Synthesis and evaluation of 6-(11C-methyl(4-(pyridin-2-yl)thiazol-2-yl)amino)benzo[d]thiazol-2(3H)-one for imaging γ-8 dependent transmembrane AMPA receptor regulatory protein by PET

Transmembrane AMPA receptor regulatory proteins (TARPs) are a recently discovered family of proteins that modulate AMPA receptors activity. Based on a potent and selective TARP subtype γ-8 antagonist, 6-(methyl(4-(pyridin-2-yl)thiazol-2-yl)amino)benzo[d]thiazol-2(3H)-one (compound 9), we perform the radiosynthesis of its ¹¹C-isotopologue 1 and conduct preliminary PET evaluation to test the feasibility of imaging TARP γ-8 dependent receptors in vivo.     

Orexin-A increases cell surface expression of AMPA receptors in the striatum

Accumulating evidence suggests that orexin signaling is involved in reward and motivation circuit functions. However, the underlying mechanisms are not yet fully understood. Here, we show that orexin-A potentiates AMPAR-mediated synaptic transmission in the striatum, possibly by regulating the surface expression of AMPARs. Primary culture of striatal neurons revealed increased surface expression of AMPARs following orexin-A treatment. The increase in surface-expressed AMPARs induced by orexin-A treatment was dependent on both ERK activation and the presence of extracellular Ca²⁺. In the corticostriatal synapses of rat brain slices, orexin-A bath-application caused a delayed increase in the AMPAR/NMDAR EPSC ratio, suggesting that orexin-A sets in motion a series of events that lead to functional alterations in the striatal circuits. Our findings provide a potential link between the activation of orexin signaling in the striatum in response to addictive substances and neural adaptations in the reward circuitry that may mediate the long-lasting addiction-related behaviors.     

Action of extracellular divalent cations on native alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptors

The effects of divalent cations on Ca2+-impermeable containing (GluR2 subunit) MPA receptors of hippocampal pyramidal neurones isolated from rat brain was studied using patch-clamping. Ca2+, Mg2+, Mn2+, Co2+, Ni2+ and Zn2+ inhibited currents induced by kainate and glutamate. Inhibition was fast, reversible and voltage independent. The rank order of activities was Ni2+ > Zn2+ > Co2+ > Ca2+ > Mn2+ > Mg2+. Cyclothiazide (0.1 mm) significantly reduced inhibition by divalent cations and 6, 7 dinitroquinoxaline-2.3-dione (DNQX). However, high concentrations of Ni2+ and DNQX inhibited AMPA receptors even in the presence of cyclothiazide. The inhibitory effect of divalent cations as well as DNQX was counteracted by an increase in agonist concentration. In the presence of divalent cations the EC50 values of kainate and glutamate were increased, but the maximal response was not changed. An increase in agonist concentration induced a parallel shift in the concentration-inhibition curve for a divalent cation. These data suggest a competitive-like type of inhibition. However, an increase in agonist concentration reduced the inhibitory action of Ni2+ less than that of DNQX. This gave evidence against direct competition between divalent cations and AMPA receptor agonists. A 'complex-competition' hypothesis was proposed to explain the inhibitory action of divalent cations; it is suggested that divalent cations form ion-agonist complexes, which compete with free agonist for agonist-binding sites on AMPA receptors.     

AMPA受体的内化及其分子机制

AMPA受体的内化不仅仅是结束它们的活性状态,受体的许多重要信号功能是与活性依赖的内化密切相关的。突触功能调节中,存在2种形式AMPA受体的内化：组构性内化和活性依赖的内化。现就AMPA受体内化的分类、过程和意义,以及活性依赖的内化的诱发因素、调节因素和内化后的去向进行综述。     

Opiate withdrawal induces dynamic expressions of AMPA receptors and its regulatory molecule CaMKIIalpha in hippocampal synapses

