Naoto Hoshi

Cyclic ADP-ribose as a second messenger revisited from a new aspect of signal transduction from receptors to ADP-ribosyl cyclase

Cyclic ADP-ribose (cADPR), an endogenous modulator of ryanodine receptor Ca(2+)-releasing channels, is found in various tissues. Cytosolic injection of cADPR induces an elevation of intracellular Ca(2+) concentrations or potentiates Ca(2+) increases. cADPR facilitates neurotransmitter or insulin release and modifies ionic currents. cADPR is synthesized by ADP-ribosyl cyclase and is metabolized by cADPR hydrolase. ADP-ribosyl cyclase activity is up-regulated by nitric oxide/cyclic GMP-dependent phosphorylation or receptor stimulation via G-proteins within membranes. These findings suggest that cADPR is a second messenger in cellular Ca(2+) signaling. However, many intriguing issues remain to be addressed before this identity is confirmed.

KCR1, a Membrane Protein That Facilitates Functional Expression of Non-inactivating K+ Currents Associates with Rat EAG Voltage-dependent K+Channels

Cerebellar granule neurons possess a non-inactivating K+ current, which controls resting membrane potentials and modulates the firing rate by means of muscarinic agonists. kcr1 was cloned from the cerebellar cDNA library by suppression cloning. KCR1 is a novel protein with 12 putative transmembrane domains and enhances the functional expression of the cerebellar non-inactivating K+ current inXenopus oocytes. KCR1 also accelerates the activation of rat EAG K+ channels expressed in Xenopusoocytes or in COS-7 cells. Far-Western blotting revealed that KCR1 and EAG proteins interacted with each other by means of their C-terminal regions. These results suggest that KCR1 is the regulatory component of non-inactivating K+ channels.

Cyclic ADP‐ribose as a potential second messenger for neuronal Ca2+ signaling

Cyclic ADP‐ribose (cADPR), a known endogenous modulator of ryanodine receptor Ca2+ releasing channels, is found in the nervous system. Injection of cADPR into neuronal cells primarily induces a transient elevation of intracellular Ca2+ concentration ([Ca2+]i), and/or secondarily potentiates [Ca2+]i increases that are the result of depolarization‐induced Ca2+ influx. Acetylcholine release from cholinergic neurons is facilitated by cADPR. cADPR modifies K+ currents or elicits Ca2+‐dependent inward currents. cADPR is synthesized by both membrane‐bound and cytosolic forms of ADP‐ribosyl cyclase in neuronal cells. cADPR hydrolase activity is weak in the membrane fraction, but high in the cytoplasm. Cytosolic ADP‐ribosyl cyclase activity is upregulated by nitric oxide/cyclic GMP‐dependent phosphorylation. Stimulation of muscarinic and β‐adrenergic receptors activates membrane‐bound ADP‐ribosyl cyclase via G proteins within membranes of neuronal tumor cells and cortical astrocytes. These findings strongly suggest that cADPR is a second messenger in Ca2+ signaling in the nervous system, although many intriguing issues remain to be addressed before this identity is confirmed.

Endomorphins inhibit high-threshold Ca2+ channel currents in rodent NG108-15 cells overexpressing μ-opioid receptors

1 Extracellular application of the novel brain peptides endomorphin 1 (EM1) and endomorphin 2 (EM2) inhibited high‐threshold Ca2+ channel currents in NGMO‐251 cells, a daughter clone of NG108‐15 mouse neuroblastoma × rat glioma hybrid cells, in which μ‐opioid receptors are overexpressed. 2 In contrast, EM1 and EM2 did not induce this inhibition in the parental NG108‐15 cells that predominantly express endogenous δ‐receptors. 3 The IC50 for EM1 and EM2 was 7.7 and 23.1 nm, respectively. 4 EM‐induced Ca2+ channel current inhibition was blocked by treatment or pretreatment of the cells with 100 μmN‐methylmaleimide or 100 ng ml−1 pertussis toxin. 5 These results show that a decrease in conductance of Ca2+ channels results following interaction of EMs with cloned μ‐receptors, which couple via Gi/Go‐type G proteins, and that EMs fulfill one of the necessary synaptic conditions for them to be identified as neurotransmitters.

