Analysis of AMPA receptor subunits expressed by single Purkinje cells using RNA polymerase chain reaction.
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Animals↗
Biomedical subjects
B Lambolez
Publications and source records attributed to B Lambolez.
In the GluR1 glutamate receptor subunit a glutamine to histidine point mutation suppresses inward rectification but not calcium permeability.
Recent papers have described glutamine to arginine point mutations of the cloned AMPA/Kainate receptor subunits that alter current-voltage relationship and suppress Ca2+ permeability, thus linking these two characteristics. We describe a glutamine to histidine mutation at the same position, which alters current-voltage relationship but retains Ca2+ permeability, thus dissociating the two properties.
Animals↗
AMPA receptor subunits expressed by single Purkinje cells.
Several subunits of the glutamate receptor of the AMPA subtype have been cloned recently. These subunits, named GluR1, GluR2, GluR3, and GluR4, exist as two splicing variants (flip and flop). We have determined the subset of AMPA receptor subunits expressed by single cerebellar Purkinje cells in culture. This was achieved by combining whole-cell patch-clamp recordings and a molecular analysis, based on the polymerase chain reaction, of the messenger RNAs harvested into the patch pipette at the end of each recording. We found that each single cell expresses the messenger RNAs encoding the following five subunits: the flip and flop versions of GluR1 and GluR2 as well as GluR3flip, GluR2 being the most abundant. In addition, GluR3flop and GluR4flip were scarcely expressed in half of these neurons, and GluR4flop was never detected.
Animals↗
Quinoxaline derivatives: structure-activity relationships and physiological implications of inhibition of N-methyl-D-aspartate and non-N-methyl-D-aspartate receptor-mediated currents and synaptic potentials.
The inhibitory potencies at excitatory amino acid (EAA) receptors of 11 quinoxaline derivatives were evaluated in two-electrode voltage-clamp recordings of Xenopus oocytes injected with rat cortex mRNA. Currents activated by kainate or (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) in Xenopus oocytes were inhibited competitively by all the quinoxaline derivatives, with apparent Ki values ranging from 0.27 to 300 microM against kainate and from 0.25 to 137 microM against AMPA. An excellent correlation was observed between inhibitory potencies of the quinoxaline derivatives against kainate and AMPA currents, in support of the contention that in this preparation these two agonists act at a single site. All 11 quinoxaline derivatives also inhibited current activated by the combination of glycine and N-methyl-D-aspartate (NMDA), apparently acting at the glycine site, and did so over a narrower range of apparent Ki values (0.37-8.1 microM). The correlation between the quinoxalines' kainate/AMPA potencies and their glycine/NMDA potencies was relatively weak. Thus, the quinoxaline derivatives were all good antagonists of glycine/NMDA currents and displayed a greater range of potencies against kainate and AMPA. The inhibitory effects of the six quinoxaline derivatives most potent in the Xenopus oocyte experiments were also tested against the excitatory postsynaptic field potential (EPSFP) recorded in the pyramidal cell dendritic field of the CA1 region of hippocampal slices after stimulation of the Schaffer collateral-commissural pathways. In slices superfused with "normal" medium (containing 1 mM Mg2+), in which the EPSFP is mediated primarily by non-NMDA receptors, IC50 values correlated closely with the Ki values against kainate/AMPA obtained in oocyte experiments but were approximately 8-fold higher. Similarly, in slices superfused with nominally Mg(2+)-free medium, in which the EPSFP is amplified due to a relief of the Mg2+ block of NMDA receptors, IC50 values correlated closely with the Ki values against glycine/NMDA obtained in oocyte experiments but were 60-fold higher. This comparison of results from the two experimental systems lends further support to the argument that hippocampal synaptic transmission is mediated postsynaptically by kainate/AMPA-type and NMDA/glycine-type EAA receptors that are pharmacologically indistinguishable from those expressed in mRNA-injected Xenopus oocytes. Furthermore, it suggests that EAA receptors in situ may be nearly saturated by high local concentrations of the endogenous ligands, a condition that would contribute substantially to the apparent non-NMDA receptor selectivity of certain quinoxaline derivatives.
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A chimeric glutamate receptor subunit: discrete changes modify the properties of the channel.
