Keisuke Tsuzuki

Glutamate receptors in the mammalian central nervous system.

Glutamate receptors (GluRs) mediate most of the excitatory neurotransmission in the mammalian central nervous system (CNS). In addition, they are involved in plastic changes in synaptic transmission as well as excitotoxic neuronal cell death that occurs in a variety of acute and chronic neurological disorders. The GluRs are divided into two distinct groups, ionotropic and metabotropic receptors. The ionotropic receptors (iGluRs) are further subdivided into three groups: alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA), kainate and N-methyl-D-aspartate (NMDA) receptor channels. The metabotropic receptors (mGluRs) are coupled to GTP-binding proteins (G-proteins), and regulate the production of intracellular messengers. The application of molecular cloning technology has greatly advanced our understanding of the GluR system. To date, at least 14 cDNAs of subunit proteins constituting iGluRs and 8 cDNAs of proteins constituting mGluRs have been cloned in the mammalian CNS, and the molecular structure, distribution and developmental change in the CNS, functional and pharmacological properties of each receptor subunit have been elucidated. Furthermore, the obtained clones have provided valuable tools for conducting studies to clarify the physiological and pathophysiological significances of each subunit. For example, the generation of gene knockout mice has disclosed critical roles of some GluR subunits in brain functions. In this article, we review recent progress in the research for GluRs with special emphasis on the molecular diversity of the GluR system and its implications for physiology and pathology of the CNS.

Molecular and Physiological Diversity of Cortical Nonpyramidal Cells

The physiological and molecular features of nonpyramidal cells were investigated in acute slices of sensory-motor cortex using whole-cell recordings combined with single-cell RT-PCR to detect simultaneously the mRNAs of three calcium binding proteins (calbindin D28k, parvalbumin, and calretinin) and four neuropeptides (neuropeptide Y, vasoactive intestinal polypeptide, somatostatin, and cholecystokinin). In the 97 neurons analyzed, all expressed mRNAs of at least one calcium binding protein, and the majority (n = 73) contained mRNAs of at least one neuropeptide. Three groups of nonpyramidal cells were defined according to their firing pattern. (1) Fast spiking cells (n = 34) displayed tonic discharges of fast action potentials with no accommodation. They expressed parvalbumin (n = 30) and/or calbindin (n = 19) mRNAs, and half of them also contained transcripts of at least one of the four neuropeptides. (2) Regular spiking nonpyramidal cells (n = 48) displayed a firing behavior characterized by a marked accommodation and presented a large diversity of expression patterns of the seven biochemical markers. (3) Finally, a small population of vertically oriented bipolar cells, termed irregular spiking cells (n = 15), fired bursts of action potentials at an irregular frequency. They consistently co-expressed calretinin and vasoactive intestinal polypeptide. Additional investigations of these cells showed that they also co-expressed glutamic acid decarboxylase and choline acetyl transferase. Our results indicate that neocortical nonpyramidal neurons display a large diversity in their firing properties and biochemical patterns of co-expression and that both characteristics could be correlated to define discrete subpopulations.

Permeation of calcium through excitatory amino acid receptor channels in cultured rat hippocampal neurones.