Probucol aggravates long QT syndrome associated with a novel missense mutation M124T in the N-terminus of HERG.

Patients with LQTS (long QT syndrome) with a mutation in a cardiac ion channel gene, leading to mild-to-moderate channel dysfunction, may manifest marked QT prolongation or torsade de pointes only upon an additional stressor. A 59-year-old woman had marked QT prolongation and repeated torsade de pointes 3 months after initiation of probucol, a cholesterol-lowering drug. We identified a single base substitution in the HERG gene by genetic analysis. This novel missense mutation is predicted to cause an amino acid substitution of Met(124)-->Thr (M124T) in the N-terminus. Three other relatives with this mutation also had QT prolongation and one of them had a prolonged QT interval and torsade de pointes accompanied by syncope after taking probucol. We expressed wild-type HERG and HERG with M124T in Xenopus oocytes and characterized the electrophysiological properties of these HERG channels and the action of probucol on the channels. Injection of the M124T mutant cRNA into Xenopus oocytes resulted in expression of functional channels with markedly smaller amplitude. In both HERG channels, probucol decreased the amplitude of the HERG tail current, decelerated the rate of channel activation, accelerated the rate of channel deactivation and shifted the reversal potential to a more positive value. The electrophysiological study indicated that QT lengthening and cardiac arrhythmia in the two present patients were due to inhibition of I(Kr) (rapidly activating delayed rectifier K(+) current) by probucol, in addition to the significant suppression of HERG current in HERG channels with the M124T mutation.

Subtype-specific coupling with ADP-ribosyl cyclase of metabotropic glutamate receptors in retina, cervical superior ganglion and NG108-15 cells

Cyclic ADP‐ribose (cADP‐ribose) is a putative second messenger or modulator. However, the role of cADP‐ribose in the downstream signals of the metabotropic glutamate receptors (mGluRs) is unclear. Here, we show that glutamate stimulates ADP‐ribosyl cyclase activity in rat or mouse crude membranes of retina via group III mGluRs or in superior cervical ganglion via group I mGluRs. The retina of mGluR6‐deficient mice showed no increase in the ADP‐ribosyl cyclase level in response to glutamate. GTP enhanced the initial rate of basal and glutamate‐stimulated cyclase activity. GTP‐γ‐S also stimulated basal activity. To determine whether the coupling mode of mGluRs to ADP‐ribosyl cyclase is a feature common to individual cloned mGluRs, we expressed each mGluR subtype in NG108‐15 neuroblastomau2003×u2003glioma hybrid cells. The glutamate‐induced stimulation of the cyclase occurs preferentially in NG108‐15 cells over‐expressing mGluRs1, 3, 5, and 6. Cells expressing mGluR2 or mGluRs4 and 7 exhibit inhibition or no coupling, respectively. Glutamate‐induced activation or inhibition of the cyclase activity was eliminated after pre‐treatment with cholera or pertussis toxin, respectively. Thus, the subtype‐specific coupling of mGluRs to ADP‐ribosyl cyclase via G proteins suggests that some glutamate‐evoked neuronal functions are mediated by cADP‐ribose.

Characterization of a novel missense mutation E637K in the pore-S6 loop of HERG in a patient with long QT syndrome

OBJECTIVEnIn a 32-year-old woman with marked QT prolongation (QTc=0.61 s) and repeated episodes of syncope, we identified a single pertinent base substitution (G to A at 1909) in HERG by genetic analysis. This novel missense mutation is predicted to cause an amino acid substitution of lysine for glutamic acid at position 637 (E637K) in the pore-S6 loop. Therefore, we investigated the role of a glutamic acid at the vicinity of the pore in HERG channels by mutating it to a lysine.nnnMETHODSnWe characterized the electrophysiological properties of the E637K mutation using a Xenopus oocyte heterologous expression system.nnnRESULTSnInjection of the E637K mutant cRNA alone into Xenopus oocytes did not result in any expression of detectable currents. Coexpression of wild-type (WT) and E637K (E637K/WT) elicited only about 30% of the control peak tail current that was expected from expression of WT alone. Kinetic analyses revealed that E637K/WT decelerated the rate of channel activation and enhanced steady-state inactivation. Furthermore, the reversal potentials at low concentrations of K+ showed a positive shift in oocytes injected with E637K/WT compared with WT alone.nnnCONCLUSIONSnThese results indicated that the E637K mutation causes apparent dominant negative suppression of WT HERG channel function and suggest that E637 at the Pore-S6 is a crucial component of the activation and inactivation gate of HERG channels.

