Kari Keinänen

A glutamate receptor channel with high affinity for domoate and kainate.

The non‐NMDA family of glutamate receptors comprises a growing number of structurally related subunits (GluR‐A to ‐D or −1 to −4; GluR‐5, −6; KA‐1). GluR‐A to ‐D appear to constitute the major AMPA receptor subtypes but the functional and pharmacological characteristics of the other subunits are unresolved. Using a mammalian expression system we demonstrate here that homomeric GluR‐5 receptors exhibit properties of a high affinity domoate (KD approximately 2 nM) and kainate (KD approximately 70 nM) binding site. For these receptors, the rank order of ligands competing with [3H]kainate binding was domoate much greater than quisqualate approximately glutamate much greater than AMPA approximately CNQX. The respective receptor channels were gated in decreasing order of sensitivity by domoate, kainate, glutamate and AMPA. In contrast to recombinantly expressed GluR‐A to ‐D channels, currents elicited at GluR‐5 receptor desensitize channels to all agonists. This property is characteristic of currents in peripheral neurons on sensory ganglia. These findings suggest the existence of at least two distinct types of non‐NMDA receptor channels, both gated by AMPA and kainate, but differing in pharmacology and current properties.

Cloning, pharmacological characteristics and expression pattern of the rat GABAA receptor α4 subunit

A cDNA of rat brain encoding the GABAA receptor α4 subunit has been cloned. Recombinant receptors composed of α4, β2 and γ2 subunits bind with high affinity the GABA agonist [3H]muscimol and the benzodiazepine ‘alcohol antagonist’ [3H]Ro 15‐4513, but fail to bind benzodiazepine agonists. The α4 subunit is expressed mainly in the thalamus, as assessed by in situ hybridization histochemistry, and may participate in a major population of thalamic GABAA receptors. The α4 mRNA is found at lower levels in cortex and caudate putamen, and is rare in cerebellum.

Structural and functional characterization of the gamma 1 subunit of GABAA/benzodiazepine receptors.

The GABAA receptor gamma 1 subunit of human, rat and bovine origin was molecularly cloned and compared with the gamma 2 subunit in structure and function. Both gamma subunit variants share 74% sequence similarity and are prominently synthesized in often distinct areas of the central nervous system as documented by in situ hybridization. When co‐expressed with alpha and beta subunits in Xenopus oocytes and mammalian cells, the gamma variants mediate the potentiation of GABA evoked currents by benzodiazepines and help generate high‐affinity binding sites for these drugs. However, these sites show disparate pharmacological properties which, for receptors assembled from alpha 1, beta 1 and gamma 1 subunits, are characterized by the conspicuous loss in affinity for neutral antagonists (e.g. flumazenil) and negative modulators (e.g. DMCM). These findings reveal a pronounced effect of gamma subunit variants on GABAA/benzodiazepine receptor pharmacology.

KCC2 interacts with the dendritic cytoskeleton to promote spine development.

The neuron-specific K-Cl cotransporter, KCC2, induces a developmental shift to render GABAergic transmission from depolarizing to hyperpolarizing. Now we demonstrate that KCC2, independently of its Cl(-) transport function, is a key factor in the maturation of dendritic spines. This morphogenic role of KCC2 in the development of excitatory synapses is mediated by structural interactions between KCC2 and the spine cytoskeleton. Here, the binding of KCC2 C-terminal domain to the cytoskeleton-associated protein 4.1N may play an important role. A more general conclusion based on our data is that KCC2 acts as a synchronizing factor in the functional development of glutamatergic and GABAergic synapses in cortical neurons and networks.

Molecular dissection of the agonist binding site of an AMPA receptor.

Two discontinuous segments (S1 and S2), separated by membrane‐associated domains, in ionotropic glutamate receptor (GluR) subunits show sequence similarity to bacterial periplasmic amino acid‐binding proteins, suggesting an evolutionary and structural relationship. Experimental evidence arguing for and against the inferred extracellular location of the S1 and S2 domains in GluRs has been presented. Here, we report that an extracellularly expressed fusion protein consisting of the S1 and S2 domains of alpha‐amino‐5‐methyl‐3‐hydroxyisoxazolone‐4‐propionate (AMPA)‐selective glutamate receptor GluR‐D joined together via a hydrophilic linker peptide specifically reproduces the AMPA‐binding properties of GluR‐D, whereas the separately expressed segments do not bind ligand. This provides direct evidence that the S1 and S2 segments of GluR‐D contain the structural determinants necessary and sufficient for selective agonist binding. Dissection of a functional neurotransmitter binding site as a soluble protein separate from the integral membrane channel will facilitate new approaches to analyse the structure of GluRs.

Specific gene transfer to neurons, endothelial cells and hematopoietic progenitors with lentiviral vectors

We present a flexible and highly specific targeting method for lentiviral vectors based on single-chain antibodies recognizing cell-surface antigens. We generated lentiviral vectors specific for human CD105+ endothelial cells, human CD133+ hematopoietic progenitors and mouse GluA-expressing neurons. Lentiviral vectors specific for CD105 or for CD20 transduced their target cells as efficiently as VSV-G pseudotyped vectors but discriminated between endothelial cells and lymphocytes in mixed cultures. CD133-targeted vectors transduced CD133+ cultured hematopoietic progenitor cells more efficiently than VSV-G pseudotyped vectors, resulting in stable long-term transduction. Lentiviral vectors targeted to the glutamate receptor subunits GluA2 and GluA4 exhibited more than 94% specificity for neurons in cerebellar cultures and when injected into the adult mouse brain. We observed neuron-specific gene modification upon transfer of the Cre recombinase gene into the hippocampus of reporter mice. This approach allowed targeted gene transfer to many cell types of interest with an unprecedented degree of specificity.