1. N‐methyl‐D‐aspartate (NMDA)‐, quisqualate‐ and kainate‐induced currents were recorded in cultured rat hippocampal neurones using the whole‐cell voltage‐clamp technique. To isolate the inward currents carried by Ca2+ and other divalent cations (Sr2+, Ba2+, Mn2+ and Mg2+), both Na+ and K+ in the control external solution were replaced with the impermeant cation N‐methylglucamine (NMG). 2. Replacement of Na+, K+ and Ca2+ with NMG abolished NMDA‐, quisqualate‐ and kinate‐induced inward currents. In Na(+)‐, K(+)‐free (abbreviated simply as Na(+)‐free) solution containing 10 mM‐Ca2+ NMDA caused prominent inward currents at ‐60 mV. In this solution with the internal solution containing 165 mM‐Cs+, the reversal potential of the NMDA‐induced current was ‐5.0 +/‐ 0.7 mV (n = 36), indicating a value of PCa/PCs = 6.2 for the ratio of the permeability coefficients of Ca2+ and Cs+ according to the constant‐field equation. 3. NMDA elicited inward current responses at ‐60 mV in Na(+)‐, Ca2(+)‐free solution containing 10 mM‐Sr2+, Ba2+, or Mn2+, but not in Na(+)‐free, 10 mM‐Mg2+ solution. On the basis of reversal potential measurements, the permeability sequence of NMDA receptor channels among the divalent cations was determined to be Ba2+ (1.2) greater than Ca2+ (1.0) greater than Sr2+ (0.8) greater than Mn2+ (0.3) much greater than Mg2+ (less than 0.02). 4. The reversal potential of the quisqualate‐induced current was more negative than ‐80 mV in Na(+)‐free, 10 mM‐Ca2+ solution, indicating a value of PCa/PCs less than 0.18. 5. Kainate‐induced current responses were classified into two types. In the type I response the reversal potential of the kainate‐induced current was more negative than ‐80 mV in Na(+)‐free, 10 mM‐Ca2+ solution, indicating that the Ca2+ permeability of this type of kainate channel is as low as that of the quisqualate channel. In the neurones which showed a type I response, there was a tendency of outward rectification in the current‐voltage plots of the kainate response in control solution. 6. In the type II response kainate caused prominent inward currents at ‐60 mV in Na(+)‐free, 10 mM‐Ca2+ solution. The reversal potential was ‐23.3 +/‐ 5.6 mV (n = 17), indicating a permeability ratio PCa/PCs = 2.3. In the neurones which showed a type II response, a remarkable inward rectification was observed in the current‐voltage plots of the kainate response in control solution. 7. Type II kainate channels showed relatively poor selectivity among divalent cations.(ABSTRACT TRUNCATED AT 400 WORDS)

Subunit composition at the single-cell level explains functional properties of a glutamate-gated channel

The diversity of known glutamate-gated channels has been markedly increased by the discovery of multiple subunits and their spliced and edited variants. These subunits can potentially form different oligomeric complexes with diverging properties. A crucial question is therefore to determine the actual subunit composition of naturally occurring glutamate receptors. We have coupled patch-clamp recordings and reverse transcription followed by PCR amplification to correlate the presence of mRNAs for each subunit and the functional properties of native glutamate receptors at the single-cell level. In a homogeneous population of functionally identified hippocampal neurons (type II) in culture bearing a glutamate receptor of the AMPA subtype with a high calcium permeability, we found that, among the multiple subunits, only two, the flop forms of GluR1 and GluR4, were expressed. In particular, GluR2 was never detected. This composition explains the uncommon properties of AMPA receptors in type II neurons.

Ca2+-Permeable AMPA Receptors Regulate Growth of Human Glioblastoma via Akt Activation

Evidence has been accumulated that glioblastoma cells release and exploit glutamate for proliferation and migration by autocrine or paracrine loops through Ca2+-permeable AMPA-type glutamate receptors. Here, we show that Ca2+ signaling mediated by AMPA receptor regulates the growth and motility of glioblastoma cells via activation of Akt. Ca2+ supplied through Ca2+-permeable AMPA receptor phosphorylated Akt at Ser-473, thereby facilitating proliferation and mobility. A dominant-negative form of Akt inhibited cell proliferation and migration accelerated by overexpression of Ca2+-permeable AMPA receptor. In contrast, introduction of a constitutively active form of Akt rescued tumor cells from apoptosis induced by the conversion of Ca2+-permeable AMPA receptor to Ca2+-impermeable receptors by the delivery of GluR2 cDNA. Therefore, Akt functions as downstream effectors for Ca2+-signaling mediated by AMPA receptor in glioblastoma cells. The activation of the glutamate-AMPA receptor-Akt pathway may contribute to the high degree of anaplasia and invasive growth of human glioblastoma. This novel pathway might give an alternative therapeutic target.

Distribution of neurones expressing inwardly rectifying and Ca(2+)‐permeable AMPA receptors in rat hippocampal slices.