Microheterogeneity in heteromultimeric assemblies formed by Shaker (Kv1) and Shaw (Kv3) subfamilies of voltage-gated K+ channels

Single K+ channels were recorded in Xenopus oocytes injected with a 1:1 mixture of mRNAs coding for NGK1 (Kvl. 2) and NGK2 (Kv3. 1a) voltage-dependent K+ channels. A new class of channels of 18 pS conductance was observed, and was designated as NGK1, 2 channels. According to their properties of activation voltages and open life times, four types of NGK1, 2 channels with microheterogeneity were detected. The results suggest that voltage-dependent NGK1 Shaker and NGK2 Shaw K+ channels, from different subfamilies, assemble to form heteromultimeric K+ channels, giving rise to a mosaic of characteristics inherited from two parental channels.

Compound heterozygosity for mutations Asp611-->Tyr in KCNQ1 and Asp609-->Gly in KCNH2 associated with severe long QT syndrome.

LQTS (long QT syndrome) is an inherited cardiac disorder characterized by prolongation of QT interval, torsades de pointes and sudden death. We have identified two heterozygous missense mutations in the KCNQ1 and KCNH2 (also known as HERG) genes [Asp611-->Tyr (D611Y) in KCNQ1 and Asp609-->Gly (D609G) in KCNH2] in a 2-year-old boy with LQTS. The aim of the present study was to characterize the contributions of the mutations in the KCNQ1 and KCNH2 genes relative to the clinical manifestations and electrophysiological properties of LQTS. Six of 11 carriers of D611Y in KCNQ1 had long QT intervals. D609G in KCNH2 was detected only in the proband. Studies on the electrophysiological alterations due to the two missense mutations revealed that the D611Y mutation in KCNQ1 did not show a significant suppression of the currents compared with wild-type, but the time constants of current activation in the mutants were increased compared with that in the wild-type. In contrast, the D609G mutation in KCNH2 showed a dominant-negative suppression. Our results suggest that the mild phenotype produced by the D611Y mutation in KCNQ1 became more serious by addition of the D609G mutation in KCNH2 in the proband.

INOSITOL TRISPHOSPHATE/CA2+ AS MESSENGERS OF BRADYKININ B2 AND MUSCARINIC ACETYLCHOLINE M1-M4 RECEPTORS IN NEUROBLASTOMA-DERIVED HYBRID CELLS

Neuroblastoma x glioma hybrid NG 108-15 and neuroblastoma x fibroblast hybrid NL308 cells possess endogenous bradykinin B2 receptors and m4 muscarinic acetylcholine receptors (mAChRs), which couple to phospholipase C and adenylate cyclase, respectively. Four genetic subtypes of mAChRs differed in their effects when stimulated in NG108-15 and NL308 cells overexpressing mAChRs. Broadly speaking, the principal effects fell into two categories: the odd-numbered receptors (m1 and m3) activated phospholipase C and increased inositol trisphosphate/Ca2+, as bradykinin did, whereas the even-numbered receptors (m2 and m4) inhibited adenylate cyclase via a pertussis toxin (PTx)-sensitive G-protein in NG108-15 cells. But all four types of NL308 cells overexpressing each m1, m2, m3 and m4 receptor activated phospholipase C, while keeping the PTx-sensitivity in m2/m4, but not in m1/m3 receptors. Coupling to ion channel effectors showed a comparable dichotomy in NG108-15 cells, while cross-activation occurred in NL308 cells.