after receptor stimulation. In contrast, Ca2+(o) solely inhibited TRPC7 currents (I(TRPC7)). Vigorous buffering of intracellular Ca2+ (Ca2+(i)) under conventional whole-cell clamp abolished the slow potentiating (i.e. accelerated activation) and inactivating effects of Ca2+(o), disclosing fast potentiation (EC50: approximately 0.4 mM) and inhibition (IC50: approximately 4 mM) of I(TRPC6) and fast inhibition (IC50: approximately 0.4 mM) of I(TRPC7). This inhibition of I(TRPC6) and I(TRPC7) seems to be associated with voltage-dependent reductions of unitary conductance and open probability at the single channel level, whereas the potentiation of I(TRPC6) showed little voltage dependence and was mimicked by Sr2+ but not Ba2+. The activation process of I(TRPC6) or its acceleration by Ca2+(o) probably involves phosphorylation by calmodulin (CaM)-dependent kinase II (CaMKII), as pretreatment with calmidazolium (3 microM), coexpression of Ca2+-insensitive mutant CaM, and intracellular perfusion of the non-hydrolysable ATP analogue AMP-PNP and a CaMKII-specific inhibitory peptide all effectively prevented channel activation. However, this was not observed for TRPC7. Instead, single CCh-activated TRPC7 channel activity was concentration-dependently suppressed by nanomolar Ca2+(i) via CaM and conversely enhanced by IP3. In addition, the inactivation time course of I(TRPC6) was significantly retarded by pharmacological inhibition of protein kinase C (PKC). These results collectively suggest that TRPC6 and 7 channels are multiply regulated by Ca2+ from both sides of the membrane through differential Ca2+-CaM-dependent and -independent mechanisms.

Animals↗
PubMed2004

Involvement of TRPM7 in cell growth as a spontaneously activated Ca2+ entry pathway in human retinoblastoma cells.

We investigated the possible involvement of the melastatin family protein TRPM7 in Ca(2+)-mediated proliferative control of human retinoblastoma (RB) cells. The growth of RB cell was facilitated by elevating the extracellular Ca(2+) concentration with a parallel increase in the magnitude of spontaneous Ca(2+) influx. Under nystatin-perforated voltage-clamp, RB cells exhibited an outward-rectifying, spontaneous cation current (I(spont)) having Ca(2+)/Mg(2+)-inhibited but -permeating properties. Various cation channel blockers inhibiting I(spont) (Gd(3+), La(3+), LOE908, 2-APB) suppressed the spontaneous Ca(2+) influx and decelerated the growth of RB cells with similar efficacies. Excision of the RB cell membrane (inside-out) into MgATP-free solution induced a 70pS single channel activity, which was effectively inhibited by millimolar concentrations of Mg(2+) or MgATP. RT-PCR and immunocytochemical experiments revealed the expression of TRPM7 mRNA and protein in RB cells, and heterologous expression of TRPM7 in HEK293 cells reproduced the key features of I(spont). In contrast, elimination of this protein from RB cells by siRNA silencing markedly reduced I(spont) density and the magnitude of spontaneous Ca(2+) influx, which was paralleled by decreased TRPM7 immunoreactivity, decelerated cell proliferation, and retarded G(1)/S cell cycle progression. These results suggest a significant regulatory role of TRPM7 for RB cell proliferation as a spontaneously activated Ca(2+) influx pathway.

Calcium↗
PubMed2004

Mutations in EFHC1 cause juvenile myoclonic epilepsy.

Juvenile myoclonic epilepsy (JME) is the most frequent cause of hereditary grand mal seizures. We previously mapped and narrowed a region associated with JME on chromosome 6p12-p11 (EJM1). Here, we describe a new gene in this region, EFHC1, which encodes a protein with an EF-hand motif. Mutation analyses identified five missense mutations in EFHC1 that cosegregated with epilepsy or EEG polyspike wave in affected members of six unrelated families with JME and did not occur in 382 control individuals. Overexpression of EFHC1 in mouse hippocampal primary culture neurons induced apoptosis that was significantly lowered by the mutations. Apoptosis was specifically suppressed by SNX-482, an antagonist of R-type voltage-dependent Ca(2+) channel (Ca(v)2.3). EFHC1 and Ca(v)2.3 immunomaterials overlapped in mouse brain, and EFHC1 coimmunoprecipitated with the Ca(v)2.3 C terminus. In patch-clamp analysis, EFHC1 specifically increased R-type Ca(2+) currents that were reversed by the mutations associated with JME.

