Biomedical subjects

Geoffrey T Swanson

Publications and source records attributed to Geoffrey T Swanson.

13 recordsLinked to original sources

PubMed2006

Total synthesis and biological evaluation of neodysiherbaine A and analogues.

Dysiherbaine (1) and its congener neodysiherbaine A (2) are naturally occurring excitatory amino acids with selective and potent agonistic activity for ionotropic glutamate receptors. We describe herein the total synthesis of 2 and its structural analogues 3-8. Advanced key intermediate 16 was employed as a branching point to assemble a series of these analogues 3-8 with respect to the C8 and C9 functionalities, which would not have been accessible through manipulations of the natural product itself. The synthesis of key intermediate 16 features (i) stereocontrolled C-glycosylation to set the C6 stereocenter, (ii) concise synthesis of the bicyclic ether skeleton through chemo- and stereoselective dihydroxylation of the exo-olefin and stereoselective epoxidation of the endo-olefin, followed by epoxide ring opening/5-exo ring closure, and (iii) catalytic asymmetric hydrogenation of enamide ester to construct the amino acid appendage. A preliminary biological evaluation of analogues for their in vivo toxicity against mice and binding affinity for glutamate receptors showed that both the type and stereochemistry of the C8 and C9 functional groups affected the subtype selectivity of dysiherbaine analogues for members of the kainic acid receptor family.

Alanine↗

PubMed2006

Intracellular trafficking of KA2 kainate receptors mediated by interactions with coatomer protein complex I (COPI) and 14-3-3 chaperone systems.

Assembly and trafficking of neurotransmitter receptors are processes contingent upon interactions between intracellular chaperone systems and discrete determinants in the receptor proteins. Kainate receptor subunits, which form ionotropic glutamate receptors with diverse roles in the central nervous system, contain a variety of trafficking determinants that promote either membrane expression or intracellular sequestration. In this report, we identify the coatomer protein complex I (COPI) vesicle coat as a critical mechanism for retention of the kainate receptor subunit KA2 in the endoplasmic reticulum. COPI subunits immunoprecipitated with KA2 subunits from both cerebellum and COS-7 cells, and beta-COP protein interacted directly with immobilized KA2 peptides containing the arginine-rich retention/retrieval determinant. Association between COPI proteins and KA2 subunits was significantly reduced upon alanine substitution of this signal in the cytoplasmic tail of KA2. Temperature-sensitive degradation of COPI complex proteins was correlated with an increase in plasma membrane localization of the homologous KA2 receptor. Assembly of heteromeric GluR6a/KA2 receptors markedly reduced association of KA2 and COPI. Finally, the reduction in COPI binding was correlated with an increased association with 14-3-3 proteins, which mediate forward trafficking of other integral signaling proteins. These interactions therefore represent a critical early checkpoint for biosynthesis of functional KARs.

14-3-3 Proteins↗

PubMed2006

Determination of binding site residues responsible for the subunit selectivity of novel marine-derived compounds on kainate receptors.

Dysiherbaine (DH) and related molecules are high-affinity, subunit-selective kainate receptor (KAR) ligands originally isolated from a marine sponge. To elucidate why DH, an agonist, and MSVIII-19, a competitive antagonist, bind selectively to glutamate receptor (GluR) 5 but not to the KA2 KAR subunit, we used molecular dynamics simulations to generate binding models that were tested experimentally in radioligand binding and electrophysiological assays. Three candidate sites, Val685, Leu735, and Ser741 in GluR5, corresponding to Ile669, Phe719, and Met725 in KA2, were predicted to underlie the distinct binding profiles of the marine toxins. Single or multiple reciprocal mutations introduced into the receptor subunits produced a variety of effects on binding affinity. Most notably, mutation of Met725 to serine in KA2 increased the affinity of DH by 350-fold; in contrast, mutation of one or more of the residues in GluR5 did not markedly alter DH binding. MSVIII-19 affinity for the KA2 subunit was significantly increased in multiple site mutants, and reciprocal mutations in the GluR5 subunit produced substantial (700-fold) reductions in MSVIII-19 affinity. Physiological characterization of the double- and triple-mutant subunits demonstrated altered functional behavior consistent with the changes in binding affinity. The results provide experimental support for the importance of these three ligand binding domain (LBD) residues and suggest steric hindrance in the KA2 subunit LBD is largely responsible for the very low affinity for the two compounds. In this study, we identified the molecular basis for subunit selectivity of these marine-derived molecules on KARs, which could facilitate the rational design of selective ligands with distinct pharmacological profiles.

