Jeremy M. Henley

Cyclothiazide unmasks AMPA‐evoked stimulation of [3H]‐L‐glutamate release from rat hippocampal synaptosomes

The effect of α‐amino‐3‐hydroxy‐5‐methylisoxazolepropionate (AMPA) on Ca2+‐sensitive, tetrodotoxin (TTX)‐insensitive K+‐stimulated [3H]‐L‐glutamate release from rat hippocampal synaptosomes was determined. AMPA in the presence, but not in the absence of cyclothiazide, a drug which blocks AMPA receptor desensitization, elicited a dose‐dependent increase in K+‐stimulated [3H]‐L‐glutamate release but had no effect on basal release. The AMPA/cyclothiazide stimulation was blocked by CNQX and by GYKI 52466, an antagonist at the cyclothiazide site. These results indicate that AMPA receptors are present on presynaptic terminals and suggest that they may play a role in the regulation of neurotransmitter release.

Interaction of Guanine Nucleotides with [3H]Kainate and 6-[3H]Cyano-7-Nitroquinoxaline-2,3-dione Binding in Goldfish Brain

Abstract— Recent reports have suggested that a major proportion of [3H]kainate binding in goldfish brain is to a novel form of G‐protein‐linked glutamate receptor. Here we confirm that guanine nucleotides decrease [3H]kainate binding in goldfish brain membranes, but that binding is also reduced to a similar extent under conditions where G‐protein modulation should be minimised. Inclusion of GTPγS resulted in an approximately twofold decrease in the affinity of [3H]kainate binding and a 50% reduction in the apparent Bmax values in both Mg2+/Na+ and Mg2+/Na+‐free buffer when assayed at 0°c. The pharmacology of [3H]kainate binding is similar to that of well‐characterised ionotropic kainate receptors but unlike that of known me‐tabotropic glutamate receptors, with neither 1S,3R‐amino‐1,3‐cyclopentanedicarboxylic acid (1S,3R‐ACPD) nor ibo‐tenic acid being effective competitors. The molecular mass of the [3H]kainate binding protein, as determined by radiation inactivation, was 40 kDa, similar to the subunit sizes of other lower vertebrate kainate binding proteins that are believed to comprise ligand‐gated ion channels. Furthermore, GTP‐γS also inhibited the binding of the non‐NMDA receptor‐selective antagonist 6‐[3H]cyano‐7‐ni‐troquinoxaline‐2,3‐dione. These data strongly suggest that the regulatory interaction between guanine nucleotides and [3H]kainate and 6‐[3H]cyano‐7‐nitroquinoxaline‐2,3‐dione binding is complex and involves competition at the agonist/antagonist binding site in addition to any G‐protein‐mediated modulation.

Localisation of glutamate receptor binding sites and mRNAs to the dorsal horn of the rat spinal cord.

The autoradiographic distribution of binding sites for [3H]AMPA, [3H]kainate, [3H]MK-801 and [3H]L-glutamate and the in situ hybridization of GluR-A, GluR-B, GluR-C and GluR-D in rat spinal cord sections were determined. All of the radioligands had similar localisations with the dorsal horn being the most intensely labelled region. In the in situ hybridization experiments only the GluR-B mRNA was detected at high levels in the spinal cord with predominant expression in the substantia gelatinosa. These results support electrophysiological evidence for the presence of glutamate activated neurones in the spinal cord and suggest that these excitatory pathways are confined mainly to the dorsal horn.

Kainate-binding proteins: phylogeny, structures and possible functions

Recent advances have demonstrated that the family of [3H]kainate-binding proteins and kainate receptors comprise a number of related polypeptides. In all the cases so far investigated, the kainate-binding proteins from non-mammalian vertebrates have M(r) values in the range of 40-50 kDa whereas mammalian kainate receptors and kainate-binding proteins have M(r) values in the order of 100 kDa. There have not, as yet, been any reports of 40-50 kDa kainate-binding proteins in mammalian CNS and, despite the cloning of increasing numbers of cDNAs encoding new kainate-binding proteins, the relationships between these two general groups of polypeptides remain unclear. Nonetheless, there is now a wealth of phylogenetic, structural and molecular biological data available about these proteins. In this review, Jeremy Henley outlines the properties and structures of kainate-binding proteins and offers some possibilities as to the roles of these often hugely abundant proteins.

Quantitative analysis of the distributions of glutamatergic ligand binding sites in goldfish brain

Goldfish brain is a widely used model system for the study of the mechanisms involved in neuronal regeneration and synaptic plasticity. Because of the proposed role of glutamate receptors in these processes we have investigated the anatomical localisations of [3H]AMPA (alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate), [3H]kainate, [3H]CNQX (6-cyano-7-nitroquinoxaline-2,3-dione) and [3H]L-glutamate binding sites in horizontal and sagittal sections. Binding sites for [3H]L-glutamate were the most widespread and both NMDA (N-methyl-D-aspartate) and non-NMDA sensitive components were detected. The density of [3H]kainate binding was very high in the cerebellum compared to other regions and in comparison with the other radioligands used. Conversely, relatively low amounts of [3H]AMPA binding were present with the telencephalon being the most densely labelled structure. [3H]CNQX binding was most densely localised in the tectum with the cerebellum also possessing high binding. In addition, there was a small population of [3H]CNQX binding sites located in the telencephalon and lobus vagi that appeared insensitive to AMPA and kainate.

