Assembly Of Receptor Subunits Research Articles

Kainate receptor channel opening and gating mechanism

Kainate receptors, a subclass of ionotropic glutamate receptors, are tetrameric ligand-gated ion channels that mediate excitatory neurotransmission1–4. Kainate receptors modulate neuronal circuits and synaptic plasticity during the development and function of the central nervous system and are implicated in various neurological and psychiatric diseases, including epilepsy, depression, schizophrenia, anxiety and autism5–11. Although structures of kainate receptor domains and subunit assemblies are available12–18, the mechanism of kainate receptor gating remains poorly understood. Here we present cryo-electron microscopy structures of the kainate receptor GluK2 in the presence of the agonist glutamate and the positive allosteric modulators lectin concanavalin A and BPAM344. Concanavalin A and BPAM344 inhibit kainate receptor desensitization and prolong activation by acting as a spacer between the amino-terminal and ligand-binding domains and a stabilizer of the ligand-binding domain dimer interface, respectively. Channel opening involves the kinking of all four pore-forming M3 helices. Our structures reveal the molecular basis of kainate receptor gating, which could guide the development of drugs for treatment of neurological disorders.

Molecular dynamics simulations of neuronal nicotinic acetylcholine receptor allosteric modulation.

The neuronal nicotinic acetylcholine receptor is a key experimental system for studying allosteric modulation, including by clinically useful drugs. Diverse physiological responses manifest from differential assembly of nicotinic receptor subunits (α2−α10 and β2−β4) into homo- or heteropentamers. An extensive body of electrophysiology data has demonstrated that the anthelmintic compounds morantel and oxantel display limited partial-agonist activity at mammalian nicotinic receptors, but are positive allosteric modulators of α3β2 receptors, while oxantel is a negative modulator of α4β2 receptors. Largely through mutation analyses, we have identified amino acid determinants of specificity for the compounds, as well as elements of the receptor extracellular domain that crosstalk between functional sites. Here, we initiated a systematic comparison of subtype specificity, based on molecular modeling and simulations of published cryo-electron microscopy structures and homology models. Preliminary results indicate differential movements in the C loop of orthosteric versus allosteric sites, involvement of key residues predicted to influence specificity in interactions with ligands, and inter- and intra-subunit functional connectivity assessed by network analysis. These studies promise to elucidate allosteric modulation mechanisms that govern site-to-site and ligand-to-gate communication in nicotinic and related receptors.

D-Aspartate consumption selectively promotes intermediate-term spatial memory and the expression of hippocampal NMDA receptor subunits

d-Aspartate (d-Asp) and d-serine (d-Ser) have been proposed to promote early-phase LTP in vitro and to enhance spatial memory in vivo. Here, we investigated the behavioural effects of chronic consumption of d-Asp and d-Ser on spatial learning of mice together with the expression of NMDA receptors. We also studied the alterations of neurogenesis by morphometric analysis of bromo-deoxyuridine incorporating and doublecortin expressing cells in the hippocampus. Our results specify a time period (3–4 h post-training), within which the animals exposed to d-Asp (but not d-Ser) show a more stable memory during retrieval. The cognitive improvement is due to elimination of transient bouts of destabilization and reconsolidation of memory, rather than to enhanced acquisition. d-Asp also protracted reversal learning probably due to reduced plasticity. Expression of GluN1 and GluN2A subunits was elevated in the hippocampus of d-Asp (but not d-Ser) treated mice. d-Asp or d-Ser did not alter the proliferation of neuronal progenitor cells in the hippocampus. The observed learning-related changes evoked by d-Asp are unlikely to be due to enhanced proliferation and recruitment of new neurones. Rather, they are likely associated with an upregulation of NMDA receptors, as well as a reorganization of receptor subunit assemblies in existing hippocampal/dentate neurons.

Expression and purification of a functional heteromeric GABAA receptor for structural studies.