Adaptive changes in brain areas following drug withdrawal are believed to contribute to drug seeking and relapse. Cocaine withdrawal alters the expression of GluR1 and GluR2/3 subunits of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors in nucleus accumbens or amygdala, but the influence of drug withdrawal on hippocampus is little known. Here, we have examined the expression of GluR1 and GluR2/3 in hippocampal membrane and synaptic fractions following repeated morphine exposure and subsequent withdrawal. Repeated morphine exposure for 12 d increased GluR1 and GluR2/3 in synaptosome but not in membrane fraction. Interestingly, CaMKIIalpha, known to be able to regulate the function of AMPA receptors, was decreased in synaptosome but not in membrane fraction; pCaMKIIalpha, the phosphorylated form of CaMKIIalpha, was increased in both fractions. However, during opiate withdrawal, GluR1 was generally reduced while GluR2/3 was prominently increased in both fractions; pCaMKIIalpha was strongly decreased immediately after withdrawal, but detectably increased in late phase of morphine withdrawal in both fractions. Importantly, the opiate withdrawal-induced increase in GluR2/3 was dependent on the activation of glucocorticoid receptors and NMDA receptors, as it was prevented by the glucocorticoid receptor antagonist RU38486, or intrahippocampal injection of the NMDA receptor antagonist AP-5 or the antagonist to NR2B-containing NMDA receptors, Ro25-6981. These findings indicate that opiate withdrawal induces dynamic expression of GluR1 and GluR2/3 subunits of AMPA receptors in hippocampal synapses, possibly revealing an adaptive process of the hippocampal functions following opiate withdrawal.     

Subunit-selective N-terminal domain associations organize the formation of AMPA receptor heteromers

The assembly of AMPA-type glutamate receptors (AMPARs) into distinct ion channel tetramers ultimately governs the nature of information transfer at excitatory synapses. How cells regulate the formation of diverse homo- and heteromeric AMPARs is unknown. Using a sensitive biophysical approach, we show that the extracellular, membrane-distal AMPAR N-terminal domains (NTDs) orchestrate selective routes of heteromeric assembly via a surprisingly wide spectrum of subunit-specific association affinities. Heteromerization is dominant, occurs at the level of the dimer, and results in a preferential incorporation of the functionally critical GluA2 subunit. Using a combination of structure-guided mutagenesis and electrophysiology, we further map evolutionarily variable hotspots in the NTD dimer interface, which modulate heteromerization capacity. This 'flexibility' of the NTD not only explains why heteromers predominate but also how GluA2-lacking, Ca(2+)-permeable homomers could form, which are induced under specific physiological and pathological conditions. Our findings reveal that distinct NTD properties set the stage for the biogenesis of functionally diverse pools of homo- and heteromeric AMPAR tetramers.     

Clustering and synaptic targeting of PICK1 requires direct interaction between the PDZ domain and lipid membranes

Protein interacting with c kinase 1 (PICK1) regulates the trafficking of receptors and ion-channels such as AMPA receptors. Traditionally, the PICK1 PDZ domain is regarded as an adaptor capable of binding to receptors trafficked by PICK1, and the lipid-binding BAR domain functions to tether PICK1 directly to membranes. Here, we show that the PICK1 PDZ domain can directly interact with lipid membranes. The PDZ domain and lipid membrane interaction is mediated by both a polybasic amino-acid cluster and a conserved 'Cys-Pro-Cys' motif located away from the peptide ligand-binding groove. Disruption of the PDZ and lipid membrane interaction totally abolished synaptic targeting of PICK1. Although mutation of the CPC motif did not affect the interaction between PICK1 and AMPA receptors, the mutant PICK1 was unable to cluster the GluR2 subunit of the receptor. In neurons, PICK1 containing the same mutation displayed dramatically compromised capacity in the trafficking of AMPA receptors. Taken together, our findings not only uncovered the novel lipid membrane-binding property of the PICK1 PDZ domain, but also provided direct evidence supporting the functional relevance of the PDZ-lipid interaction.     

Dynamics and allosteric potential of the AMPA receptor N-terminal domain

Glutamate-gated ion channels (ionotropic glutamate receptors, iGluRs) sense the extracellular milieu via an extensive extracellular portion, comprised of two clamshell-shaped segments. The distal, N-terminal domain (NTD) has allosteric potential in NMDA-type iGluRs, which has not been ascribed to the analogous domain in AMPA receptors (AMPARs). In this study, we present new structural data uncovering dynamic properties of the GluA2 and GluA3 AMPAR NTDs. GluA3 features a zipped-open dimer interface with unconstrained lower clamshell lobes, reminiscent of metabotropic GluRs (mGluRs). The resulting labile interface supports interprotomer rotations, which can be transmitted to downstream receptor segments. Normal mode analysis reveals two dominant mechanisms of AMPAR NTD motion: intraprotomer clamshell motions and interprotomer counter-rotations, as well as accessible interconversion between AMPAR and mGluR conformations. In addition, we detect electron density for a potential ligand in the GluA2 interlobe cleft, which may trigger lobe motions. Together, these data support a dynamic role for the AMPAR NTDs, which widens the allosteric landscape of the receptor and could provide a novel target for ligand development.     