1. Current‐voltage (I‐V) relationships and Ca2+ permeability of receptor channels activated by bath application of kainate, a non‐desensitizing agonist of alpha‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA) receptors, were examined in various types of neurones in hippocampal slices of 5‐ to 13‐day‐old rats by using the tight‐seal patch clamp recording technique. 2. Three types of responses were observed: type I response with outwardly rectifying I‐V relationship, type II response with I‐V relationship of marked inward rectification, and intermediate response with I‐V relationship of weaker inward rectification. Neurones with type I, type II and intermediate I‐V relationships of kainate responses were referred to as type I, type II and intermediate neurones, respectively. 3. Permeability of Ca2+ ions was estimated by the reversal potential of kainate response in the outside‐out patch in Na(+)‐free extracellular solution containing 100 mM Ca2+. The reversal potentials were ‐44.4 +/‐ 14.0 mV (mean +/‐ S.D.) for type I (n = 7), +11.8 +/‐ 3.6 mV for type II (n = 5), and ‐8.7 +/‐ 7.4 mV for the intermediate neurones (n = 7). The values of PCa/PCs, the ratios of the permeability coefficients of Ca2+ and Cs+, estimated according to the constant‐field equation were 0.08 for type I, 1.71 for type II, and 0.50 for the intermediate neurones. 4. Type II and intermediate responses were observed mainly in non‐pyramidal neurones in various areas of the hippocampus, most frequently observed in the stratum molecular of the dentate gyrus and in the stratum radiatum and the stratum lacunosum‐molecular of both the CA1 and CA3 regions. Both type II and intermediate neurones stained with biocytin had round‐ or ellipsoidal‐shaped somata and issued divergent axonal projections to the surrounding structures. 5. Excitatory postsynaptic currents (EPSCs) recorded in type II neurones had 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX)‐sensitive fast and D‐2‐amino‐5‐phosphonovalerate (APV)‐sensitive slow components. The I‐V relationship of the fast component showed a strong inward rectification, indicating that inwardly rectifying AMPA receptors are involved in excitatory synaptic transmission.

Three types of voltage-dependent calcium current in cultured rat hippocampal neurons

Voltage-dependent calcium (Ca2+) currents in cultured rat hippocampal neurons were studied with the whole-cell recording mode of the patch-clamp technique. On the basis of the voltage-dependence of activation, kinetics of inactivation and pharmacology, 3 types of Ca2+ currents were distinguished. The low-threshold Ca2+ current (Il) was activated at -60 mV, and completely inactivated during a 100-ms depolarization to -40 mV (time constant: tau = 16 +/- 1 ms). The high-threshold currents (Ih), which were activated at -20 mV, could be separated into two types. The high-threshold, fast inactivating current (Ih,f) decayed quickly during a maintained depolarization (tau = 33 +/- 3 ms at 0 mV), whereas the high-threshold, slowly inactivating current (Ih,s) decayed with a much slower time constant (tau = 505 +/- 42 ms at 0 mV). The inactivations of Ih,f and Ih,s exhibited different time- and voltage-dependencies. Nickel ions (Ni2+, 25 microM) markedly suppressed Il, but little affected Ih. Cadmium ions (Cd2+, 10 microM) almost completely suppressed Ih, but left a small amount of Il. Lanthanum ions (La3+, 10 microM) almost completely suppressed both Il and Ih. Ih,s was sensitive to block by the dihydropyridine antagonist nicardipine (10 microM).

Input- and subunit-specific AMPA receptor trafficking underlying long-term potentiation at hippocampal CA3 synapses

