Altered mRNA Editing and Expression of Ionotropic Glutamate Receptors after Kainic Acid Exposure in Cyclooxygenase-2 Deficient Mice

Kainic acid (KA) binds to the AMPA/KA receptors and induces seizures that result in inflammation, oxidative damage and neuronal death. We previously showed that cyclooxygenase-2 deficient (COX-2−/−) mice are more vulnerable to KA-induced excitotoxicity. Here, we investigated whether the increased susceptibility of COX-2−/− mice to KA is associated with altered mRNA expression and editing of glutamate receptors. The expression of AMPA GluR2, GluR3 and KA GluR6 was increased in vehicle-injected COX-2−/− mice compared to wild type (WT) mice in hippocampus and cortex, whereas gene expression of NMDA receptors was decreased. KA treatment decreased the expression of AMPA, KA and NMDA receptors in the hippocampus, with a significant effect in COX-2−/− mice. Furthermore, we analyzed RNA editing levels and found that the level of GluR3 R/G editing site was selectively increased in the hippocampus and decreased in the cortex in COX-2−/− compared with WT mice. After KA, GluR4 R/G editing site, flip form, was increased in the hippocampus of COX-2−/− mice. Treatment of WT mice with the COX-2 inhibitor celecoxib for two weeks decreased the expression of AMPA/KA and NMDAR subunits after KA, as observed in COX-2−/− mice. After KA exposure, COX-2−/− mice showed increased mRNA expression of markers of inflammation and oxidative stress, such as cytokines (TNF-α, IL-1β and IL-6), inducible nitric oxide synthase (iNOS), microglia (CD11b) and astrocyte (GFAP). Thus, COX-2 gene deletion can exacerbate the inflammatory response to KA. We suggest that COX-2 plays a role in attenuating glutamate excitotoxicity by modulating RNA editing of AMPA/KA and mRNA expression of all ionotropic glutamate receptor subunits and, in turn, neuronal excitability. These changes may contribute to the increased vulnerability of COX-2−/− mice to KA. The overstimulation of glutamate receptors as a consequence of COX-2 gene deletion suggests a functional coupling between COX-2 and the glutamatergic system

The Biochemistry, Ultrastructure, and Subunit Assembly Mechanism of AMPA Receptors

The AMPA-type ionotropic glutamate receptors (AMPA-Rs) are tetrameric ligand-gated ion channels that play crucial roles in synaptic transmission and plasticity. Our knowledge about the ultrastructure and subunit assembly mechanisms of intact AMPA-Rs was very limited. However, the new studies using single particle EM and X-ray crystallography are revealing important insights. For example, the tetrameric crystal structure of the GluA2cryst construct provided the atomic view of the intact receptor. In addition, the single particle EM structures of the subunit assembly intermediates revealed the conformational requirement for the dimer-to-tetramer transition during the maturation of AMPA-Rs. These new data in the field provide new models and interpretations. In the brain, the native AMPA-R complexes contain auxiliary subunits that influence subunit assembly, gating, and trafficking of the AMPA-Rs. Understanding the mechanisms of the auxiliary subunits will become increasingly important to precisely describe the function of AMPA-Rs in the brain. The AMPA-R proteomics studies continuously reveal a previously unexpected degree of molecular heterogeneity of the complex. Because the AMPA-Rs are important drug targets for treating various neurological and psychiatric diseases, it is likely that these new native complexes will require detailed mechanistic analysis in the future. The current ultrastructural data on the receptors and the receptor-expressing stable cell lines that were developed during the course of these studies are useful resources for high throughput drug screening and further drug designing. Moreover, we are getting closer to understanding the precise mechanisms of AMPA-R-mediated synaptic plasticity

Structural features of phenol derivatives determining potency for activation of chloride currents via α(1) homomeric and α(1)β heteromeric glycine receptors

1. Phenol derivatives constitute a family of neuroactive compounds. The aim of our study was to identify structural features that determine their modulatory effects at glycine receptors. 2. We investigated the effects of four methylated phenol derivatives and two halogenated analogues on chloride inward currents via rat α(1) and α(1)β glycine receptors, heterologously expressed in HEK 293. 3. All compounds potentiated the effect of a submaximal glycine concentration in both α(1) homomeric and α(1)β glycine receptors. While the degree of maximum potentiation of the glycine 10 μM effect in α(1)β receptors was not different between the compounds, the halogenated compounds achieved half-maximum potentiating effects in the low μM range – at more than 20-fold lower concentrations compared with their nonhalogenated analogues (P<0.0001). The coactivating effect was over-ridden by inhibitory effects at concentrations >300 μM in the halogenated compounds. Neither the number nor the position of the methyl groups significantly affected the EC(50) for coactivation. 4. Only the bimethylated compounds 2,6 and 3,5 dimethylphenol (at concentrations >1000 μM) directly activated both α(1) and α(1)β receptors up to 30% of the maximum response evoked by 1000 μM glycine. 5. These results show that halogenation in the para position is a crucial structural feature for the potency of a phenolic compound to positively modulate glycine receptor function, while direct activation is only seen with high concentrations of compounds that carry at least two methyl groups. The presence of the β subunit is not required for both effects

Kinetics of desensitization and recovery from desensitization for human α4β2-nicotinic acetylcholine receptors stably expressed in SH-EP1 cells

AIM: Studies were conducted to define the kinetics of the onset of and recovery from desensitization for human α4β2-nicotinic acetylcholine receptors (nAChR) heterologously expressed in the SH-EP1 human epithelial cell line. METHODS: Whole-cell patch clamp recordings were performed to evaluate α4β2-nAChR currents. RESULTS: Application of 0.1 μmol/L nicotine or 1 mmol/L acetylcholine (ACh) for 1 s or longer induced two phases, with time constants of ∼70 and ∼700 ms, for the onset of α4β2-nAChR desensitization. For a given duration of agonist exposure, recovery from desensitization induced by nicotine was slower than recovery from ACh-induced desensitization. Comparisons with published reports indicate that time constants for the recovery of α4β2-nAChRs from desensitization are smaller than those for the recovery of human muscle-type nAChRs(1) from desensitization produced by the same concentrations and durations of exposure to an agonist. Moreover, the extent of human α4β2-nAChR desensitization and rate of recovery are the same, regardless of whether they are measured using whole-cell recording or based on published findings(2) using isotopic ion flux assays; this equality demonstrates the equivalent legitimacy of these techniques in the evaluation of nAChR desensitization. Perhaps most significantly, recovery from desensitization also was best fit to a biphasic process. Regardless of whether it was fit to single or double exponentials, however, half-times for recovery from desensitization grew progressively longer with an increased duration of agonist exposure during the desensitizing pulse. CONCLUSION: These findings indicate the existence of α4β2-nAChRs in many distinctive states of desensitization, as well as the induction of progressively deeper states of desensitization with the increased duration of agonist exposure