The GABA-gated chloride channels of the Cys-loop receptor family, known as GABAA receptors, function as the primary gatekeepers of fast inhibitory neurotransmission in the central nervous system. Formed by the pentameric arrangement of five identical or homologous subunits, GABAA receptor subtypes are defined by the subunit composition that shape ion channel properties. An understanding of the structural basis of distinct receptor properties has been hindered by the absence of high resolution structural information for heteromeric assemblies. Robust heterologous expression and purification protocols of high expressing receptor constructs are vital for structural studies. Here, we describe a unique approach to screen for well-behaving and functional GABAA receptor subunit assemblies by using the Xenopus oocyte as an expression host in combination with fluorescence detection size exclusion chromatography (FSEC). To detect receptor expression, GFP fusions were introduced into the α1 subunit isoform. In contrast to expression of α1 alone, co-expression with the β subunit promoted formation of monodisperse assemblies. Mutagenesis experiments suggest that the α and β subunits can tolerate large truncations in the non-conserved M3/M4 cytoplasmic loop without compromising oligomeric assembly or GABA-gated channel activity, although removal of N-linked glycosylation sites is negatively correlated with expression level. Additionally, we report methods to improve GABAA receptor expression in mammalian cell culture that employ recombinant baculovirus transduction. From these methods we have identified a well-behaving minimal functional construct for the α1/β1 GABAA receptor subtype that can be purified in milligram quantities while retaining high affinity agonist binding activity.

Phosphorylation of the kainate receptor (KAR) auxiliary subunit Neto2 at serine 409 regulates synaptic targeting of the KAR subunit GluK1

Synaptic strength at excitatory synapses is determined by the presence of glutamate receptors (i.e. AMPA, NMDA, and kainate receptors) at the synapse. Synaptic strength is modulated by multiple factors including assembly of different receptor subunits, interaction with auxiliary subunits, and post-translational modifications of either the receptors or their auxiliary subunits. Using mass spectrometry, we found that the intracellular region of neuropilin and tolloid-like proteins (Neto) 1 and Neto2, the auxiliary subunits of kainate receptor (KARs), are phosphorylated by multiple kinases in vitro Specifically, Neto2 was phosphorylated at serine 409 (Ser-409) by Ca2+/calmodulin-dependent protein kinase II (CaMKII) and protein kinase A (PKA) both in vitro and in heterologous cells. Interestingly, we observed a substantial increase in Neto2 Ser-409 phosphorylation in the presence of CaMKII, and this phosphorylation was reduced in the presence of the KAR subunit GluK1 or GluK2. We also found endogenous phosphorylation of Neto2 at Ser-409 in the brain. Moreover, Neto2 Ser-409 phosphorylation inhibited synaptic targeting of GluK1 because, unlike WT Neto2 and the phosphodeficient mutant Neto2 S409A, the Neto2 S409D phosphomimetic mutant impeded GluK1 trafficking to synapses. These results support a molecular mechanism by which Neto2 phosphorylation at Ser-409 helps restrict GluK1 targeting to the synapse.

Understanding Nicotinic Receptor Assembly in the Endoplasmic Reticulum with Single Molecule Fluorescence Microscopy

Neuronal nicotinic acetylcholine receptors (nAChRs) are pentameric ligand-gated ion channels that are expressed throughout the central and peripheral nervous systems. Some subtypes of these channels have been shown to upregulate in the presence of nicotine resulting in changes in expression levels and stoichiometry of receptors on the plasma membrane. We have developed a method capable of isolating intact and functional nAChRs from the endoplasmic reticulum using cell-derived vesicles. These nanoscale vesicles enable us to isolate single receptors in their native membrane providing a method to perform organelle specific single molecule stoichiometry measurements. I will explain the progress we have made using these methods to determine that nicotine and several other nAChR ligands alter the assembly of these receptors. Some ligands, including nicotine and its metabolites, lead to increased fractions of high sensitivity alpha4 beta2 nAChRs and others lead to higher fractions of the low sensitivity receptor. Additionally, many of these ligands lead to changes in receptor trafficking. Our results indicate that nAChR ligands act in the endoplasmic reticulum to assist in the assembly of nascent receptor subunits.

