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Optimal expression of cloned NMDAR1/NMDAR2A heteromeric glutamate receptors: a biochemical characterization.

ABSTRACT: The N-methyl-D-aspartate R1 (NMDAR1) and NMDAR2A subunits were expressed transiently either alone or in combination in human embryonic kidney (HEK) 293 cells. The biochemical and pharmacological properties of the cloned receptors were compared with those of adult rat brain NMDA receptors using both immunological methods with a newly developed anti-NMDAR2A-(1435-1445) antibody and [3H]MK801 radioligand binding activity. Anti-NMDAR2A-(1435-1445) antibodies recognized specifically four immunoreactive species with M(r)s of 180,000, 122,000, 97,000 and 54,000 in rat brain, but only a single band of M(r) 180,000 in HEK 293 cells singly transfected with plasmid pCISNMDAR2A. N-deglycosylation of HEK cell membranes yielded a 165,000-M(r) immunoreactive species, which is in agreement with the size predicted from the cDNA sequence for the mature NMDAR2A subunit. Co-expression of NMDAR1 and NMDAR2A subunits in HEK 293 cells resulted in cell death. Thus conditions were established for the optimum expression of heteromeric receptors in viable cells, including a requirement for DL-2-amino-5-phosphonopentanoic acid (AP5) in the culture medium post-transfection. Cells transfected with pCISNMDAR1 and pCISNMDAR2A combined yielded a 10-fold increase in the number of [3H]MK801 binding sites compared with single subunit expression. MK801 had similar affinity for the expressed receptors as for those found in adult rat and mouse brain. These results demonstrate that the NMDAR1 and NMDAR2A receptor subunits co-assemble to form a heteromeric complex with properties similar to those of the native receptors of adult mammalian forebrain. Furthermore, the conditions reported for maximal transient expression provide a basis for further structure-activity studies.

SUBMITTER: Cik M

PROVIDER: S-EPMC1137775 | biostudies-other | 1993 Dec

REPOSITORIES: biostudies-other

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Project description:(1) UNC-49B and UNC-49C are gamma-aminobutyric acid (GABA) receptor subunits encoded by the Caenorhabditis elegans unc-49 gene. UNC-49B forms a homomeric GABA receptor, or can co-assemble with UNC-49C to form a heteromeric receptor. The pharmacological properties of UNC-49B homomers and UNC-49B/C heteromers were investigated in Xenopus oocytes. (2) The UNC-49 subunits are most closely related to the bicuculline- and benzodiazepine-insensitive RDL GABA receptors of insects. Consistent with this classification, bicuculline (10 micro M) did not inhibit, nor did diazepam (10 micro M) enhance UNC-49B homomeric or UNC-49B/C heteromeric receptors. (3) The UNC-49C subunit strongly affects the pharmacology of UNC-49B/C heteromeric receptors. UNC-49B homomers were much more picrotoxin sensitive than UNC-49B/C heteromers (IC(50)=0.9+/-0.2 micro M and 166+/-42 micro M, respectively). Pentobarbitone enhancement was greater for UNC-49B homomers compared to UNC-49B/C heteromers. Propofol (50 micro M) slightly enhanced UNC-49B homomers but slightly inhibited UNC-49B/C heteromers. Penicillin G (10 mM) inhibited UNC-49B homomers less strongly than UNC-49B/C heteromers (30% compared to 53% inhibition, respectively). (4) Several aspects of UNC-49 pharmacology were unusual. Picrotoxin sensitivity strongly correlates with dieldrin sensitivity, yet UNC-49B homomers were highly dieldrin resistant. The enhancing neurosteroid pregnanolone (5beta-pregnan-3alpha-ol-20-one; 10 micro M) strongly inhibited both UNC-49 receptors. Alphaxalone (10 micro M), another enhancing neurosteroid, did not affect UNC-49B homomers, but slightly inhibited UNC-49B/C heteromers. (5) UNC-49 subunits and mammalian GABA(A) receptor alpha, beta, and gamma subunit classes all share roughly the same degree of sequence similarity. Thus, although they are most similar to other invertebrate GABA receptors, the UNC-49 receptors share significant structural and pharmacological overlap with mammalian GABA(A) receptors.

Project description:BackgroundGlutamate racemase (MurI) is a cofactor-independent enzyme that is essential to the bacterial peptidoglycan biosynthesis pathway and has therefore been considered an attractive target for the development of antimicrobial drugs. While in our previous study the essentiality of the murI gene was shown in Streptococcus mutans, the primary aetiologic agent of human dental caries, studies on S. mutans MurI have not yet provided definitive results. This study aimed to produce and characterize the biochemical properties of the MurI from the S. mutans UA159 genome.MethodsStructure characterization prediction and multiple sequence alignment were performed by bioinformatic analysis. Recombinant His6-tagged S. mutans MurI was overexpressed in the expression vector pColdII and further purified using a Ni2+ affinity chromatography method. Protein solubility, purity and aggregation state were analyzed by SDS-PAGE, Western blotting, native PAGE and SEC-HPLC. Kinetic parameters were assessed by a circular dichroism (CD) assay. Kinetic constants were calculated based on the curve fit for the Michaelis-Menten equation. The effects of temperature and pH on enzymatic activity were determined by a series of coupled enzyme reaction mixtures.ResultsThe glutamate racemase gene from S. mutans UA159 was amplified by PCR, cloned and expressed in Escherichia coli BL21 (DE3). The 264-amino-acid protein, as a mixture of dimeric and monomeric enzymes, was purified to electrophoretic homogeneity. In the CD assay, S. mutans MurI displayed unique kinetic parameters (K m, d-Glu?l-Glu = 0.3631 ± 0.3205 mM, V max, d-Glu?l-Glu = 0.1963 ± 0.0361 mM min-1, k cat, d-Glu?l-Glu = 0.0306 ± 0.0065 s-1, k cat/K m, d-Glu?l-Glu = 0.0844 ± 0.0128 s-1 mM-1, with d-glutamate as substrate; K m, l-Glu?d-Glu = 0.8077 ± 0.5081 mM, V max, l-Glu?d-Glu = 0.2421 ± 0.0418 mM min-1, k cat , l - Glu?d-Glu = 0.0378 ± 0.0056 s-1, k cat/K m, l-Glu?d-Glu = 0.0468 ± 0.0176 s-1 mM-1, with l-glutamate as substrate). S. mutans MurI possessed an assay temperature optimum of 37.5 °C and its optimum pH was 8.0.ConclusionThe findings of this study provide insight into the structure and biochemical traits of the glutamate racemase in S. mutans and supply a conceivable guideline for employing glutamate racemase in anti-caries drug design.

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Optimal expression of cloned NMDAR1/NMDAR2A heteromeric glutamate receptors: a biochemical characterization.

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