Animals↗
PubMed2004

Ca2+ store-independent augmentation of [Ca2+]i responses to G-protein coupled receptor activation in recombinantly TRPC5-expressed rat pheochromocytoma (PC12) cells.

Mammalian homologues of the Drosophila canonical transient receptor potential (trp) protein (TRPC) have been implicated to function as receptor-operated Ca(2+) channels (ROCs) or store-operated Ca(2+) channels (SOCs). To determine the role of TRPC5 protein in neural cells, TRPC5 was recombinantly expressed in rat pheochromocytoma cells (PC12) and changes in intracellular Ca(2+) concentration ([Ca(2+)](i)) and Na(+) concentration ([Na(+)](i)) were analyzed. TRPC1 and TRPC3 mRNAs were endogenously expressed in PC12 cells. In TRPC5-expressed cells (TRPC5-cells), the resting [Ca(2+)](i) and [Na(+)](i) were significantly higher than those in control cells. The [Ca(2+)](i) increases induced by bradykinin and uridine 5'-triphosphate were significantly larger in TRPC5-cells. TRPC5 expression did not change in store-operated Ca(2+) entry elicited by thapsigarigin. TRPC5-cells showed larger inward current and increase of [Na(+)](i) in response to BK than control cells. These results suggest that TRPC5 channels expressed in PC12 cells function as ROCs activated by G-protein/phospholipase C coupled receptors, but not as SOCs.

Animals↗
PubMed2004

Novel real-time sensors to quantitatively assess in vivo inositol 1,4,5-trisphosphate production in intact cells.

Real-time observation of messenger molecules in individual intact cells is essential for physiological studies of signaling mechanisms. We have developed a novel inositol 1,4,5-trisphosphate (IP(3)) sensor based on the pleckstrin homology (PH) domain from phospholipase C (PLC) delta. The environmentally sensitive fluorophore 6-bromoacetyl-2-dimethyl-aminonaphtalene was conjugated to the genetically introduced cysteine at the mouth of the IP(3) binding pocket for enhanced IP(3) selectivity and for rapid and direct visualization of intracellular IP(3) > or = 0.5 microM as fluorescence emission decreased. The probe, tagged with arginine-rich sequences for efficient translocation into various cell types, revealed a major contribution of Ca2+ influx to PLC-mediated IP(3) production that boosts Ca2+ release from endoplasmic reticulum. Thus, our IP(3) probe was extremely effective to quantitatively assess real-time physiological IP(3) production via those pathways formed only in the intact cellular configuration.

Animals↗
PubMed2004

Polyglutamine repeats of spinocerebellar ataxia 6 impair the cell-death-preventing effect of CaV2.1 Ca2+ channel--loss-of-function cellular model of SCA6.

Spinocerebellar ataxia (SCA) 6 is caused by small expansion of a polyglutamine sequence, encoded by CAG trinucleotide repeats, at the C-terminal end of the human CaV2.1 (P/Q-type) Ca2+ channel alpha12.1 subunit and it manifests itself as slowly progressive cerebellar ataxia. To elucidate the pathogenic mechanisms underlying SCA6, we introduced CAG repeats of various lengths into the Ca2+ channel alpha12.1 subunit cDNA and expressed them in baby hamster kidney cells stably expressing the auxiliary subunits (alpha2delta and beta4). The occurrence of cell death differed between cells transfected with the normal and mutant Ca2+ channels under the condition of serum starvation plus potassium-induced depolarization, and Cdk inhibition elucidated the differences more clearly. The CaV2.1 (P/Q-type) Ca2+ channel-specific blocker omega-agatoxin IVA abolished the cell-death-preventing effect of the normal Ca2+ channel. Together with our previous finding that the polyglutamine expansion in SCA6 interferes with the Ca2+ channel to reduce Ca2+ influx, these results indicate that impaired function of the mutant Ca2+ channels rendered them unable to prevent cell death.

Animals↗