Characterization of the functional role of the N-glycans in the AMPA receptor ligand-binding domain

The ligand-binding domains of AMPA receptor subunits carry two conserved N-glycosylation sites. In order to gain insight into the functional role of the corresponding N-glycans, we examined how the elimination of glycosylation at these sites (N407 and N414) affects the ligand-binding characteristics, structural stability, cell-surface expression, and channel properties of homomeric GluR-D (GluR4) receptor and its soluble ligand-binding domain (S1S2). GluR-D S1S2 protein expressed as a secreted protein in insect cells was found to be glycosylated at N407 and N414. No major differences in the ligand-binding properties were observed between the 'wild-type' S1S2 and non-glycosylated N407D/N414Q double mutant, or between S1S2 proteins expressed in the presence or absence of tunicamycin, an inhibitor of N-glycosylation. Purified glycosylated and non-glycosylated S1S2 proteins also showed similar thermostabilities as determined by CD spectroscopy. Full-length homomeric GluR-D receptor with N407D/N414Q mutation was expressed on the surface of HEK293 cells like the wild-type GluR-D. In outside-out patches, GluR-D and the N407D/N414Q mutant produced similar rapidly desensitizing current responses to glutamate and AMPA. We therefore report that the two conserved ligand-binding domain glycans do not play any major role in receptor-ligand interactions, do not impart a stabilizing effect on the ligand-binding domain, and are not critical for the formation and surface localization of homomeric GluR-D AMPA receptors in HEK293 cells.     

PTEN regulates AMPA receptor-mediated cell viability in iPS-derived motor neurons

Excitatory transmission in the brain is commonly mediated by the α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors. In amyotrophic lateral sclerosis (ALS), AMPA receptors allow cytotoxic levels of calcium into neurons, contributing to motor neuron injury. We have previously shown that oculomotor neurons resistant to the disease process in ALS show reduced AMPA-mediated inward calcium currents compared with vulnerable spinal motor neurons. We have also shown that PTEN (phosphatase and tensin homolog deleted on chromosome 10) knockdown via siRNA promotes motor neuron survival in models of spinal muscular atrophy (SMA) and ALS. It has been reported that inhibition of PTEN attenuates the death of hippocampal neurons post injury by decreasing the effective translocation of the GluR2 subunit into the membrane. In addition, leptin can regulate AMPA receptor trafficking via PTEN inhibition. Thus, we speculate that manipulation of AMPA receptors by PTEN may represent a potential therapeutic strategy for neuroprotective intervention in ALS and other neurodegenerative disorders. To this end, the first step is to establish a fibroblast–iPS–motor neuron in vitro cell model to study AMPA receptor manipulation. Here we report that iPS-derived motor neurons from human fibroblasts express AMPA receptors. PTEN depletion decreases AMPA receptor expression and AMPA-mediated whole-cell currents, resulting in inhibition of AMPA-induced neuronal death in primary cultured and iPS-derived motor neurons. Taken together, our results imply that PTEN depletion may protect motor neurons by inhibition of excitatory transmission that represents a therapeutic strategy of potential benefit for the amelioration of excitotoxicity in ALS and other neurodegenerative disorders.     

AMPA受体失敏和快速兴奋性突触传递

ＡＭＰＡ受体是离子型谷氨酸受体中重要的一类亚型,在中枢神经系统内主要介导快速的兴奋性突触传递。近年来,ＡＭＰＡ受体独特的失敏特性逐渐被阐明,已经确定了一些特异调节ＡＭＰＡ受体失敏的化合物。大量的生理学和药理学证据表明,ＡＭＰＡ受体失敏在快速兴奋性突触传递中起着重要的作用,对单个突触的传递效率、神经元的整合功能和突触的可塑性均有影响。     

