Jahanshah Amin

Key features of heat shock regulatory elements

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.

Ketamine, But Not Phencyclidine, Selectively Modulates Cerebellar GABAA Receptors Containing α6 and δ Subunits

Phencyclidine (PCP) and ketamine are dissociative anesthetics capable of inducing analgesia, psychomimetic behavior, and a catatonic state of unconsciousness. Despite broad similarities, there are notable differences between the clinical actions of ketamine and PCP. Ketamine has a lower incidence of adverse effects and generally produces greater CNS depression than PCP. Both noncompetitively inhibit NMDA receptors, yet there is little evidence that these drugs affect GABAA receptors, the primary target of most anesthetics. α6β2/3δ receptors are subtypes of the GABAA receptor family and are abundantly expressed in granular neurons within the adult cerebellum. Here, using an oocyte expression system, we show that at anesthetically relevant concentrations, ketamine, but not PCP, modulates α6β2δ and α6β3δ receptors. Additionally, at higher concentrations, ketamine directly activates these GABAA receptors. Comparatively, dizocilpine (MK-801 [(+)-5-methyl-10,11-dihydro-5H-dibenzo [a,d] cyclohepten-5,10-imine maleate]), a potent noncompetitive antagonist of NMDA receptors that is structurally unrelated to PCP, did not produce any effect on α6β2δ receptors. Of the recombinant GABAA receptor subtypes examined (α1β2, α1β2γ2, α1β2δ, α4β2γ2, α4β2δ, α6β2γ2, α6β2δ, and α6β3δ), the actions of ketamine were unique to α6β2δ and α6β3δ receptors. In dissociated granule neurons and cerebellar slice recordings, ketamine potentiated the GABAergic conductance arising from α6-containing GABAA receptors, whereas PCP showed no effect. Furthermore, ketamine potentiation was absent in cerebellar granule neurons from transgenic functionally null α6−/− and δ−/−mice. These findings suggest that the higher CNS depressant level achieved by ketamine may be the result of its selective actions on α6β2/3δ receptors.

Sustained Nicotine Exposure Differentially Affects α3β2 and α4β2 Neuronal Nicotinic Receptors Expressed in Xenopus Oocytes

Abstract: To determine whether prolonged exposure to nicotine differentially affects α3β2 versus α4β2 nicotinic receptors expressed in Xenopus oocytes, oocytes were coinjected with subunit cRNAs, and peak responses to agonist, evoked by 0.7 or 7 µM nicotine for α4β2 and α3β2 receptors, respectively, were determined before and following incubation for up to 48 h with nanomolar concentrations of nicotine. Agonist responses of α4β2 receptors decreased in a concentration‐dependent manner with IC50 values in the 10 nM range following incubation for 24 h and in the 1 nM range following incubation for 48 h. In contrast, responses of α3β2 receptors following incubation for 24–48 h with 1,000 nM nicotine decreased by only 50–60%, and total ablation of responses could not be achieved. Attenuation of responses occurred within the first 5 min of nicotine exposure and was a first‐order process for both subtypes; half‐lives for inactivation were 4.09 and 2.36 min for α4β2 and α3β2 receptors, respectively. Recovery was also first‐order for both subtypes; half‐lives for recovery were 21 and 7.5 h for α4β2 and α3β2 receptors, respectively. Thus, the responsiveness of both receptors decreased following sustained exposure to nicotine, but α4β2 receptors recovered much slower. Results may explain the differential effect of sustained nicotine exposure on nicotinic receptor‐mediated neurotransmitter release.

Ecdysterone receptor is a sequence-specific transcription factor involved in the developmental regulation of heat shock genes.

Purification of ecdysterone receptor from Drosophila melanogaster to apparent homogeneity is reported. Purified receptor binds specifically to several sequences in the promoters of the developmentally active hsp27 and hsp23 heat shock genes that were previously implied in ecdysterone regulation of the genes and that share limited homology among themselves and with mammalian steroid receptor binding sites. Some of these elements confer ecdysterone regulation on a basal promoter in transfected cells, acting in a synergistic fashion. Transcription in vitro of promoters containing such elements is stimulated up to 100-fold by added purified ecdysterone receptor, depending on receptor dosage and the number of elements present. Transcriptional enhancement requires sequence-specific binding of receptor to template promoters which facilitates the formation of a preinitiation complex. Ecdysterone stimulates DNA binding of the receptor in vitro.

