A cDNA encoding a novel member of the G-protein-coupled receptor (GCR) superfamily. an ATP receptor, has been isolated from an embryonic chtck whole brain cDNA hbrary by hybridizatton screenmg. The encoded protein has a sequence of 362 amino acids (41 kDa) and shares no more than 27% amino acid identity with any known GCR. When expressed as a complementary RNA (cRNA) m Xenopus oocytes a slowly-developing inward current was observed in response to application of ATP. The pharmacology of this expressed protem defines it as a PZv purinoceptor.

The genes of the ionotropic ␥-aminobutyric acid receptor (GABR) subunits have shown an unusual chromosomal clustering, but only now can this be fully specified by analyses of the human genome. We have characterized the genes encoding the 18 known human GABR subunits, plus one now located here, for their precise locations, sizes, and exon/intron structures. Clusters of 17 of the 19, distributed between five chromosomes, are specified in detail, and their possible significance is considered. By applying search algorithms designed to recognize sequences of all known GABRtype subunits in species from man down to nematodes, we found no new GABR subunit is detectable in the human genome. However, the sequence of the human orthologue of the rat GABR 3 receptor subunit was uncovered by these algorithms, and its gene could be analyzed. Consistent with those search results, orthologues of the ␤4 and ␥4 subunits from the chicken, not cloned from mammals, were not detectable in the human genome by specific searches for them. The relationships are consistent with the mammalian subunit being derived from the ␤ line and ⑀ from the ␥ line, with mammalian loss of ␤4 and ␥4. In their structures the human GABR genes show a basic pattern of nine coding exons, with six different genomic mechanisms for the alternative splicing found in various subunits. Additional noncoding exons occur for certain subunits, which can be regulatory. A dicysteine loop and its exon show remarkable constancy between all GABR subunits and species, of deduced functional significance.

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.

cDNAs encoding three splice variants of the P2X2 receptor were isolated from rat cerebellum. The first variant has a serine/proline-rich segment deleted from the intracellularly located carboxyl-terminal domain of the P2X2 subunit. The second and third variants have the splice site in the second half of the predicted first transmembrane domain. Either a 12-amino acid insertion or a six-amino acid deletion occurs at this position. cRNAs for these isoforms of the P2X2 subunit were injected into Xenopus laevis oocytes and tested for function. ATP evoked inward currents only with the splice variant [designated P2X2(b)] having the 69-amino acid deletion. The potencies of various agonists at the homomeric P2X2(b) receptor were not significantly different from those at the P2X2(a) homomeric channel. However, the P2X2(b) receptor showed significantly lower antagonist sensitivity. In contrast to the nondesensitizing P2X2(a) receptor, prolonged application of ATP produced a more rapid desensitization of the P2X2(b) receptor. When the P2X2(a) and P2X2(b) receptor responses were recorded in transfected mammalian cells, this difference was again found. The change in desensitization may be determined by proline/serine-rich segments and/or phosphorylation motifs that are removed from the tail region in formation of the P2X2(b) subunit. In situ hybridization of the three newly isolated isoforms of the P2X2 subunit was performed at the macroscopic and cellular levels; transcripts for two of them [P2X2(b) and p2x2(c)] but not the third [p2x2(d)], which carries the 12-amino acid addition, were present in many structures in the neonatal rat brain and on sensory and sympathetic ganglia. mRNA for the p2x2(d) splice variant was present only in the nodose ganglion, at a low level.