GluR1 and GluR2 are two highly homologous subunits of the glutamate AMPA receptor but with different functional properties. In ligand gated channels the transmembrane domain II is thought to form the wall of the ionic pore and determine the electrical properties. A chimeric AMPA receptor subunit was constructed by replacing the region comprising transmembrane domains I and II in GluR1 by the corresponding region of GluR2. Alone or forming an heteromer with GluR1, the resulting chimera has the properties of GluR2. Sequence comparison suggests that an arginine at position 600 in the chimera instead of a glutamine in GluR1 is responsible for these properties.
Amino Acid Sequence↗
Electrophysiological and pharmacological properties of GluR1, a subunit of a glutamate receptor-channel expressed in Xenopus oocytes.
A cDNA clone encoding an excitatory amino acid receptor was isolated from a rat brain cDNA library by Hollmann et al. (Nature, 342 (1989) 643-648). In Xenopus oocytes, this clone, GluR1, expressed a functional receptor-channel activated by kainate (KA), domoate (D), glutamate and quisqualate (QA). The apparent affinity (EC50) for QA (0.1 microM) was higher than that for KA (50 microM). The maximal response to QA was about 1/10 of that to KA. QA inhibited the KA induced current. The N-methyl-D-aspartate (non-NMDA) receptor antagonist 6,7-dinitroquinoxaline-2,3 dione (DNQX) competitively blocked the effects of both agonists. Currents induced by KA, QA and D in oocytes expressing GluR1 showed identical voltage sensitivities. GluR1 and KA receptor-channels expressed from rat striatum poly(A)+ RNA showed the same ionic selectivity, being permeable mostly to Na+ and K+. The current-voltage relationships of GluR1 showed a strong inward rectification, whereas those of KA receptor-channels expressed from poly(A)+ RNA from various rat brain regions were more linear.
Animals↗
Reduction of desensitization of a glutamate ionotropic receptor by antagonists.
The glutamate receptor channel subtype that responds to both quisqualate (QA) and alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionate (AMPA) was expressed in Xenopus oocytes injected with rat cerebral cortex mRNA. Voltage-clamp current responses to QA, AMPA, and glutamate (GLU) exhibited a rapid increase followed by a decrease to a desensitized steady state (DS). Perfusion with high agonist concentrations produced smaller DS responses than perfusion with low concentrations. During the DS, the current was increased by lowering of the concentration of agonist or by application of low concentrations of a competitive antagonist, 6,7-dinitroquinoxaline-2,3-dione (DNQX). This paradoxical increase of the agonist-induced currents during the DS was also observed in cultured Purkinje cells with another competitive antagonist, 6-cyano-7-nitro-quinoxaline-2,3-dione (CNQX). Dose-response curves obtained in oocytes were bell shaped, with a negative slope for high concentrations of QA. DNQX shifted these bell-shaped curves to the right. Together, these results indicate that the agonists are able to reversibly inhibit the AMPA receptor. The classical desensitization model of Katz and Thesleff [J. Physiol. (Lond.) 138:63-80 (1957)] cannot account for our observations.
6-Cyano-7-nitroquinoxaline-2,3-dione↗
First use of a beta-carboline as photoaffinity label for the benzodiazepine receptor.
Photolabelling of benzodiazepine receptors isolated from rat cortex with a new beta-carboline-type photoaffinity label, ethyl 6-azido-beta-carboline-3-carboxylase, at 254 nm produced a 42% decrease in the maximal number of propyl beta-carboline-3-carboxylate binding sites but practically no decrease in the number of flunitrazepam binding sites. Moreover, the binding affinity of ethyl beta-carboline-3-carboxylase was diminished 11-fold by photolabelling while that of diazepam was diminished less than 2-fold. These results provide additional evidence that beta-carbolines and benzodiazepines bind to discrete sites on the benzodiazepine receptor.
Affinity Labels↗
Concanavalin A potentiates NMDA-evoked responses in the Xenopus oocyte expression system.
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Animals↗
Benzodiazepine agonists protect a histidine residue from modification by diethyl pyrocarbonate whereas propyl beta-carboline does not.
The pH sensitivity of benzodiazepine binding suggests that a histidine residue may be present in, or close to the benzodiazepine binding site. This was confirmed by the selective modification of histidine residues using diethyl pyrocarbonate which was found to block both benzodiazepine and beta-carboline binding. In order to assess whether this histidine residue is located in or adjacent to the benzodiazepine and beta-carboline binding sites, experiments were performed using either benzodiazepine or beta-carboline to protect against diethyl pyrocarbonate treatment. It was found that benzodiazepine agonists, but not propyl beta-carboline protect the benzodiazepine binding sites from diethyl pyrocarbonate modification.
Animals↗