α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid (AMPA) Receptor Channels Lacking the N-terminal Domain

Ionotropic glutamate receptor (iGluR) subunits contain a ∼400-residue extracellular N-terminal domain (“X domain”), which is sequence-related to bacterial amino acid-binding proteins and to class C G-protein-coupled receptors. The X domain has been implicated in the assembly, transport to the cell surface, allosteric ligand binding, and desensitization in various members of the iGluR family, but its actual role in these events is poorly characterized. We have studied the properties of homomeric α-amino-3-hydroxy-5-methylisoxazolepropionate (AMPA)-selective GluR-D glutamate receptors carrying N-terminal deletions. Our analysis indicates that, surprisingly, transport to the cell surface, ligand binding properties, agonist-triggered channel activation, rapid desensitization, and allosteric potentiation by cyclothiazide can occur normally in the complete absence of the X domain (residues 22–402). The relatively intact ligand-gated channel function of a homomeric AMPA receptor in the absence of the X domain indirectly suggests more subtle roles for this domain in AMPA receptors, e.g. in the assembly of heteromeric receptors and in synaptic protein interactions.

Selective Binding of Synapse-associated Protein 97 to GluR-A α-Amino-5-hydroxy-3-methyl-4-isoxazole Propionate Receptor Subunit Is Determined by a Novel Sequence Motif

A family of four closely related PDZ domain-containing membrane-associated guanylate kinase homologues (MAGUKs) is involved in the regulation of the amount and functional state of ionotropic glutamate receptors in excitatory synapses. To understand the mechanisms that determine the specificity of these interactions, we examined the structural basis of the highly selective association between the ionotropic GluR subunit GluR-A and synapse-associated protein 97 (SAP97). The C terminus of GluR-A bound to the PDZ domains of SAP97, but not to those of three related MAGUKs, PSD-93, PSD-95, and SAP102. Experiments with single PDZ domains indicated that the strongest contribution was by the second PDZ domain. Unexpectedly, mutation analysis of the GluR-A C terminus revealed that a tripeptide sequence SSG at position −9 to −11 plays an essential role in this binding, in addition to a C-terminal type I PDZ binding motif (leucine at C terminus and threonine at the −2 position). Analysis of the in vitroMAGUK-binding properties of a GluR-D mutant with a one-residue deletion at the C terminus provides further support for the view that an SSG sequence located N-terminally from a type I PDZ binding motif can mediate selective binding to SAP97 and suggest the existence of a novel variation of the PDZ domain-peptide interaction.

AMPA receptors and bacterial periplasmic amino acid-binding proteins share the ionic mechanism of ligand recognition

In order to identify key structural determinants for ligand recognition, we subjected the ligand‐binding domain of the α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazole propionic acid (AMPA)‐selective glutamate receptor GluR‐D subunit to site‐directed mutagenesis. Based on the analysis of the [3H]AMPA‐binding properties of the mutated binding sites, we constructed a revised three‐dimensional model of the ligand‐binding site, different in many respects from previously published models. In particular, our results indicate that the residues Arg507 and Glu727 represent the structural and functional correlates of Arg77 and Asp161 in the homologous bacterial lysine/ornithine/arginine‐binding protein and histidine‐binding protein, and directly interact with the α‐carboxyl and α‐amino group of the bound ligand, respectively. In contrast, Glu424, implicated previously in ionic interactions with the α‐amino group of the agonist, is unlikely to have such a role in ligand binding. Our results indicate that glutamate receptors share with the bacterial polar amino acid‐binding proteins the fundamental mechanism of amino acid recognition.

Isoform-Specific Early Trafficking of AMPA Receptor Flip and Flop Variants

Flip and flop splice variants of AMPA receptor subunits are expressed in distinct but partly overlapping patterns and impart different desensitization kinetics to cognate receptor channels. In the absence of specific antibodies, isoform-specific differences in trafficking or localization of native flip and flop subunits remain uncharacterized. We report that in several transfected cell lines, transport of homomeric glutamate receptor (GluR)-Dflop receptors is largely blocked at the endoplasmic reticulum (ER) exit, whereas GluR-Dflip undergoes complex glycosylation and reaches the plasma membrane at >10× higher levels than GluR-Dflop, as determined by immunofluorescence, patch-clamp recordings and biochemical assays. The transport difference between flip and flop is independent of activity, is primarily determined by amino acid residue 780 (Leu in flop, Val in flip), and is manifested even in the secretion of the soluble ligand-binding domain, suggesting it is independent of oligomerization. Coexpression with stargazin or with the flip isoform rescues the surface expression of GluR-Dflop near to the level exhibited by GluR-Dflip. Our results demonstrate that the extracellular flip/flop region, via interactions with ER luminal splice form-specific protein(s), plays a hitherto unappreciated and important role in AMPA-receptor trafficking.