We have isolated a 1785-bp complementary DNA (cDNA) encoding the murine P2X U receptor subunit from NTW8 mouse microglial cells. The encoded protein has 80% and 85% homology to the human and rat P2X U subunits, respectively. Functional properties of the heterologously expressed murine P2X U homomeric receptor broadly resembled those of the P2X U receptor in the native cell line. However, marked phenotypic differences were observed between the mouse receptor, and the other P2X U receptor orthologues isolated with respect to agonist and antagonist potencies, and the kinetics of formation of the large aqueous pore.z 1998 Federation of European Biochemical Societies.
In the transmitter-gated ion channel class of receptors, the members of which are all believed to be heterooligomers, the number and arrangement of the subunits are only known with any certainty for the nicotinic acetylcholine receptor from Torpedo electric fish . That receptor has been shown to possess a pentameric rosette structure, with five homologous subunits (a2ßyS) arranged to enclose the central ion channel . The data were obtained by electron image analysis of two-dimensional receptor arrays, which form as a consequence of that receptor's exceptionally high abundance in the Torpedo membranes and are therefore not attainable for other receptors. We have applied another direct approach to determine the quaternary structure of native ionotropic GABA receptors. We have purified those receptors from porcine brain cortex and analysed the rotational symmetry of isolated receptors visualized by electron microscopy . The results show the receptor to have a pentameric structure with a central water-filled pore, which can now be said to be characteristic of the entire superfamily . Key Words: GABAA receptor-Electron microscopy-Image analysis-Quaternary structure. J. Neurochem . 62, 815-818 (1994) .
1 The aim of this study was to functionally characterize the recombinant mouse P2X 4 receptor and to compare its pharmacological properties with those of the human and rat orthologues. 2 Whole cell recordings were made from rafts of HEK-293 cells stably expressing recombinant mouse, rat or human P2X 4 receptors, using Cs-aspartate containing electrodes (3 ± 8 MO) in a HEPES-buered extracellular medium. 3 The agonist potency of ATP at the three species orthologues was similar, with mean EC 50 values of 2.3 mM, 1.4 mM and 5.5 mM, respectively. 4 Adenosine-5'-tetraphosphate (AP4) acted as a partial agonist with respect to ATP at the mouse and human P2X 4 receptors (EC 50 =2.6 and 3.0 mM), but was signi®cantly less potent at the rat orthologue (EC 50 =20.0 mM). a,b-methylene adenosine-5'-triphosphate (a,b-meATP) also acted as a partial agonist, producing 29% of the maximum response at the mouse P2X 4 and 24% at the human P2X 4 receptor. 5 In contrast to the other species orthologues, a,b-meATP failed to elicit a signi®cant agonist response at rat P2X 4 receptors, and was found to act as an antagonist, with an IC 50 of 4.6 mM, against 10 mM ATP. 6 Mouse P2X 4 receptors were found to be sensitive to the antagonist, pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) (IC 50 =10.5 mM), as were human P2X 4 receptors (IC 50 =9.6 mM). The rat receptor however, showed a low sensitivity to PPADS (IC 50 4100 mM). 7 All three orthologues were relatively suramin-insensitive (IC 50 4100 mM) and insensitive to 1-[N,O-Bis(5-isoquinoline sulphonyl)benzyl]-2-(4-phenylpiperazine)ethyl]-5-isoquinoline sulphonamide (KN-62; IC 50 43 mM). 8 Our results suggest that the pharmacological properties of the mouse receptor are most similar to the human P2X 4 receptor, and dier markedly from the rat receptor.
Polyclonal antibodies have been raised against the GABA/benzodiazepine receptor purified to homogeneity from bovine cerebral cortex in deoxycholate and Triton X-100 media. Radioimmunoassay was applied to measure specific antibody production using the 125I-labelled gamma-aminobutyric acid (GABA)/benzodiazepine receptor as antigen. The antibodies specifically immunoprecipitated the binding sites for [3H]muscimol and for [3H]flunitrazepam from purified preparations. In addition, when a 3-[(3-cholamidopropyl)dimethylammonio] 1-propanesulphonate (CHAPS) extract of bovine brain membranes was treated with the antibodies, those sites as well as the [3H]propyl-beta-carboline-3-carboxylate binding, the [35S]t-butylbicyclophosphorothionate binding (TBPS), the barbiturate-enhanced [3H]flunitrazepam binding, and the GABA-enhanced [3H]flunitrazepam binding were all removed together into the immunoprecipitate. Western blot experiments showed that these antibodies recognise the alpha-subunit of the purified GABA/benzodiazepine receptor. These results further support the existence in the brain of a single protein, the GABAA receptor, containing a set of regulatory binding sites for benzodiazepines and chloride channel modulators.
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