Structural insights into phenylethanolamines high-affinity binding site in NR2B from binding and molecular modeling studies

<p>Abstract</p> <p>Background</p> <p>Phenylethanolamines selectively bind to NR2B subunit-containing <it>N</it>-methyl-<it>D</it>-aspartate-subtype of ionotropic glutamate receptors and negatively modulate receptor activity. To investigate the structural and functional properties of the ifenprodil binding domain on the NR2B protein, we have purified a soluble recombinant rat NR2B protein fragment comprising the first ~400 amino acid amino-terminal domain (ATD2B) expressed in <it>E. coli</it>. Spectral measurements on refolded ATD2B protein demonstrated specific binding to ifenprodil. We have used site-directed mutagenesis, circular dichroism spectroscopy and molecular modeling to obtain structural information on the interactions between critical amino acid residues and ifenprodil of our soluble refolded ATD2B proteins. Ligand-induced changes in protein structure were inferred from changes in the circular dichroism spectrum, and the concentration dependence of these changes was used to determine binding constants for ifenprodil and its analogues.</p> <p>Results</p> <p>Ligand binding of ifenprodil, RO25,6981 and haloperidol on soluble recombinant ATD2B determined from circular dichroism spectroscopy yielded low-to-high micromolar equilibrium constants which concurred with functional IC₅₀measurement determined in heterologously expressed NR1/NR2B receptors in <it>Xenopus </it>oocytes. Amino acid residue substitutions of Asp101, Ile150 and Phe176 with alanine residue within the ATD2B protein altered the recombinant protein dissociation constants for ifenprodil, mirroring the pattern of their functional phenotypes. Molecular modeling of ATD2B as a clam-shell-like structure places these critical residues near a putative ligand binding site.</p> <p>Conclusion</p> <p>We report for the first time biochemical measurements show that the functional measurements actually reflect binding to the ATD of NR2B subunit. Insights gained from this study help advance the theory that ifenprodil is a ligand for the ATD of NR2B subunit.</p

UNDERSTANDING THE CLINICAL RELEVANCE OF MUTATIONS AFFECTING THE ANGIOTENSIN CONVERTING ENZYME

Ph.DDOCTOR OF PHILOSOPH

Medial Septum Modulates Cellular Response Induced in Hippocampus on Microinjection of Cholinergic Agonists into Hypothalamic Lateral Supramammillary Nucleus

Cholinergic mechanisms in supramammillary nucleus (SuM), especially the lateral SuM (lSuM) modulates septo-hippocampal neural activity. The lSuM, as compared to the contiguous medial SuM (mSuM) has relatively dense projections to hippocampus and cingulate cortex (Cg). In the present study, we have investigated whether the effects of cholinergic activation of SuM on hippocampal and cortical neural activities involve a cooperative interaction with the medial septum (MS). Microinjection of the broad-spectrum cholinergic agonist, carbachol, or the cholinergic-nicotinic receptor agonist, nicotine, into the lSuM and the mSuM in urethane anesthetized rat evoked a similar pattern of hippocampal theta rhythm. Despite that, only the lSuM microinjections resulted in an increase in expression of c-Fos-like immunoreactivity (c-Fos-ir) in neurons, including interneurons, of the ipsilateral hippocampus with a very dense expression in dentate gyrus. Likewise, a robust induction of c-Fos-ir was also observed in the ipsilateral Cg. Inhibition of the MS with muscimol pre-treatment attenuated both carbachol-evoked c-Fos-ir and theta activation. The findings indicate that cholinergic–nicotinic mechanisms in lSuM evoke not only neural activation via the ascending synchronizing pathway but also an MS-modulated expression of the plasticity-related molecule c-Fos in cortical regions that are strongly innervated by the lSuM

New insights into the not-so-new NR3 subunits of N-methyl-D-aspartate receptor: localization, structure, and function,”Molecular Pharmacology

Number of tables: 0 Number of references: 98 No. of words in Abstract: 152 Nonstandard Abbreviations: NMDA, N-methyl-D-aspartate; AMPA, α-amino-3-hydroxy-5-methyl-4-isoazolepropionic acid; kainite, 2-carboxy-3-carboxymethyl-4-isopropenylpyrrolidine; ATD, aminoterminal domain; CTD, cytoplasmic terminal domain; S1S2, ligand binding domain; RT-PCR, reverse transcription polymerase chain reaction; PSD, postsynaptic density; HEK, Human embryonic kidney; CNQX, 6-cyano-7-nitroquinoxaline-2,3-dione; DCS, D-cycloserine; ACPC, 1-aminocyclopropane-1-carboxylic acid; ACBC, 1-aminocyclobutane-1-carboxylic acid; SCAM, substituted cysteine accessibility method; 5,7-DCKA, 5,7-dichlorokynurenic acid; SP, signal peptide; aa, amino acids; bp, base pairs

Medial Septum Modulates Cellular Response Induced in Hippocampus on Microinjection of Cholinergic Agonists into Hypothalamic Lateral Supramammillary Nucleus

Yves Dauge est sénateur d’Indre et Loire (membre de la commission de la culture, de l'éducation et de la communication, membre du groupe d'études sur le patrimoine architectural). Inspecteur général de l’équipement, ancien maire de Chinon, il a été délégué interministériel à la ville et au développement social urbain de 1988 à 1991. Il a conduit la délégation française au Vème Forum Urbain Mondial à Rio. Il anime avec les services du Ministère des Affaires Etrangères et Européennes, le nouvea..

Medial Septum Modulates Cellular Response Induced in Hippocampus on Microinjection of Cholinergic Agonists into Hypothalamic Lateral Supramammillary Nucleus

10.3389/fnana.2017.00079Frontiers in Neuroanatomy117

Fluorogenic probes to monitor cytosolic phospholipase A(2) activity

10.1039/c6cc09305aCHEMICAL COMMUNICATIONS53111813-181