Alanine↗

PubMed2005

Divergent pharmacological activity of novel marine-derived excitatory amino acids on glutamate receptors.

Kainate receptors show a particular affinity for a variety of natural source compounds, including dysiherbaine (DH), a potent agonist derived from the marine sponge Dysidea herbacea. In this study, we characterized the pharmacological activity and structural basis for subunit selectivity of neodysiherbaine (neoDH) and MSVIII-19, which are natural and synthetic analogs of DH, respectively. NeoDH and MSVIII-19 differ from DH in the composition of two functional groups that confer specificity and selectivity for ionotropic glutamate receptors. In radioligand binding assays, neoDH displayed a 15- to 25-fold lower affinity relative to that of DH for glutamate receptor (GluR)5 and GluR6 kainate receptor subunits but a 7-fold higher affinity for kainate (KA)2 subunits, whereas MSVIII-19 displaced [(3)H]kainate only from GluR5 subunits but not GluR6 or KA2 subunits. NeoDH was an agonist for kainate and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors in patch-clamp recordings; in contrast, MSVIII-19 acted as a potent antagonist for homomeric GluR5 receptor currents with weaker activity on other kainate and AMPA receptors. Neither neoDH nor MSVIII-19 activated group I metabotropic GluRs. Homology modeling suggests that two critical amino acids confer the high degree of selectivity between the dysiherbaine analogs and the GluR5 and KA2 subunits. In summary, these data describe the pharmacological activity of two new compounds, one of which is a selective GluR5 receptor antagonist that will be of use for understanding native receptor function and designing more selective ligands for kainate receptors.

Alanine↗

PubMed2004

Ligand binding is a critical requirement for plasma membrane expression of heteromeric kainate receptors.

Intracellular trafficking of ionotropic glutamate receptors is controlled by multiple discrete determinants in receptor subunits. Most such determinants have been localized to the cytoplasmic carboxyl-terminal domain, but other domains in the subunit proteins can play roles in modulating receptor surface expression. Here we demonstrate that formation of an intact glutamate binding site also acts as an additional quality-control check for surface expression of homomeric and heteromeric kainate receptors. A key ligand-binding residue in the KA2 subunit, threonine 675, was mutated to either alanine or glutamate, which eliminated affinity for the receptor ligands kainate and glutamate. We found that plasma membrane expression of heteromeric GluR6/KA2(T675A) or GluR6/KA2(T675E) kainate receptors was markedly reduced compared with wild-type GluR6/KA2 receptors in transfected HEK 293 and COS-7 cells and in cultured neurons. Surface expression of homomeric KA2 receptors lacking a retention/retrieval determinant (KA2-R/A) was also reduced upon mutation of Thr-675 and elimination of the ligand binding site. KA2 Thr-675 mutant subunits were able to co-assemble with GluR5 and GluR6 subunits and were degraded at the same rate as wild-type KA2 subunit protein. These results suggest that glutamate binding and associated conformational changes are prerequisites for forward trafficking of intracellular kainate receptors following multimeric assembly.

Animals↗

PubMed2004

Attenuated plasticity of postsynaptic kainate receptors in hippocampal CA3 pyramidal neurons.

Kainate receptor-mediated components of postsynaptic currents at hippocampal mossy fiber synapses have markedly slower kinetics than currents arising from AMPA receptors. Here, we demonstrate that other aspects of kainate and AMPA receptor function at this synapse are distinct; in particular, kainate receptor currents are less sensitive to short- and long-term increases in presynaptic strength. EPSCs arising predominantly from AMPA receptors exhibited well characterized paired-pulse facilitation, frequency facilitation, and NMDA receptor-independent long-term potentiation, whereas isolated kainate receptor synaptic currents (KA-EPSCs) exhibited attenuated facilitation and long-term potentiation. In addition, KA-EPSCs varied in their sensitivity to a low-affinity competitive antagonist, suggestive of a synaptic heterogeneity greater than that of EPSCs comprised predominantly of AMPA receptors. These data suggest that the proportional contribution of AMPA and kainate receptors to ensemble synaptic currents will vary depending on the firing frequency of mossy fiber afferents. These synaptic features may be a mechanism for limiting activation of kainate receptors at mossy fiber synapses, which has been shown to be involved in seizurogenic firing of the CA3 network.

