Yasue Yamada

Modulation of the Channel Activity of the ε2/ζ1-Subtype N-Methyl d-Aspartate Receptor by PSD-95

A channel-associated protein PSD-95 has been shown to induce clustering of N-methyld-aspartate (NMDA) receptors, interacting with the COOH terminus of the ε subunit of the receptors. The effects of PSD-95 on the channel activity of the ε2/ζ1 heteromeric NMDA receptor were examined by injection of PSD-95 cRNA into Xenopus oocytes expressing the NMDA receptors. Expression of PSD-95 decreased the sensitivity of the NMDA receptor channels to l-glutamate. Mutational studies showed that the interaction between the COOH terminus of the ε2 subunit of the NMDA receptor and the second PSD-95/Dlg/Z0-1 domain of PSD-95 is critical for the decrease in glutamate sensitivity. It is known that protein kinase C markedly potentiates the channel activity of the NMDA receptor expressed in oocytes. PSD-95 inhibited the protein kinase C-mediated potentiation of the channels. Thus, we demonstrated that PSD-95 functionally modulates the channel activity of the ε2/ζ1 NMDA receptor. PSD-95 makes signal transmission more efficient by clustering the channels at postsynaptic sites. In addition to this, our results suggest that PSD-95 plays a protective role against neuronal excitotoxicity by decreasing the glutamate sensitivity of the channels and by inhibiting the protein kinase C-mediated potentiation of the channels.

Differential modulation of NR1‐NR2A and NR1‐NR2B subtypes of NMDA receptor by PDZ domain‐containing proteins

The PSD‐95/Dlg/ZO‐1 (PDZ) domain‐containing proteins MALS and PSD‐95 localize to post‐synaptic densities and bind the COOH‐termini of NR2 subunits of the NMDA receptor. The effects of MALS‐2 and PSD‐95 on the channel activity of NMDA receptors were compared using the Xenopus oocyte expression system. Both MALS‐2 and PSD‐95 increased the current response of the NR1‐NR2B receptor to l‐glutamate. In contrast, the current response of the NR1‐NR2A receptor was increased by PSD‐95 but not by MALS‐2. MALS‐2 had no effect either on the potentiation of NR1‐NR2A or NR1‐NR2B channel activity by protein kinase C, or on Src‐mediated potentiation of NR1‐NR2A activity, whereas PSD‐95 almost completely inhibited the effects of these protein kinases. Construction of chimeras of MALS‐2 and PSD‐95 revealed that the first two PDZ domains and two NH2‐terminal cysteine residues are essential for the inhibitory effects of PSD‐95 on protein kinase C‐mediated potentiation of NR1‐NR2A and NR1‐NR2B channel activity, respectively. The second of the three PDZ domains of PSD‐95 was required for its inhibition of Src‐mediated potentiation of NR1‐NR2A activity. These results indicate that the NR1‐NR2A and NR1‐NR2B receptors are modulated differentially by MALS‐2 and PSD‐95, and that similar regulatory effects of PSD‐95 on these receptors are achieved by distinct mechanisms.

Increase of adenylate kinase isozyme 1 protein during neuronal differentiation in mouse embryonal carcinoma P19 cells and in rat brain primary cultured cells.

Abstract: Adenylate kinase (AK), which catalyzes the equilibrium reaction among AMP, ADP, and ATP, is considered to participate in the homeostasis of energy metabolism in cells. Among three vertebrate isozymes, AK isozyme 1 (AK1) is present prominently in the cytosol of skeletal muscle and brain. When mouse embryonal carcinoma P19 cells were differentiated by retinoic acid into neural cells, the amount of AK1 protein and enzyme activity increased about fivefold concomitantly with neurofilament (NF). Double‐immunofluorescence staining showed that both AK1 and NF were located in neuronal processes as well as the perinuclear regions in neuron‐like cells, but not in glia‐like cells. The amount of brain‐type creatine kinase increased only twofold during P19 differentiation. The AK isozyme 2, which was not detected in adult mouse brain, was found in P19 cells and did not increase during the differentiation. Mitochondrial AK isozyme 3, which uses GTP instead of ATP as a phosphate donor, was increased significantly. Immunohistochemical analysis with the primary cultured cells from rat cerebral cortex showed similar cellular localization of AK1 to those observed with differentiated P19 cells. These results suggest an important role of this enzyme in neuronal functions and in neuronal differentiation.