Nicotinic Acetylcholine Receptors as Targets for Tobacco Cessation Therapeutics: Cutting-Edge Methodologies to Understand Receptor Assembly and Trafficking.

Tobacco dependence is a chronic relapsing disorder and nicotine, the primary alkaloid in tobacco, acts at nicotinic receptors to stimulate dopamine release in brain, which is responsible for the reinforcing properties of nicotine, leading to addiction. Although the majority of tobacco users express the desire to quit, only a small percentage of those attempting to quit are successful using the currently available pharmacotherapies. Nicotine upregulates the number of specific nicotinic receptors on the neuronal cell surface. An increase in receptor trafficking or preferential stoichiometric assembly of receptor subunits involves changes in assembly, endoplasmic reticulum export, vesicle transport, decreased degradation, desensitization, enhanced maturation of functional pentamers, and pharmacological chaperoning. Understanding these changes on a mechanistic level is important to the development of nicotinic receptors as drug targets. For this reason, cutting-edge methodologies are being developed and employed to pinpoint distinct changes in localization, assembly, export, vesicle trafficking, and stoichiometry in order to further understand the physiology of these receptors and to evaluate the action of novel therapeutics for smoking cessation.

Etomidate uniquely modulates the desensitization of recombinant α1β3δ GABAA receptors

Central GABAA receptors mediate GABAergic phasic and tonic inhibition. While synaptic αβγ GABAA receptors primarily mediate phasic inhibition, extrasynaptic αβδ receptors play an important role in mediating tonic inhibition. Etomidate is a general anesthetic that produces its effects by enhancing GABAA receptor activity. We previously showed that etomidate modulates the gating of oocyte-expressed αβγ and αβδ receptors with similar overall allosteric impact, but different pharmacological patterns. In αβγ receptors, etomidate enhances apparent GABA sensitivity (reduces GABA EC50), modestly increases maximal GABA efficacy, and slows current deactivation without affecting desensitization (Zhong et al., 2008). In αβδ receptors characterized by low GABA efficacy, etomidate dramatically increases responses to both low and maximal GABA. The effects of etomidate on desensitization and deactivation of αβδ receptors are unknown. To investigate the kinetic effects of etomidate on α1β3δ receptors of defined subunit arrangement, we expressed concatenated trimer (β3-α1-δ) and dimer (β3-α1) GABAA receptor subunit assemblies in human embryonic kidney (HEK)293T cells and recorded whole-cell voltage-clamp currents during rapid external solution exchanges. As expected, etomidate substantially increased maximal GABA-induced currents and prolonged deactivation. Moreover, desensitization was significantly decreased by etomidate. During prolonged GABA applications, etomidate enhanced steady-state currents more than peak currents. Thus, etomidate enhances tonic GABAergic inhibition through extrasynaptic αβδ receptors by both augmenting gating and reducing desensitization.

GABAA Receptor α and γ Subunits Shape Synaptic Currents via Different Mechanisms

Synaptic GABAA receptors (GABAARs) mediate most of the inhibitory neurotransmission in the brain. The majority of these receptors are comprised of α1, β2, and γ2 subunits. The amygdala, a structure involved in processing emotional stimuli, expresses α2 and γ1 subunits at high levels. The effect of these subunits on GABAAR-mediated synaptic transmission is not known. Understanding the influence of these subunits on GABAAR-mediated synaptic currents may help in identifying the roles and locations of amygdala synapses that contain these subunits. Here, we describe the biophysical and synaptic properties of pure populations of α1β2γ2, α2β2γ2, α1β2γ1 and α2β2γ1 GABAARs. Their synaptic properties were examined in engineered synapses, whereas their kinetic properties were studied using rapid agonist application, and single channel recordings. All macropatch currents activated rapidly (<1 ms) and deactivated as a function of the α-subunit, with α2-containing GABAARs consistently deactivating ∼10-fold more slowly. Single channel analysis revealed that the slower current decay of α2-containing GABAARs was due to longer burst durations at low GABA concentrations, corresponding to a ∼4-fold higher affinity for GABA. Synaptic currents revealed a different pattern of activation and deactivation to that of macropatch data. The inclusion of α2 and γ1 subunits slowed both the activation and deactivation rates, suggesting that receptors containing these subunits cluster more diffusely at synapses. Switching the intracellular domains of the γ2 and γ1 subunits substantiated this inference. Because this region determines post-synaptic localization, we hypothesize that GABAARs containing γ1 and γ2 use different mechanisms for synaptic clustering.