Animals↗

PubMed2004

A C-terminal determinant of GluR6 kainate receptor trafficking.

Intracellular trafficking of ionotropic glutamate receptors is regulated predominantly by determinants in the cytoplasmic C-terminal domain of the subunit proteins. Although AMPA receptors are found at the vast majority of excitatory synapses, synaptic kainate receptors exhibit a much more restricted distribution, suggesting that specific mechanisms exist for selective trafficking of these receptor proteins. In this report, we define a critical forward trafficking motif that is necessary for surface expression of the glutamate receptor 6 (GluR6) kainate receptor as well as chimeric proteins containing only the GluR6 C-terminal domain. The trafficking determinant was identified by tracking surface expression of green fluorescent protein-tagged GluR6 receptors with confocal immunofluorescence in COS-7 cells and cultured neurons and patch-clamp electrophysiology in human embryonic kidney 293 cells. Serial truncation and alanine site mutagenesis of the GluR6 subunit C terminus localized the critical motif to a seven amino acid stretch of predominantly basic residues. Alanine mutation of the trafficking motif reduced kainate receptor current amplitudes by >90% and resulted in retention of the mutated receptors in the endoplasmic reticulum. This forward trafficking domain is the first such identified for kainate receptors.

Amino Acid Motifs↗

PubMed2003

Cell surface expression of GluR5 kainate receptors is regulated by an endoplasmic reticulum retention signal.

Kainate receptors (KARs) are mediators of excitatory neurotransmission in the mammalian central nervous system, and their efficient targeting and trafficking is critical for normal synaptic function. A key step in the delivery of KARs to the neuronal plasma membrane is the exit of newly assembled receptors from the endoplasmic reticulum (ER). Here we report the identification of a novel ER retention signal in the alternatively spliced C-terminal domain of the GluR5-2b subunit, which controls receptor trafficking in both heterologous cells and neurons. The ER retention motif consists of a critical arginine (Arg-896) and surrounding amino acids, disruption of which promotes ER exit and surface expression of the receptors, as well as altering their physiological properties. The Arg-896-mediated ER retention of GluR5 is regulated by a mutation that mimics phosphorylation of Thr-898, but not by PDZ interactions. Furthermore, two positively charged residues (Arg-900 and Lys-901) in the C terminus were also found to regulate ER export of the receptors. Taken together, our results identify novel trafficking signals in the C-terminal domain of GluR5-2b and demonstrate that alternative splicing is an important mechanism regulating KAR function.

Alternative Splicing↗

PubMed2003

Distribution of kainate receptor subunits at hippocampal mossy fiber synapses.

Kainate receptors function as mediators of postsynaptic currents and as presynaptic modulators of synaptic transmission at mossy fiber synapses. Despite intense research into the physiological properties of mossy fiber kainate receptors, their subunit composition in the presynaptic and postsynaptic compartments is unclear. Here we describe the distribution of kainate receptor subunits in mossy fiber synapses using subunit-selective antibodies and knock-out mice. We provide morphological evidence for the presynaptic localization of KA1 and KA2 receptor subunits at mossy fiber synapses. Immunogold staining for KA1 and KA2 was commonly seen at synaptic contacts and in vesicular structures. Postsynaptic labeling in dendritic spines was also observed. Although KA1 predominantly showed presynaptic localization, KA2 was concentrated to a greater degree on postsynaptic membranes. Both subunits coimmunoprecipitated from hippocampal membrane extracts with GluR6 but not GluR7 subunits. These results demonstrate that KA1 and KA2 subunits are localized presynaptically and postsynaptically at mossy fiber synapses where they most likely coassemble with GluR6 subunits to form functional heteromeric kainate receptor complexes.

Animals↗

PubMed2003

Multiple trafficking signals regulate kainate receptor KA2 subunit surface expression.