PSD-95 eliminates Src-induced potentiation of NR1/NR2A-subtype NMDA receptor channels and reduces high-affinity zinc inhibition

The channel activity of NMDA receptors is regulated by phosphorylation by protein kinases and by interaction with other proteins. Recombinant NR1/NR2A subtype NMDA receptor channels are potentiated by the protein tyrosine kinase Src, an effect which is mediated by a reduction in the high‐affinity, voltage‐independent Zn2+ inhibition. However, it has been reported that Src‐induced potentiation of NMDA receptor currents in hippocampus neurons is not mediated by a reduction in Zn2+ inhibition. The post‐synaptic density protein PSD‐95 interacts with the C‐terminus of NR2 subunits of the NMDA receptor. Here we demonstrate that PSD‐95 eliminates the Src‐induced potentiation of NR1/NR2A channels expressed in oocytes and reduces the sensitivity of the channels to Zn2+. Our results reveal that the absence of Src‐induced potentiation of PSD‐95‐coupled NR1/NR2A channels is not to due to the reduced sensitivity of these channels to Zn2+. These results indicate that PSD‐95 functionally modulates NR1/NR2A channels and explain why Src‐induced potentiation of NMDA receptor currents in hippocampus neurons is not mediated by a reduction in Zn2+ inhibition.

Activation of channel activity of the NMDA receptor-PSD-95 complex by guanylate kinase-associated protein (GKAP)

The channel‐associated protein PSD‐95 functionally modulates NMDA receptor channels, interacting with the channels via PDZ domain of PSD‐95. PSD‐95 also interacts with guanylate kinase‐associated protein (GKAP) through the guanylate kinase‐like domain of PSD‐95. Here we report that GKAP markedly potentiates the channel activity of the receptor‐PSD‐95 complex. However, GKAP had no effect on basic properties of the channels nor on PSD‐95‐induced changes in channel properties. Thus, GKAP affects the channel activity of the NMDA receptor via PSD‐95 quantitatively, which may make signal transmission more efficient at postsynaptic sites.

Nucleotide sequence and expression of the gutQ gene within the glucitol operon of Escherichia coli

The glucitol operon in Escherichia coli is known to consist of five structural genes with the order: gut O P A B D M R. We have sequenced downstream from gutR and have identified an open reading frame encoding a water-soluble protein (223 amino acids; molecular weight = 23,562) with a putative ATP binding site. Expression of this protein in a maxicell system has been demonstrated. A repetitive extragenic palindromic (REP) sequence capable of forming stem-loop structures follows gutQ in the downstream, presumptive intercistronic region. The function of the Gut Q protein is not known.

Pb2+ reduces the current from NMDA receptors expressed in Xenopus oocytes.

We compared the effects of Pb2+ on four types of NMDA receptors expressed in Xenopus ooeytes. Pb2+ reduced the currents evoked by glutamate and glycine. The K i values of the receptors, ε1/ζ1, ε2/ζ1, ε3/ζ1 and ε4/ζ1, were 39, 34, 54 and 42 μM, respectively, and their Hill coefficients were 0.53, 4.6, 0.52 and 0.37, respectively. The ε2/ζ1 receptor that was inhibited in the presence of over 30 μM Pb2+ was not recovered to the control level after a Pb2+ washout for over 30 min, suggesting that ε2/ζ1 is responsible for the chronic Pb2+ intoxication in the nervous system.

Characterization of the gntT gene encoding a high-affinity gluconate permease in Escherichia coli.

We characterized the gntT gene encoding a high-affinity gluconate permease of Escherichia coli K-12. Primer extension and lacZ-operon fusion analyses revealed that gntT has one strong and two weak promoters, all of which are regulated positively by cAMP-CRP and negatively by GntR. The weak promoters became constitutive when separated from the upstream region including the strong promoter that overlaps a putative GntR-binding sequence. Gluconate-specific uptake activity was observed with cells harboring the gntT plasmid clone, which was enhanced by the presence of gntK encoding gluconate kinase.

322 The different effects of neuromuscular blocking agents on adult- and fetal-types of nicotinic acetylcholine receptors

The detailed distribution of glucocorticoid receptor (GR) immunoreactivity and mRNA was investigated by immunohistochemistry and in situ hybridization in conjunction with the use of specific polyclonal antibody and fluorescein-labeled probe. We compared the distribution of GR immunoreactivity with that of mRNA in the adult rat brain. GR immunoreactive and mRNA-containing cells were widely distributed throughout the brain from the olfactory bulb of the forebrain to the gracilecuneate nuclei of the medulla oblongata. GR immunoreactivity and mRNA showed the similar distributional pattern, but in the hippocampus the differential distribution wis noted. The ubiquitous occurrence in the whole brain indicated the multi-modal function of GR, but the present study provides the groundwork for the elucidation of the role of GR in brain function, by categorizing the distributional pattern of GR.