Insights into the activation mechanism of ρ1 GABA receptors obtained by coexpression of wild type and activation-impaired subunits

To gain insight into the activation mechanism of homomeric ligand-gated receptor—channels, we examined human homomeric ρ1 GABA receptors with fewer than the normal number of agonist binding sites. This was accomplished by coexpressing different ratios of wild type and activation-impaired ρ1 subunits. Dose—response relations from oocytes coexpressing wild type and mutant subunits were comprised of two components in terms of GABA sensitivity; one ‘wild type’-like and the other ‘mutant’-like. Applying the binomial hypothesis to subunit coassembly enabled us to correlate these two components of the GABA dose—response relations to the underlying chimaeric receptor subtypes. We demonstrate that the receptors activate near normal provided that they are comprised of at least three wild type subunits. Our data are consistent with five equivalent and independent GABA binding sites of which only three need bind GABA to open the pore. The two additional binding sites may increase the GABA sensitivity of the ρ1 receptor and, when bound by agonist, stabilize the open state.

Rat α6β2δ GABAA receptors exhibit two distinct and separable agonist affinities

The onset of motor learning in rats coincides with exclusive expression of GABAA receptors containing α6 and δ subunits in the granule neurons of the cerebellum. This development temporally correlates with the presence of a spontaneously active chloride current through α6‐containing GABAA receptors, known as tonic inhibition. Here we report that the coexpression of α6, β2, and δ subunits produced receptor–channels which possessed two distinct and separable states of agonist affinity, one exhibiting micromolar and the other nanomolar affinities for GABA. The high‐affinity state was associated with a significant level of spontaneous channel activity. Increasing the level of expression or the ratio of β2 to α6 and δ subunits increased the prevalence of the high‐affinity state. Comparative studies of α6β2δ, α1β2δ, α6β2γ2, α1β2γ2 and α4β2δ receptors under equivalent levels of expression demonstrated that the significant level of spontaneous channel activity is uniquely attributable to α6β2δ receptors. The pharmacology of spontaneous channel activity arising from α6β2δ receptor expression corresponded to that of tonic inhibition. For example, GABAA receptor antagonists, including furosemide, blocked the spontaneous current. Further, the neuroactive steroid 5α‐THDOC and classical glycine receptor agonists β‐alanine and taurine directly activated α6β2δ receptors with high potency. Specific mutation within the GABA‐dependent activation domain (βY157F) impaired both low‐ and high‐affinity components of GABA agonist activity in α6βY157Fδ receptors, but did not attenuate the spontaneous current. In comparison, a mutation located between the second and third transmembrane segments of the δ subunit (δR287M) significantly diminished the nanomolar component and the spontaneous activity. The possibility that the high affinity state of the α6β2δ receptor modulates the granule neuron activity as well as potential mechanisms affecting its expression are discussed.

Organization of the Drosophila melanogaster hsp70 heat shock regulation unit.

Expression from the Drosophila melanogaster hsp70 promoter was controlled by a regulatory unit that was composed of two sequence elements that resembled the heat shock consensus sequence. The unit functioned in both orientations and at different distances from downstream promoter sequences. Each element of the unit alone was essentially inactive. Association of two elements resulted in a dramatic increase of transcription from the hsp70 promoter. This synergistic effect was independent of the relative orientation of the elements and, to a large extent, of the distance between them. Duplication of a region containing only one element also yielded a highly active, heat-regulated promoter. Genes with three to five elements were three to four times more active than those with a single regulatory unit.

Rat alpha6beta2delta GABAA receptors exhibit two distinct and separable agonist affinities.