The kainate receptor subunit KA2 does not form functional homomeric channels despite its structural similarity to the functional glutamate receptor 5-7subunits and high agonist binding affinity in in vitro assays. In this study, we first demonstrate that homomeric KA2 receptors fail to reach the plasma membrane and then identify the molecular mechanisms preventing surface expression. Specifically, we show that KA2 subunits form homooligomeric receptors that are confined to the endoplasmic reticulum (ER). We then demonstrate that, in both heterologous expression systems and primary neurons, the intracellular retention of KA2 is not caused by subunit misfolding but, rather, is mediated through discrete protein trafficking signals, including an arginine-rich ER retention/retrieval motif and a di-leucine endocytic sequence in the C terminus of the KA2 subunit. Disruption of these motifs results in ER exit and surface expression of KA2 homomeric receptors that remain nonfunctional. Furthermore, our data suggest that the ER retention/retrieval signal in KA2 is sterically shielded during heteromeric assembly, allowing delivery of functional heteromeric receptors to the plasma membrane. Taken together, our results illustrate novel regulatory mechanisms that control the intracellular trafficking and surface expression of kainate receptors.

Amino Acid Motifs↗

PubMed2003

Loss of kainate receptor-mediated heterosynaptic facilitation of mossy-fiber synapses in KA2-/- mice.

Multimeric assemblies of kainate (KA) receptor subunits form glutamate-gated ion channels that mediate EPSCs and function as presynaptic modulators of neurotransmitter release at some central synapses. The KA2 subunit is a likely constituent of many neuronal kainate receptors, because it is widely expressed in most neurons in the CNS. We have studied the effect of genetic ablation of this receptor subunit on synaptic transmission at the mossy-fiber-CA3 pyramidal cell synapse in hippocampal slices, where kainate receptors are localized to both presynaptic and postsynaptic sites. We found that both postsynaptic and presynaptic mossy-fiber kainate receptor function is altered in neurons from KA2-/- mice. The presynaptic facilitatory autoreceptor, which modulates glutamate release from mossy-fiber terminals, had a reduced affinity for exogenous agonists and synaptic glutamate. Although presynaptic facilitation attributable to homosynaptic glutamate release was normal at mossy-fiber synapses in KA2-/- neurons, heterosynaptic kainate receptor-mediated facilitation resulting from the spillover of glutamate from CA3 collateral synapses was absent. Consistent with a decrease in glutamate affinity of the receptor, the half-decay of the postsynaptic kainate-mediated EPSC was shorter in the knock-out mice. These results identify the KA2 subunit as a determinant of kainate receptor function at presynaptic and postsynaptic mossy-fiber kainate receptors.

Analysis of Variance↗

PubMed2002

Trans-synaptic Eph receptor-ephrin signaling in hippocampal mossy fiber LTP.

The site of induction of long-term potentiation (LTP) at mossy fiber-CA3 synapses in the hippocampus is unresolved, with data supporting both pre- and postsynaptic mechanisms. Here we report that mossy fiber LTP was reduced by perfusion of postsynaptic neurons with peptides and antibodies that interfere with binding of EphB receptor tyrosine kinases (EphRs) to the PDZ protein GRIP. Mossy fiber LTP was also reduced by extracellular application of soluble forms of B-ephrins, which are normally membrane-anchored presynaptic ligands for the EphB receptors. The application of soluble ligands for presynaptic ephrins increased basal excitatory transmission and occluded both tetanus and forskolin-induced synaptic potentiation. These findings suggest that PDZ interactions in the postsynaptic neuron and trans-synaptic interactions between postsynaptic EphB receptors and presynaptic B-ephrins are necessary for the induction of mossy fiber LTP.

Adaptor Proteins, Signal Transducing↗

PubMed2002

Differential activation of individual subunits in heteromeric kainate receptors.

Neuronal kainate receptors are assembled from subunits with dissimilar specificities for agonists and antagonists. The composite biophysical behavior of heteromeric kainate receptors is determined by intersubunit interactions whose nature is unclear. Here we use dysiherbaine, a selective kainate receptor agonist, to show that GluR5 subunits assembled in heteromeric GluR5/KA-2 kainate receptor complexes can gate current without concomitant activation of their partner KA-2 subunits. A long-lasting interaction between dysiherbaine and GluR5 subunits elicits a tonic current from GluR5/KA-2 receptors; subsequent cooperative gating of KA-2 subunits can be elicited by both agonists, such as glutamate, and some classically defined antagonists, such as CNQX. This study demonstrates that each type of subunit within a heteromeric kainate receptor contributes a distinct conductance upon activation by agonist binding, and therefore provides insight into the biophysical function of ionotropic glutamate receptors.

6-Cyano-7-nitroquinoxaline-2,3-dione↗