RIC-3 differentially modulates α4β2 and α7 nicotinic receptor assembly, expression, and nicotine-induced receptor upregulation

BackgroundRecent work has shown that the chaperone resistant to inhibitors of acetylcholinesterase (RIC-3) is critical for the folding, maturation and functional expression of a variety of neuronal nicotinic acetylcholine receptors. α7 nicotinic receptors can only assemble and functionally express in select lines of cells, provided that RIC-3 is present. In contrast, α4β2 nicotinic receptors can functionally express in many cell lines even without the presence of RIC-3. Depending on the cell line, RIC-3 has differential effects on α4β2 receptor function – enhancement in mammalian cells but inhibition in Xenopus oocytes. Other differences between the two receptor types include nicotine-induced upregulation. When expressed in cell lines, α4β2 receptors readily and robustly upregulate with chronic nicotine exposure. However, α7 nicotinic receptors appear more resistant and require higher concentrations of nicotine to induce upregulation. Could the coexpression of RIC-3 modulate the extent of nicotine-induced upregulation not only for α7 receptors but also α4β2 receptors? We compared and contrasted the effects of RIC-3 on assembly, trafficking, protein expression and nicotine-induced upregulation on both α7 and α4β2 receptors using fluorescent protein tagged nicotinic receptors and Förster resonance energy transfer (FRET) microscopy imaging.ResultsRIC-3 increases assembly and cell surface trafficking of α7 receptors but does not alter α7 protein expression in transfected HEK293T cells. In contrast, RIC-3 does not affect assembly of α4β2 receptors but increases α4 and β2 subunit protein expression. Acute nicotine (30 min exposure) was sufficient to upregulate FRET between α4 and β2 subunits. Surprisingly, when RIC-3 was coexpressed with α4β2 receptors nicotine-induced upregulation was prevented. α7 receptors did not upregulate with acute nicotine in the presence or absence of RIC-3.ConclusionsThese results provide interesting novel data that RIC-3 differentially regulates assembly and expression of different nicotinic receptor subunits. These results also show that nicotine-mediated upregulation of α4β2 receptors can be dynamically regulated by the presence of the chaperone, RIC-3. This could explain a novel mechanism why high affinity α4β2 receptors are upregulated in specific neuronal subtypes in the brain and not others.

Modulation of Delta Subunit-Containing GABAA Receptors by Etomidate Depends on Subunit Arrangement

