Browsing by Subject "glutamate receptors"
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Item Cellular mechanisms that modulate kainate receptor trafficking and assembly(2007-06-08) Pornpun Vivithanaporn; Geoffrey T. Swanson; Xiaodong Cheng; Lisa A. Elferink; Kenneth M. Johnson; John MarshallKainate receptors (KARs) in the mammalian brain play a variety of physiological roles that require selective assembly, intracellular trafficking, and synaptic targeting. Cytoplasmic and non-cytoplasmic determinants that modulate KAR expression at the plasma membrane have been recently characterized. The intracellular trafficking determinants are presumed to bind to chaperone proteins, but these proteins have not been identified for any KAR subunit. Here we identified two chaperone proteins that associated with the carboxy terminus but differently regulated the localization of KA2 receptors. We found that coatomer protein complex I (COPI) subunits interacted with KA2 subunits at the arginine-based ER retention/retrieval motif and these associations were decreased in heteromeric GluR6a/KA2 receptors. Disruptions of COPI and KA2 associations by alanine mutations at the arginine-rich domain and elimination of COPI vesicles were correlated with the increased expression at the plasma membrane of KA2 receptors, indicating that COPI proteins regulated the ER localization of receptors. KA2 receptors also co-precipitated with three isoforms of 14-3-3 proteins; only the KA2 and 14-3-3zeta association was correlated with higher plasma membrane expression of receptors. In addition to identifying cytoplasmic chaperone systems, we were interested in understanding the nature of a recently described trafficking checkpoint in non-cytoplasmic regions. Mutations in domains typically involved in glutamate binding and ion permeation disrupt expression of KARs at the plasma membrane. A conformational change of receptors after glutamate binding is proposed to permit egress of KARs from the ER. We mutated critical amino acid residues in the extracellular linker domain of GluR6a subunits and found that desensitization rates were only weakly correlated with plasma membrane expression levels. Alteration of these residues impaired other stages of receptor biosyntheis including assembly and degradation of mutated receptors. We found that mutations at the transduction linker collectively altered subunit assembly, degradation, desensitization and a post-assembly stage. Our characterizations of chaperone proteins and mechanisms that regulate intracellular trafficking provide a better understanding of cellular controls in the early stages of KAR biosynthesis.Item A structure-function analysis of the marine toxin neodysiherbaine and its synthetic analogs on kainate receptors(2008-08-15) Laura Leanne Lash; Geoffrey T. Swanson; Li-Yen Mae Huang; Kenneth M. Johnson; Joel P. Gallagher; Anis ContractorKainate receptor antagonists show efficacy in multiple animal models of neuropathologies, including pain and epilepsy. These receptors are selectively targeted by a number of natural and synthetic ligands. The goals of this project were to identify compounds with unique specificity for kainate receptors and to gain insight into structural determinants of affinity and activity of the marine toxin dysiherbaine, DH. DH is a high-affinity kainate receptor agonist and the most potent convulsant excitatory amino acid identified to date. The structure of DH is unique relative to other kainate receptor agonists because it has a glutamate backbone attached to a tetra-substituted hydrofuropyran ring system with two functional groups, at the C8 and C9 positions, that are important determinants for selectivity. Here, four groups of structural analogs were screened. Among the structural analogs characterized in the current study, 2,4-epi-neoDH, displayed a novel pharmacological profile at kainate receptors. In radioligand binding assays, 2,4-epi-neoDH showed micromolar binding affinity to GluR5(Q)-2a and GluR6a subunits. Electrophysiology recordings revealed it acted as an antagonist at these receptors, without detectable activity at AMPA receptors. This molecule represents the first antagonist for GluR5 and GluR6 receptors without concurrent activity at AMPA receptors. Our characterization of the series of structural analogs revealed they exhibit diverse binding affinities and a range of seizure-inducing potencies. It was determined that their seizure-inducing potency correlates with their binding affinity for GluR5 receptors, suggesting they induce seizure behavior through direct activation of GluR5 kainate receptors in the brain. Additionally, the C8 hydroxyl group of the naturally occurring analog, neodysiherbaine, was identified as a critical determinant for divergent affinity for GluR6 versus GluR7 subunits. Site-directed mutagenesis revealed two amino acid residues in the GluR6 binding cavity that are important for the selectivity. Our studies reveal insight into the structural requirements of DH and analogs for their diverse activity at kainate receptors. These studies offer a foundation for design of selective kainate receptor ligands and provide pharmacological tools for studying structure and function of these receptors.