Phospholipase A2 enhances [3H]AMPA binding to a putative homomeric GluR-B receptor in the rat spinal cord

[3H]AMPA and [3H]kanine binding to rat spinal cord was localised most densely in the substantia gelatinosa of the dorsal horn. Phospholipase A2 elicited a dose‐dependent increase in specific [3H]AMPA binding but not in [3H]kainate binding. The enhancement of [3H]AMPA binding was blocked by bromophenacyi bromide and at 0C and was not mimicked by arachidonic acid. Since GluR‐B is the only AMPA receptor subunit detectable in the rat spinal cord, and recombinant homomeric GluR‐B assemblies do not bind [3H]kainate, these results suggest phospholipase A2 may modulate [3H]AMPA binding to putative homomeric GluR‐B receptors.

In vitro translation and membrane topology of rat recombinant mGluR 1α

The structure and post-translational processing of the metabotropic glutamate receptor 1 alpha (mGluR1 alpha) was analysed by in vitro cell-free translation, protease protection and deglycosylation. We show that mGluR1 alpha can be synthesized in the rabbit-reticulocyte translation system to yield a predominant polypeptide product with an apparent molecular weight of 142 kDa. In the presence of dog-pancreatic microsomes this polypeptide was processed to an apparent molecular weight of 147 kDa. Treatment with the enzyme peptide-N-glycosidase F (PNGF) demonstrated that the increase in the apparent molecular weight of the processed translation product was due to N-linked glycosylation. Addition of the non-selective protease, proteinase K; resulted in the loss of this 147 kDa band and the appearance of a protected fragment of approx 92 kDa. A carboxy-terminal deletion mutant of mGluR1 alpha was almost completely protected from protease action. These data show that the amino terminal of mGluR1 alpha is translocated into the lumen of the endoplasmic reticulum and will consequently be located extracellularly when targeted to the plasma membrane. The data presented here on mGluR1 alpha indicates the potential of in vitro translation and protease protection in the study of the molecular structure and processing of glutamate receptors.

Characterisation of an Allosteric Modulatory Protein Associated with α‐[3H]Amino‐3‐Hydroxy‐5‐Methylisoxazolepropionate Binding Sites in Chick Telencephalon: Effects of High‐Energy Radiation and Detergent Solubilisation

Abstract: α‐[3H]Amino‐3‐hydroxy‐5‐methylisoxazolepro‐pionate ([3H]AMPA) binds to 1‐day‐old chick telencephalon membranes with KD and Bmax values of 138 nM and 2.56 pmol/mg of protein, respectively. High‐energy radiation bombardment of intact frozen telencephalon resulted in a biphasic inactivation curve for [3H]AMPA binding. At a 5.8‐Mrad radiation dose, the affinity of [3H]AMPA binding was increased (54 nM), but there was no apparent alteration in the Bmax value (2.76 pmol/mg of protein). We attribute this phenomenon to the inactivation of a high molecular weight modulatory protein that down‐regulates the affinity of [3H]AMPA binding. The estimated molecular masses of the AMPA binding site and of the modulatory component were 59 and 108 kDa, respectively. Solubilisation with n‐octyl‐β‐glucopyranoside resulted in an increase in the Bmax (4.7 pmol/ mg of protein) with no pronounced alteration in the affinity (109 nM) of [3H]AMPA binding. However, the solubilisation‐induced increase in Bmax did not occur in telencephalon irradiated before solubilisation. In contrast, the increase in affinity induced by radiation treatment was still detected in solubilised extracts. These results suggest that the number and affinity of [3H]AMPA sites in chick telencephalon are closely regulated and that the modulatory systems involved are affected by both irradiation and solubilisation.

Autoradiographic distribution of glutamatergic ligand binding sites inXenopus brain: evidence for intracellular [3H]AMPA binding sites

The binding of a series of [3H]glutamatergic ligands was determined by receptor autoradiography of membrane homogenate pellets and horizontal sections of Xenopus brain. Consistent with previous reports that a unitary glutamate receptor is present in Xenopus CNS, the radioligands showed similar densities of binding sites in the membrane homogenate pellets. Furthermore, [3H]kainate binding was completely displaced by AMPA or CNQX, [3H]AMPA binding was completely displaced by kainate or CNQX and [3H]CNQX binding was completely displaced by AMPA or kainate. However, in whole brain sections there were apparently 2- to 5-fold more [3H]AMPA and [3H]CNQX than [3H]kainate sites. The absence of these extra sites in broken-washed membrane preparations suggests that the additional [3H]AMPA and [3H]CNQX binding may be due to cytosolic sites. The observation that all [3H]AMPA and [3H]CNQX binding in the brain sections is displaced by kainate indicates that the putative cytosolic sites are sensitive to relatively high concentrations of kainate but that they differ from the previously characterised Xenopus CNS unitary receptors.

Autoradiographic localisations of glutamatergic ligand binding sites in Xenopus brain

In the Xenopus central nervous system the binding sites for [3H]kainate and [3H]alpha-amino-3-hydroxy-5-methylisoxazolepropionate [( 3H]AMPA) are highly localised and the distributions of both ligands are largely coincident. The telencephalon was the most strongly labelled area with a relatively uniform distribution of binding sites. In addition, the infundibulum and the cerebellum were also heavily labelled. Areas containing a lower density of binding sites included the septum, the thalamus and the optic lobes. [3H]Kainate binding was potently inhibited by 1 microM AMPA in the presence of 0.1 M KSCN and [3H]AMPA binding was blocked by 1 microM kainate. These data are consistent with the hypothesis that kainate and AMPA bind to the same site on a single protein entity and that this unitary AMPA/KA binding protein may constitute the predominant type of excitatory amino acid receptor in the Xenopus brain.