Background: GABAA receptors are allosterically modulated by a variety of compounds including etomidate and tetrahydrodeoxycorticosterone (THDOC). Extrasynaptic αβδ GABAA receptors mediate tonic inhibition, and may be more sensitive to modulators than synaptic αβγ receptors. Studies of concatenated subunit assemblies suggests that the subunit arrangement of αβδ may vary (1). We compared the modulation by etomidate and THDOC on α1β3δ receptors with different subunit arrangements, and to α1β3γ2L receptors. Methods: Plasmids encoding concatenated GABAA receptor subunit assemblies and free subunits were obtained from Prof. Erwin Sigel (Bern, Switzerland). Xenopus oocytes were used to express β3-α1-δ/β3-α1 and β3-α1-δ/α1-β3 receptors as well as free subunit mixtures. Effects of etomidate (3 μM) or THDOC (1 μM) on maximal GABA-activated receptor currents were measured using two-microelectrode voltage-clamp electrophysiology. Etomidate enhancement of β3-α1-δ/β3-α1 was assessed over a broad [GABA] range, and analyzed with an allosteric model.Results: High GABA elicited small currents in oocytes expressing β3-α1-δ/β3-α1 and β3-α1-δ/α1-β3 receptors. THDOC produced similar large current enhancements in β3-α1-δ/β3-α1 and β3-α1-δ/α1-β3 receptors. However, etomidate produced a greater current enhancement in β3-α1-δ/α1-β3 receptors than in β3-α1-δ/β3-α1. Etomidate increased the maximal response of β3-α1-δ/β3-α1 receptors to GABA and produced a small leftward shift in GABA EC50.Discussion: using concatenated receptor constructs resulted in evidence of δ subunit incorporation that was not seen using free subunit mRNAs. GABA is a weak partial agonist in α1β3δ receptors, while etomidate and THDOC modulate via different sites. Etomidate efficacy in α1β3δ is similar to that in α1β3γ receptors with homologous subunit arrangement, and interactions at β3/β3 interfaces may be more efficacious than those at β3/α1 interfaces.Support: NIH (R01GM89745 and P01GM58448)Reference:1) Kaur KH et al, J. Biol Chem 2009;284:7889-96

Plasticity of the Asialoglycoprotein Receptor Deciphered by Ensemble FRET and Single-Molecule Counting PALM

The composition of many multi-subunited receptor complexes at the plasma membrane is still debated as well as the impact of changes in the receptor subunit composition. Receptor plasticity, involving changes in stoichiometry and receptor function, has been difficult to address. Here, we introduce new spectroscopic tools to dissect receptor subunit assembly of the Asialoglycoprotein Receptor. Using this highly tractable receptor model system we show that the Asialoglycoprotein Receptor can assemble into distinct oligomers at the plasma membrane and that differential subunit assembly dictates receptor specificity. With ensemble analyses of Fluorescence Resonance Energy Transfer (FRET) and analytical modeling, we quantified receptor subunit homo- and hetero-oligomerization in the living cell. Furthermore, we established single-molecule counting using Photoactivated Localization Microscopy (PALM), visualizing an asymmetric receptor subunit assembly on the single-molecule level. Our results define a probability hierarchy of oligomerization driven by different molecular motifs that entails distinct co-existent receptor subunit assemblies. The Asialoglycoprotein Receptor is involved in the clearance of thrombogenic material. The variety of potential ligands and the propensity of the subunits to form distinct oligomers may explain previous inconsistent results and underscore the importance of deciphering oligomerization in the single cell and on the single-molecule level.

Transmembrane Region of N-Methyl-d-aspartate Receptor (NMDAR) Subunit Is Required for Receptor Subunit Assembly

N-Methyl-D-aspartate receptors (NMDARs), one of three main classes of ionotropic glutamate receptors, play major roles in synaptic plasticity, synaptogenesis, and excitotoxicity. Unlike non-NMDA receptors, NMDARs are thought to comprise obligatory heterotetrameric complexes mainly composed of GluN1 and GluN2 subunits. When expressed alone in heterogenous cells, such as HEK293 cells, most of the NMDAR subunits can neither leave the endoplasmic reticulum (ER) nor be expressed in the cell membrane because of the ER retention signals. Only when NMDARs are heteromerically assembled can the ER retention signals be masked and NMDARs be expressed in the surface membrane. However, the mechanisms underlying NMDAR assembly remain poorly understood. To identify regions in subunits that mediate this assembly, we made a series of truncated or chimeric cDNA constructs. Using FRET measurement in living cells combined with immunostaining and coimmunoprecipitation analysis, we examined the assembly-determining domains of NMDAR subunits. Our results indicate that the transmembrane region of subunits is necessary for the assembly of NMDAR subunits, both for the homodimer and the heteromer.