The onset of motor learning in rats coincides with exclusive expression of GABAA receptors containing α6 and δ subunits in the granule neurons of the cerebellum. This development temporally correlates with the presence of a spontaneously active chloride current through α6‐containing GABAA receptors, known as tonic inhibition. Here we report that the coexpression of α6, β2, and δ subunits produced receptor–channels which possessed two distinct and separable states of agonist affinity, one exhibiting micromolar and the other nanomolar affinities for GABA. The high‐affinity state was associated with a significant level of spontaneous channel activity. Increasing the level of expression or the ratio of β2 to α6 and δ subunits increased the prevalence of the high‐affinity state. Comparative studies of α6β2δ, α1β2δ, α6β2γ2, α1β2γ2 and α4β2δ receptors under equivalent levels of expression demonstrated that the significant level of spontaneous channel activity is uniquely attributable to α6β2δ receptors. The pharmacology of spontaneous channel activity arising from α6β2δ receptor expression corresponded to that of tonic inhibition. For example, GABAA receptor antagonists, including furosemide, blocked the spontaneous current. Further, the neuroactive steroid 5α‐THDOC and classical glycine receptor agonists β‐alanine and taurine directly activated α6β2δ receptors with high potency. Specific mutation within the GABA‐dependent activation domain (βY157F) impaired both low‐ and high‐affinity components of GABA agonist activity in α6βY157Fδ receptors, but did not attenuate the spontaneous current. In comparison, a mutation located between the second and third transmembrane segments of the δ subunit (δR287M) significantly diminished the nanomolar component and the spontaneous activity. The possibility that the high affinity state of the α6β2δ receptor modulates the granule neuron activity as well as potential mechanisms affecting its expression are discussed.

Genes for Drosophila small heat shock proteins are regulated differently by ecdysterone.

Genes for small heat shock proteins (hsp27 to hsp22) are activated in late third-instar larvae of Drosophila melanogaster in the absence of heat stress. This regulation has been simulated in cultured Drosophila cells in which the genes are activated by the addition of ecdysterone. Sequence elements (HERE) involved in ecdysterone regulation of the hsp27 and hsp23 genes have been defined by transfection studies and have recently been identified as binding sites for ecdysterone receptor. We report here that the hsp27 and hsp23 genes are regulated differently by ecdysterone. The hsp27 gene is activated rapidly by ecdysterone, even in the absence of protein synthesis. In contrast, high-level expression of the hsp23 gene begins only after a lag of about 6 h, is dependent on the continuous presence of ecdysterone, and is sensitive to low concentrations of protein synthesis inhibitors. Transfection experiments with reporter constructs show that this difference in regulation is at the transcriptional level. Synthetic hsp27 or hsp23 HERE sequences confer hsp27- or hsp23-type ecdysterone regulation on a basal promoter. These findings indicate that the hsp27 gene is a primary, and the hsp23 gene is mainly a secondary, hormone-responsive gene. Ecdysterone receptor is implied to play a role in the regulation of both genes.

Determinants for the DNase I-hypersensitive chromatin structure 5′ to a human hsp70 gene

A DNase I hypersensitive site was detected in chromatin formed over a human hsp70 gene segment after amplification in COS7 cells. Deletion mutant analysis was used to evaluate the sequence requirements for this chromatin structure. Determinants sufficient to form the hypersensitive site are contained in a 280 base-pair sequence corresponding approximately to the region that is hypersensitive. Deletion of sequences from either end of this region resulted in reduced hypersensitivity, suggesting that multiple genetic elements contribute to the formation of this chromatin structure. As has been reported for other heat shock genes, the hypersensitive chromatin structure is present prior to heat treatment and does not change in intensity or position after heat shock, in spite of the fact that hsp70 gene expression is completely dependent on heat induction. Sequence requirements for hypersensitivity were generally similar to those for heat-induced gene expression when mutant plasmids were tested at low copy number (e.g. in HeLa cells or in COS cells without amplification); however, deletion of sequences between -223 and -162 with respect to the start of transcription abolished the hypersensitive site but had no effect on gene expression. A barrier to exonuclease III digestion was detected within this region (near an imperfect inverted repeat sequence centered at position -202), suggesting that proteins are tightly bound to the DNA at this location.