The predominant inhibitory neurotransmitter of the brain, GABA (gamma-aminobutyric acid), activates chloride-selective ion pores integral to the receptor complex. Subunits comprising the presumed hetero-pentameric GABA channel have been cloned, but little information is available on the domains important for activation. Rat wild-type or mutated alpha 1-, beta 2- and gamma 2-subunits (designated alpha, beta and gamma) were coexpressed in Xenopus oocytes and examined electrophysiologically. We report here the identification of two separate and homologous domains of the beta-subunit, each of which contributes a tyrosine and threonine essential for activation by GABA. Conservative substitution of each of these four amino acids dramatically decreased GABA channel sensitivity to activation by GABA and the GABA agonist muscimol. These substitutions, however, did not impair activation by the barbiturate pentobarbital, indicating these two different classes of agonists activate GABA channels through distinct mechanisms. We also present evidence suggesting that the two identified domains of the beta-subunit contribute a major component of the GABA receptor.

The promoters of heat shock protein genes are among the best-studied inducible eucaryotic promoters. Regions responsible for heat regulation have been identified previously by deletion experiments with several different heat shock genes. In this paper the critical importance of two novel features of heat shock regulatory elements was investigated. First, the elements were modular and, as a consequence, displayed a characteristic 5-nucleotide periodicity produced by multiple GAA blocks that were arranged in alternating orientations and at 2-nucleotide intervals. Functional heat shock regulatory elements appeared to include three or more of these blocks. Second, the nucleotides at the two positions immediately upstream from GAA segments played an important role in defining the competence of regulatory elements.Heat shock protein (hsp) genes occur in all cell types examined so far and are typically silent at the temperature of normal growth but are expressed at exceedingly high levels at elevated temperatures or in cells suffering from other types of stress (36). Drosophila melanogaster hsp70 genes encoding a major hsp of 70 kilodaltons (kDa) were the first hsp genes to be introduced into a variety of different cell types, such an NIH 3T3 cells (9), monkey COS cells (24, 30), Xenopus oocytes (3, 43), Drosophila cells (6,7,10,11,19,20), and others (see reference 27 for review). In most cell types, the genes were expressed in a heat-regulated fashion. A region located about 45 to 65 nucleotides upstream from the transcription start site of hsp70 genes was found to be essential for heat regulation in monkey cells and Xenopus oocytes (3,24,30). A second region containing related sequences, between about -65 and -90, was required in addition to the above region for high activity in Drosophila cells (1,11,39).Comparison of the -45 to -65 sequence of Drosophila hsp70 genes and of analogous sequences in other hsp genes led to the establishment of a heat shock consensus sequence, CNNGAANNTTCNNG, (31), where N is any nucleotide.Factors binding to this type of sequence have been identified (17,28,37,45,47,48), and two groups have purified such factors from Drosophila nuclear extracts and have shown that they are specifically involved in transcriptional activation of hsp genes (28,45,48).Since the sequences described above are the only elements specifically required for heat regulation and are also binding sites for hsp gene-specific transcription factors, they are of central importance for the understanding of heat shock regulation. Earlier studies in this laboratory (2) as well as by another group (40) had indicated that the heat shock consensus sequence that had been accepted for many years as the prototype heat shock regulatory sequence did not by itself function as a regulatory element. We have attempted here to define experimentally the nature of heat shock regulatory elements.MATERIALS AND METHODS Plasmid constructions. Plasmids ( Fig. 1 and 2) were derived from constructs D88 and D50 (2). In the latter constructs, D. m...

Benzodiazepines (BZs) act on gamma-aminobutyric acid type A (GABAA) receptors such as alpha1beta2gamma2 through key residues within the N-terminal region of alpha subunits, to render their sedative and anxiolytic actions. However, the molecular mechanisms underlying the BZs' other clinical actions are not known. Here we show that, with low concentrations of GABA, diazepam produces a biphasic potentiation for the alpha1beta2gamma2-receptor channel, with distinct components in the nanomolar and micromolar concentration ranges. Mutations at equivalent residues within the second transmembrane domains (TM2) of alpha, beta and gamma subunits, proven important for the action of other anesthetics, abolish the micromolar, but not the nanomolar component. Converse mutation of the corresponding TM2 residue and a TM3 residue within rho1 subunits confers diazepam sensitivity on homo-oligomeric rho1-receptor channels that are otherwise insensitive to BZs. Thus, specific and distinct residues contribute to a previously unresolved component (micromolar) of diazepam action, indicating that diazepam can modulate the GABAA-receptor channel through two separable mechanisms.

SUMMARYBenzodiazepines (BZs) exert their therapeutic effects in the mammalian central nervous system at least in part by modulating the activation of ␥-aminobutyric acid (GABA)-activated chloride channels. To gain further insight into the mechanism of action of BZs on GABA receptors, we have been investigating structural determinants required for the actions of the BZ diazepam (dzp) on recombinant ␣1␤2␥2 GABA A receptors. Sitedirected mutagenesis was used to introduce point mutations into the ␣1 and ␥2 GABA A receptor subunits. Wild-type and mutant GABA A receptors were then expressed in Xenopus laevis oocytes or human embryonic kidney 293 (HEK 293) cells and studied using two-electrode voltage-clamp and ligandbinding techniques. With this approach, we identified two tyrosine residues on the ␣1 subunit (Tyr159 and Tyr209) that when mutated to serine, dramatically impaired modulation by dzp. The Y209S substitution resulted in a Ͼ7-fold increase in the EC 50 for dzp, and the Y159S substitution nearly abolished dzp-mediated potentiation. Both of these mutations abolished binding of the high affinity BZ receptor antagonist [ 3 H]Ro 15-1788 to GABA A receptors expressed in HEK 293 cells. These tyrosine residues correspond to two tyrosines of the ␤2 subunit (Tyr157 and Tyr205) previously postulated to form part of the GABA-binding site. Mutation of the corresponding tyrosine residues on the ␥2 subunit produced only a slight increase in the EC 50 for dzp (ϳ2-fold) with no significant effect on the binding affinity of [3 H]Ro 15-1788. These data suggest that Tyr159 and Tyr209 of the ␣1 subunit may be components of the BZ-binding site on ␣1␤2␥2 GABA A receptors.BZs are frequently prescribed as anxiolytics, sedatives, anticonvulsants, and muscle relaxants (1-3). It is now generally accepted that these compounds exert their therapeutic effects, at least partly, by interacting with GABA A receptors in the brain (2-8). Thus, a substantial effort has been directed at understanding the molecular mechanism by which BZs modulate GABA A receptor function (9 -12).Molecular cloning studies (13-15) have revealed multiple classes and isoforms of GABA A receptor subunits in the mammalian brain (␣1-6, ␤1-4, ␥1-3, ␦). This diversity of ␣, ␤, and ␥ subunits allows the expression of a vast number of structurally unique GABA A receptor subtypes with distinct pharmacologies. Studies using exogenous expression, photoaffinity labeling, chimeric subunits, and site-directed mutagenesis have indicated that the ␣ subunit contributes a major component of the BZ-binding site and, depending on the subtype, can confer either BZ1 or BZ2 pharmacology on the GABA A receptor (16 -23). In particular, a histidine residue at position 101 (22) and a glycine residue at position 200 (21) have been implicated in BZ binding to the GABA receptor complex (Fig. 1).Although the ␣ subunit seems to form part of the BZbinding site, the presence of a ␥ subunit is essential for the normal modulatory actions of BZs on GABA A receptors (19,24,25; although see Ref. 26). The...