Antigenic Structure of the Human Muscle Nicotinic Acetylcholine Receptor Main Immunogenic Region

The main immunogenic region on the alpha1 subunits of muscle nicotinic acetylcholine receptors provokes half or more of the autoantibodies in myasthenia gravis and its animal model. Many of these autoantibodies depend on the native conformation of the receptor for their ability to bind with high affinity. We mapped this region and explained the conformation dependence of its epitopes by making chimeras in which sequences of human muscle alpha1 subunits were replaced in human neuronal alpha7 subunits or Aplysia acetylcholine binding protein. These chimeras also revealed that the main immunogenic region can play a major role in promoting conformational maturation and, consequently, assembly of receptor subunits.

Formation of NR1/NR2 and NR1/NR3 Heterodimers Constitutes the Initial Step in N-Methyl-D-aspartate Receptor Assembly

N-Methyl-D-aspartate (NMDA) receptors are tetrameric protein complexes composed of the glycine-binding NR1 subunit with a glutamate-binding NR2 and/or glycine-binding NR3 subunit. Tri-heteromeric receptors containing NR1, NR2, and NR3 subunits reconstitute channels, which differ strikingly in many properties from the respective glycine- and glutamate-gated NR1/NR2 complexes and the NR1/NR3 receptors gated by glycine alone. Therefore, an accurate oligomerization process of the different subunits has to assure proper NMDA receptor assembly, which has been assumed to occur via the oligomerization of homodimers. Indeed, using fluorescence resonance energy transfer analysis of differentially fluorescence-tagged subunits and blue native polyacrylamide gel electrophoresis after metabolic labeling and affinity purification revealed that the NR1 subunit is capable of forming homo-oligomeric aggregates. In contrast, both the NR2 and the NR3 subunits formed homo- and hetero-oligomers only in the presence of the NR1 subunit indicating differential roles of the subunits in NMDA receptor assembly. However, co-expression of the NR3A subunit with an N-terminal domain-deleted NR1 subunit (NR1(DeltaNTD)) abrogating NR1 homo-oligomerization did not affect NR1/NR3A receptor stoichiometry or function. Hence, homo-oligomerization of the NR1 subunit is not essential for proper NR1/NR3 receptor assembly. Because identical results were obtained for NR1(DeltaNTD)/NR2 NMDA receptors (Madry, C., Mesic, I., Betz, H., and Laube, B. (2007) Mol. Pharmacol., 72, 1535-1544) and NR1-containing hetero-oligomers are readily formed, we assume that heterodimerization of the NR1 with an NR3 or NR2 subunit, which is followed by the subsequent association of two heterodimers, is the key step in determining proper NMDA receptor subunit assembly and stoichiometry.

Monitoring Ligand‐Dependent IL‐5 Receptor Activation on Cells Using Fluorescence Resonance Energy Transfer (FRET)

Interleukin-5 (IL-5) is important in a number of pathophysiological states, including allergy, asthma, hypereosinophilia syndromes, myeloid leukemia, and certain infectious diseases. IL-5 exerts its biological functions through binding to its receptor, which is composed of subunits α and βc. To date, the mechanism of receptor subunit interactions in the cellular context is poorly understood. We used fluorescence resonance energy transfer (FRET) to measure the interactions of IL-5R subunits in intact cells, including the extent of preassembly and the effects of IL-5 stimulation. Phoenix cells coexpressing IL-5Rα and βc fused to cyan or yellow fluorescent proteins (CFP or YFP) were used to evaluate protein-protein interactions by FRET before and after IL-5 treatment and in the presence or absence of the IL-5Rα inhibitor AF17121. We observed FRET between IL-5R βc chains in the absence of ligand, demonstrating that βc receptor subunits exist in preformed homomeric receptor complexes. In the presence of ligand, we observed dose-dependent FRET between IL-5Rα and βc consistent with heteromeric receptor subunit assembly. Ligand-dependent heteromeric subunit assembly, but not βc self-assembly, was inhibited by the IL-5Rα inhibitor AF17121. Taken together, these results are consistent with a model in which βc molecules exist in the cell membrane as dimers prior to ligand binding, and where binding of cytokine ligand by IL-5Rα leads to recruitment of βc dimers into heteromeric complexes.