谷氨酸下调培养海马神经元AMPA受体G1uR2亚单位的表达

目的 研究在癫痫发病过程中,谷氨酸对AMPA受体G1uR2亚单位表达变化的影响。方法 用RT-PCR和Western Blot方法观察谷氨酸诱导培养大鼠海马神经元AMPA受体G1uR2亚单位mRNA和蛋白的表达变化。结果 在谷氨酸刺激后2h,8h,12h,培养海马神经元G1uR2 mRNA和蛋白表达明显下降,与对照组相比,差异有显著性（P〈0．05）,而非NMDA受体拮抗剂CNQX能阻断此变化。结论 在癫痫等疾病中,谷氨酸能通过激活AMAP／KA受体下调AMPA受体G1uR2亚单位的表达,参与发病过程。     

谷氨酸下调培养海马神经元AMPA 受体GluR2亚单位的表达

目的研究在癫痫发病过程中,谷氨酸对AMPA受体Glu R2亚单位表达变化的影响。方法用RT-PCR和Western Blot方法观察谷氨酸诱导培养大鼠海马神经元AMPA受体Glu R2亚单位mRNA和蛋白的表达变化。结果在谷氨酸刺激后2h,8h,12h,培养海马神经元Glu R2mRNA和蛋白表达明显下降,与对照组相比,差异有显著性(P<0.05),而非NMDA受体拮抗剂CNQX能阻断此变化。结论在癫痫等疾病中,谷氨酸能通过激活AMAP/KA受体下调AMPA受体GluR2亚单位的表达,参与发病过程。     

Ultrastructural localisation and differential agonist-induced regulation of AMPA and kainate receptors present at the presynaptic active zone and postsynaptic density

Activity-dependent changes in ionotropic glutamate receptors at the postsynaptic membrane are well established and this regulation plays a central role in the expression of synaptic plasticity. However, very little is known about the distributions and regulation of ionotropic receptors at presynaptic sites. To determine if presynaptic receptors are subject to similar regulatory processes we investigated the localisation and modulation of AMPA (GluR1, GluR2, GluR3) and kainate (GluR6/7, KA2) receptor subunits by ultrasynaptic separation and immunoblot analysis of rat brain synaptosomes. All of the subunits were enriched in the postsynaptic fraction but were also present in the presynaptic and non-synaptic synaptosome fractions. AMPA stimulation resulted in a marked decrease in postsynaptic GluR2 and GluR3 subunits, but an increase in GluR6/7. Conversely, GluR2 and GluR3 increased in the presynaptic fraction whereas GluR6/7 decreased. There were no significant changes in any of the compartments for GluR1. NMDA treatment decreased postsynaptic GluR1, GluR2 and GluR6/7 but increased presynaptic levels of these subunits. NMDA treatment did not evoke changes in GluR3 localisation. Our results demonstrate that presynaptic and postsynaptic subunits are regulated in opposite directions by AMPA and NMDA stimulation.     

Gating motions underlie AMPA receptor secretion from the endoplasmic reticulum

Ion channel biogenesis involves an intricate interplay between subunit folding and assembly. Channel stoichiometries vary and give rise to diverse functions, which impacts on neuronal signalling. AMPA glutamate receptor (AMPAR) assembly is modulated by RNA processing. Here, we provide mechanistic insight into this process. First, we show that a single alternatively spliced residue within the ligand-binding domain alters AMPAR secretion from the ER. Local contacts differ between Leu758 of the GluR2-flop splice form as compared with the flip-specific Val758, which is transmitted globally to alter resensitization kinetics. Detailed biochemical and functional analysis of mutants suggest that AMPARs sample the gating cascade prior to ER export. Irreversibly locking the receptor within various states of the cascade severely attenuates ER transit. Alternative RNA processing by contrast, shifts equilibria between transition states reversibly and thereby modulates secretion kinetics. These data reveal how RNA processing tunes AMPAR biogenesis, and imply that gating transitions in the ER determine iGluR secretory traffic.     