Hippocampal CA3 pyramidal neurons receive synaptic inputs from both mossy fibres (MFs) and associational fibres (AFs). Long‐term potentiation (LTP) at these synapses differs in its induction sites and N‐methyl‐D‐aspartate receptor (NMDAR) dependence. Most evidence favours the presynaptic and postsynaptic mechanisms for induction of MF LTP and AF LTP, respectively. This implies that molecular and functional properties differ between MF and AF synapses at both presynaptic and postsynaptic sites. In this study, we focused on the difference in the postsynaptic trafficking of α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid receptors (AMPARs) between these synapses. To trace the subunit‐specific trafficking of AMPARs at each synapse, GluR1 and GluR2 subunits were introduced into CA3 pyramidal neurons in hippocampal organotypic cultures using the Sindbis viral expression system. The electrophysiologically‐tagged GluR2 AMPARs, produced by the viral‐mediated transfer of the unedited form of GluR2 (GluR2Q), were inserted into both MF and AF postsynaptic sites in a neuronal activity‐independent manner. Endogenous Ca2+‐impermeable AMPARs at these synapses were replaced with exogenous Ca2+‐permeable receptors, and Ca2+ influx via the newly expressed postsynaptic AMPARs induced NMDAR‐independent LTP at AF synapses. In contrast, no GluR1 AMPAR produced by the gene transfer was constitutively incorporated into AF postsynaptic sites, and only a small amount into MF postsynaptic sites. The synaptic trafficking of GluR1 AMPARs was triggered by the activity of Ca2+/calmodulin‐dependent kinase II or high‐frequency stimulation to induce LTP at AF synapses, but not at MF synapses. These results indicate that MF and AF postsynaptic sites possess distinct properties for AMPAR trafficking in CA3 pyramidal neurons.

Heat shock protein 90 (Hsp90) chaperone complex inhibitor, radicicol, potentiated radiation-induced cell killing in a hormone-sensitive prostate cancer cell line through degradation of the androgen receptor.

Until now, there has not been enough information on how androgens or androgen deprivation may influence the response of cancer cells to radiation. In this study, the effect of dihydrotestosterone (DHT) on cellular proliferative activity and radiosensitivity was examined in a hormone-sensitive human prostate cancer cell line, LNCaP. In addition, the study also examined how a heat shock protein 90 (Hsp90) chaperone complex inhibitor modified the effect of DHT on the radiosensitivity of the cells, because binding of the androgen receptor (AR) to Hsp90 is required to maintain the stability and functioning of AR. The hormone-sensitive human prostate cancer cell line, LNCaP, was used. Radicicol was used as one of the known Hsp90 chaperone complex inhibitors, and the cells were incubated in the presence of this compound at a concentration of 500 nM. Cellular radiosensitivity was determined by the clonogenic assay; the changes in the protein expression were examined by Western blotting or immunofluorescence. DHT at a concentration of 1 nM caused enhancement of the proliferative activity and reduction of the radiosensitivity of the cells. Radicicol at a concentration of 500 nM abolished the DHT-induced decrease in cellular radiosensitivity and potentiated the radiation-induced cell killing synergistically. Consistent with the changes in the cellular radiosensitivity, radicicol degraded AR, Raf-1 and HER2/neu via reduced binding of AR to Hsp90, although selective degradation of HER2/neu caused by Herceptin, a monoclonal antibody against HER2, did not affect the cellular radiosensitivity. The results suggest that the Hsp90 chaperone complex may be a potential molecular target for potentiation of radiation-induced cell killing in a hormone-sensitive prostate cancer cell line.

Ion permeation properties of the cloned mouse ϵ2/ζ1 NMDA receptor channel

Abstract The heteromeric mouse ϵ2/ζ1 N- methyl - d - aspartate (NMDA) receptor was expressed in Xenopus oocytes, and its channel properties were studied using both the outside-out patch-clamp and two-microelectrode voltage-clamp techniques. In the cloned receptor channel, permeation properties of monovulent and divalent cations, and voltage-dependent block by Mg2+ were similar to those reported previously in the native NMDA receptor channels. The sequence of single-channel conductances for alkali metals was Rb+ > Cs+ ≈ K+ > Na+ > Li+, whereas the sequence of relative permeabilities was Cs+ > Rb+ > K+ ≈ Na+ > Li+. The single-channel conductances measured in isotonic Ca2+, Sr2+ and Ba2+ solutions were almost equal, and approximately one-fifth of the value in the Na+ solution, although the permeabilities for these alkaline earth cations were higher than for Na+. It is likely that Ca2+, Sr2+ and Ba2+ would enter the NMDA receptor channel more easily than Na+, but would bind to a site in the channel more tightly, the net effect being a reduced value of the current.