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A cDNA clone for human catechol-Omethyltransferase (hCOMT; S-adenosyl-L-methionine:catechol O-methyltransferase; EC 2.1.1.6) was isolated from a human hepatoma cell line (Hep G2) cDNA library by hybridization screening with a porcine cDNA probe. The cDNA clone was sequenced and found to have an insert of 1226 nucleotides. The deduced primary structure of hCOMT is composed of 271 amino acid residues with the predicted molecular mass of 30 kDa. At its N terminus it has a hydrophobic segment of 21 amino acid residues that may be responsible for insertion of hCOMT into the endoplasmic reticulum membrane. The primary structure of hCOMT exhibits high homology to the porcine partial cDNA sequence (93%). The deduced amino acid sequence contains two tryptic peptide sequences ( Catechol-O-methyltransferase (COMT; S-adenosyl-L-methionine; catechol O-methyltransferase; EC 2.1.1.6) is an enzyme that catalyzes the transfer of a methyl group from S-adenosyl-L-methionine to the m-hydroxy group of catecholamine neurotransmitters (dopamine, noradrenaline, adrenaline), their metabolites, and L-dopa, thereby inactivating them. The enzyme has a broad substrate specificity accepting as substrate also catechol steroids, a-methyldopa, and apomorphine (1). It is widely distributed in various cerebral and extracerebral tissues of all mammalian species (2), including erythrocytes (3). The occurrence of at least two distinct isoforms of COMT has been demonstrated, of which one is soluble (S-COMT) and the other membrane-bound

Two a subunits of the GABAA receptor in rat brain have been identified by molecular cloning. The deduced polypeptide sequences share major characteristics with other chemically gated ion channel proteins. One polypeptide represents the rat homologue of the a3 subunit previously cloned from bovine brain [14], while the other polypeptide is a yet unknown subunit, termed ct5. When coexpressed with the 81 subunit in Xenopus oocytes the receptors containing the a5 subunit revealed a higher sensitivity to GABA than receptors expressed from al + /I1 subunits or a3 + 81 subunits (K, = 1 PM, 13 PM and 14 PM, respectively). The a5 subunit was expressed only in a few brain areas such as cerebral cortex, hippocampal formation and olfactory bulb granular layer as shown by in situ hybridization histochemistry. Since the mRNA of the a5 subunit was colocalized with the al and a3 subunits only in cerebral cortex and in the hippocampal formation the a5 subunit may be part of distinct GABAA receptors in neuronal populations within the olfactory bulb.GABA* receptor heterogeneity; GABA* receptor isoform aS/fil; GABA, receptor isoform a3/,91; Hybridization histochemistry, in situ; Subunit expression; (Xenopus oocytes)

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An in situ hybridization technique was used to determine the distribution in rat brain of RNA homologous to cDNA clones encoding the a and .8 subunits of the rat brain GABAA yaminobutyrate receptor. The subunit proteins were mapped in adjacent sections autoradiographically and immmunohistochemically. Many brain areas containing high densities of GABAA receptors showed strong hybridization signals with both the a-and the 1subunit antisense RNA probe-e.g., cerebral cortex, hippocampus, and cerebellum. On a cellular level, a dense dendritic localization of GABAA receptors was correlated with a strong hybridization in the corresponding somata-e.g., in mitral cells of the olfactory bulb, pyramidal cells of hippocampus, granule cells of the dentate gyrus, and Purkinje and granule cells of the cerebellum. In some brain areas-e.g., substantia nigra--the intensity of the hybridization signal with the ,&subunit probe was much weaker than that with the a-subunit probe, whereas the inverse ratio of hybridization intensity was found in others-e.g., in bed nucleus. This regional heterogeneity in the hybridization pattern may reflect regional differences in RNA stability, transcription rate, or subunit composition. The results open the way for studies on the regulation of GABAA-receptor gene expression in normal and pathological brain in situ.The most abundant inhibitory neurotransmitter in the brain, ytaminobutyrate (GABA), facilitates chloride conductance of neuronal membranes by means ofGABAA receptors (1, 2). The function of GABAA receptors can be allosterically modulated, in particular by drugs acting at the benzodiazepine receptor (BZR) (3, 4); these drugs are widely used in the treatment of anxiety, insomnia, and epilepsy. The GABAA receptor complex is a heterooligomer consisting of a and p subunits (5-7) that not only contain the binding sites for GABA, BZR ligands, and other drugs (5-9) but also form the chloride channel (10).The identification of neurons that express the GABAA receptor is of therapeutic interest, since these cells propagate the drug effects elicited at the BZR. Such neurons have been identified electrophysiologically by their response to GABA and BZR ligands (1, 2). In addition, the distribution of the receptor protein helped to identify some GABA-recipient neurons (11, 12). However, an extensive mapping of neurons that express the GABAA receptor complex has not been possible. In situ hybridization histochemistry was therefore used to study the expression of mRNA homologous to the aand p-subunit cDNAs of the GABAA receptor in rat brain. The hybridization pattern was compared to the known regional, cellular, and subcellular distribution of the GABAA receptor protein in the rat central nervous system (11)(12)(13)(14).MATERIALS AND METHODS Preparation of Tissue. Male rats [specific pathogen-free (SPF) albino, Fullinsdorf, Switzerland] weighing 120-130 g were killed by transcardiac perfusion for 20-30 min with ice-cold phosphate-buffered saline containing as fixative either 4% (wt/vol) paraformal...