AMPA and Kainate Receptors in Turtle Retina: An Immunocytochemical Study

Summary 1. Glutamate is one of the main neurotransmitters in the retina. Its effects are mediated by a large number of ionotropic and metabotropic receptors. 2. The distribution of ionotropic AMPA receptor subunits GluR1–4, kainate receptor subunits GluR5–7 and KA2, as well as delta receptors 1–2 was studied in turtle retina. Indirect immunofluorescence was used to localize the different receptor subunits viewed using light microscopy. 3. Results show that all subunits, with excerption of GluR1 and GluR5, are widely distributed in the turtle retina. 4. They are mainly located in the both plexiform layers of the retina where punctate staining, a sign for synaptic localization, is observed. 5. The vast majority of the subunits possess specific pattern of staining that allow to suppose that they are involved in different retinal circuits. 6. It can be assumed that the GluR2/3 and GluR6/7 subunits are expressed on the dendrites of a subpopulation of bipolar cells that are immunopositive for α-isoform of protein kinase C (PKCα). The GluR2/3 and GluR6/7 subunits are most probably used by the same PKCα immunopositive bipolar cells in their synaptic contacts with the third-order retinal neurons, the amacrine and ganglion cells.     

Lymphocyte activation and capping of hormone receptors

In this study both a ligand-dependent treatment [concanavalin A (Con A)] and a ligand-independent treatment [high-voltage pulsed galvanic stimulation (HVPGS)] have been used to initiate lymphocyte activation via a transmembrane signaling process. Our results show that both treatments cause the exposure of two different hormone [insulin and interleukin-2 (IL-2)] receptors within the first 5 min of stimulation. When either insulin or IL-2 is present in the culture medium, the stimulated lymphocytes undergo the following responses: (1) increased free intracellular Ca2+ activity; (2) aggregation of insulin or IL-2 receptors into patch/cap structures; (3) tyrosine-kinase-specific phosphorylation of a 32-kd membrane protein; and finally (4) induction of DNA synthesis. Further analysis indicates that hormone receptor capping is inhibited by (1) cytochalasin D, suggesting the involvement of microfilaments; (2) sodium azide, indicating a requirement for ATP production; and (3) W-5, W-7, and W-12 drugs, implying a need for Ca2+/calmodulin activity. Treatment with these metabolic or cytoskeletal inhibitors also prevents both the tyrosine-kinase-specific protein phosphorylation and DNA synthesis which normally follow hormone receptor capping. Double immunofluorescence staining shows that actomyosin, Ca2+/calmodulin, and myosin light-chain kinase are all closely associated with the insulin and IL-2 receptor cap structures. These findings strongly suggest that an actomyosin-mediated contractile system (regulated by Ca2+, calmodulin, and myosin light-chain kinase in an energy-dependent manner) is required not only for the collection of insulin and IL-2 receptors into patch and cap structures but also for the subsequent activation of tyrosine kinase and the initiation of DNA synthesis. We, therefore, propose that the exposure and subsequent patching/capping of at least one hormone receptor are required for the activation of mouse splenic T-lymphocytes.     

The regulation of AMPA receptor-binding sites

A wide variety of mechanisms have been identified that can regulate the α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)-receptor complex. Modulation has been shown to occur at the nucleic acid level via RNA editing and alternative splicing. At the posttranslational level, processes such as phosphorylation, glycosylation, chemical modification of reactive groups on the receptor proteins, interaction with a putative receptor-associated modulatory protein, and changes in the lipid environment have been reported to regulate receptor binding and function. In this review, we discuss general aspects of the cell biology, pharmacology, and function of AMPA receptors. In particular, we focus on some factors shown to modulate agonist binding and discuss possible molecular mechanisms underlying the regulation observed.     

Role of free fatty acid receptors in the regulation of energy metabolism

Free fatty acids (FFAs) are energy-generating nutrients that act as signaling molecules in various cellular processes. Several orphan G protein-coupled receptors (GPCRs) that act as FFA receptors (FFARs) have been identified and play important physiological roles in various diseases. FFA ligands are obtained from food sources and metabolites produced during digestion and lipase degradation of triglyceride stores. FFARs can be grouped according to ligand profiles, depending on the length of carbon chains of the FFAs. Medium- and long-chain FFAs activate FFA1/GPR40 and FFA4/GPR120. Short-chain FFAs activate FFA2/GPR43 and FFA3/GPR41. However, only medium-chain FFAs, and not long-chain FFAs, activate GPR84 receptor. A number of pharmacological and physiological studies have shown that these receptors are expressed in various tissues and are primarily involved in energy metabolism. Because an impairment of these processes is a part of the pathology of obesity and type 2 diabetes, FFARs are considered as key therapeutic targets. Here, we reviewed recently published studies on the physiological functions of these receptors, primarily focusing on energy homeostasis.