Molecular properties of P2X receptors

P2X receptors for adenosine tri-phosphate (ATP) are a distinct family of ligand-gated cation channels with two transmembrane domains, intracellular amino and carboxy termini and a large extracellular ligand binding loop. Seven genes (P2X(1-7)) have been cloned and the channels form as either homo or heterotrimeric channels giving rise to a wide range of phenotypes. This review aims to give an account of recent work on the molecular basis of the properties of P2X receptors. In particular, to consider emerging information on the assembly of P2X receptor subunits, channel regulation and desensitisation, targeting, the molecular basis of drug action and the functional contribution of P2X receptors to physiological processes.

Ubiquilin-1 Regulates Nicotine-induced Up-regulation of Neuronal Nicotinic Acetylcholine Receptors

Chronic exposure to nicotine, as in tobacco smoking, up-regulates nicotinic acetylcholine receptor surface expression in neurons. This up-regulation has been proposed to play a role in nicotine addiction and withdrawal. The regulatory mechanisms behind nicotine-induced up-regulation of surface nicotinic acetylcholine receptors remain to be determined. It has recently been suggested that nicotine stimulation acts through increased assembly and maturation of receptor subunits into functional pentameric receptors. Studies of muscle nicotinic acetylcholine receptors suggest that the availability of unassembled subunits in the endoplasmic reticulum can be regulated by the ubiquitin-proteosome pathway, resulting in altered surface expression. Here, we describe a role for ubiquilin-1, a ubiquitin-like protein with the capacity to interact with both the proteosome and ubiquitin ligases, in regulating nicotine-induced up-regulation of neuronal nicotinic acetylcholine receptors. Ubiquilin-1 interacts with unassembled alpha3 and alpha4 subunits when coexpressed in heterologous cells and interacts with endogenous nicotinic acetylcholine receptors in neurons. Coexpression of ubiquilin-1 and neuronal nicotinic acetylcholine receptors in heterologous cells dramatically reduces the expression of the receptors on the cell surface. In cultured superior cervical ganglion neurons, expression of ubiquilin-1 abolishes nicotine-induced up-regulation of nicotinic acetylcholine receptors but has no effect on the basal level of surface receptors. Coimmunostaining shows that the interaction of ubiquilin-1 with the alpha3 subunit draws the receptor subunit and proteosome into a complex. These data suggest that ubiquilin-1 limits the availability of unassembled nicotinic acetylcholine receptor subunits in neurons by drawing them to the proteosome, thus regulating nicotine-induced up-regulation.

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.

Aberrant Formation of Glutamate Receptor Complexes in Hippocampal Neurons of Mice Lacking the GluR2 AMPA Receptor Subunit

The number and type of receptors present at the postsynaptic membrane determine the response to the neurotransmitter released from the presynaptic terminal. Because most neurons receive multiple and distinct synaptic inputs and contain several different subtypes of receptors stimulated by the same neurotransmitter, the assembly and trafficking of receptors in neurons is a complex process involving many levels of regulation. To investigate the mechanism that neurons use to regulate the assembly of receptor subunits, we studied a GluR2 knock-out mouse. GluR2 is a critical subunit that controls calcium permeability of AMPA receptors and is present in most native AMPA receptors. Our data indicate that in the absence of GluR2, aberrant receptor complexes composed of GluR1 and GluR3 are formed in the hippocampus, and that there is an increased number of homomeric GluR1 and GluR3 receptors. We also show that these homomeric and heteromeric receptors are less efficiently expressed at the synapse. Our results show that GluR2 plays a critical role in controlling the assembly of AMPA receptors, and that the assembly of subunits may reflect the affinity of one subunit for another or the stability of intermediates in the assembly process. Therefore, GluR1 may have a greater preference for GluR2 than it does for GluR3.