␥-Aminobutyric acid (GABA) type A receptors mediate fast inhibitory synaptic transmission and have been implicated in responses to sedative͞hypnotic agents (including neuroactive steroids), anxiety, and learning and memory. Using gene targeting technology, we generated a strain of mice deficient in the ␦ subunit of the GABA type A receptors. In vivo testing of various behavioral responses revealed a strikingly selective attenuation of responses to neuroactive steroids, but not to other modulatory drugs. Electrophysiological recordings from hippocampal slices revealed a significantly faster miniature inhibitory postsynaptic current decay time in null mice, with no change in miniature inhibitory postsynaptic current amplitude or frequency. Learning and memory assessed with fear conditioning were normal. These results begin to illuminate the novel contributions of the ␦ subunit to GABA pharmacology and sedative͞hypnotic responses and behavior and provide insights into the physiology of neurosteroids.

GABA A receptors are ligand-gated chloride ion channels that are presumed to be pentamers composed of ␣, ␤, and ␥ subunits. The subunit stoichiometry, however, is controversial, and the subunit arrangement presently is not known. In this study the ratio of subunits in recombinant ␣1␤3␥2 receptors was determined in Western blots from the relative signal intensities of antibodies directed against the N terminus or the cytoplasmic loop of different subunits after the relative reactivity of these antibodies had been determined with GABA A receptor subunit chimeras composed of the N-terminal domain of one and the remaining part of the other subunit. Via this method a subunit stoichiometry of two ␣ subunits, two ␤ subunits, and one ␥ subunit was derived. Similar experiments investigating the composition of ␣1␤3 receptors expressed on the surface of human embryonic kidney (HEK) 293 cells cotransfected with ␣1 and ␤3 subunits resulted in a stoichiometry of two ␣ and three ␤ subunits. Density gradient centrifugation studies indicated that combinations of ␣1␤3␥2 or ␣1␤3 subunits expressed in HEK 293 cells are able to form pentamers, whereas combinations of ␣1␥2 or ␤3␥2 subunits predominantly form heterodimers. These results provide valuable information on the mechanism of GABA A receptor assembly and support the conclusion that GABA A receptors are pentamers in which a total of four alternating ␣ and ␤ subunits are connected by a ␥ subunit. Key words: GABA A receptor; stoichiometry; assembly; subunit arrangement; human embryonic kidney 293 cells; chimeric subunits; density gradient centrifugation; Western blotGABA, the major inhibitory transmitter in the CNS, mediates fast synaptic inhibition by opening the chloride ion channel intrinsic to the GABA A receptor. This receptor is a hetero-oligomeric protein and the site of action of a variety of pharmacologically and clinically important drugs, such as benzodiazepines, barbiturates, steroids, anesthetics, and convulsants (Sieghart, 1995). So far, six ␣, three ␤, three ␥, one ␦, and two subunits of these receptors, as well as several alternatively spliced isoforms of some of these subunits, have been identified in mammalian brain (Macdonald and Olsen, 1994;Sieghart, 1995). Expression studies have indicated that an ␣, a ␤, and a ␥ subunit have to combine to produce GABA A receptors with a pharmacology resembling that of receptors found in the brain and that, depending on the subunits used for transfection of cells, receptors with distinct pharmacological and electrophysiological properties do arise (Sieghart, 1995). Overall it is assumed, however, that a total of five subunits have to combine to form functional GABA A receptors (Nayeem et al., 1994).A variety of subunit-specific antibodies has been raised to investigate the subunit composition of GABA A receptors. Immunocytochemical studies demonstrating the colocalization of subunits in GABA A receptor clusters on neuronal membranes (Fritschy et al., 1992;Caruncho and Costa, 1994;Fritschy and Möhler, 1995;Somogyi et al., 199...

The ability of peptide‐N4‐(N‐acetyl‐β‐glucosaminyl)asparagine amidase F (PNGase F) from Flavobacterium meningosepticum and PNGase A from sweet almonds to deglycosylate N‐glycopeptides and N‐glycoproteins from plants was compared. Bromelain glycopeptide and horseradish peroxidase‐C glycoprotein, which contain xylose linked β1 → 2 to β‐mannose and fucose linked α1 → 3 to the innermost N‐acetylglucosamine, were used as substrates. In contrast to PNGase A, the enzyme from F. meningosepticum did not act upon these substrates even at concentrations 100‐fold higher than required for complete deglycosylation of commonly used standard substrates. After removal of α1 → 3‐linked fucose from the plant glycopeptide and glycoprotein by mild acid hydrolysis, they were readily degraded by PNGase F at moderate enzyme concentrations. Hence we conclude that α1 → 3 fucosylation of the inner N‐acetylglucosamine impedes the enzymatic action of PNGase F. Knowledge of this limitation of the deglycosylation potential of PNGase F may turn it from a pitfall into a useful experimental tool.