Phosphorylation of the GABAA receptor by cAMP-dependent protein kinase and by protein kinase C: Analysis of the substrate domain

Previous work has shown that the GABA_A-receptor (GABA_A-R) could be phosphorylated by cAMP-dependent protein kinase (PKA), protein kinase C (PKC), and a receptor associated kinase. However, no clear picture has yet emerged concerning the particular subunit subtypes of the GABA_A-R that were phosphorylated by PKA and PKC. In the present report we show that an antibody raised against a 23 amino acid polypeptide corresponding to a sequence in the putative intracellular loop of the 1 subunit of the receptor blocks the in vitro phosphorylation of the purified receptor by PKA and PKC. Moreover, N-terminal sequence analysis of the principal phosphopeptide fragment obtained after proteolysis of the receptor yielded a sequence that corresponds to the 3 subunit of the receptor. Such data provide additional support for our hypothesis (Browning et al., 1990, Proc. Natl. Acad. Sci. USA 87:1315–1317) that both PKA and PKC phosphorylate the -subunit of the GABA_A-R.Special issue dedicated to Dr. Paul Greengard.     

The influence of GABA on cells in the gustatory region of the hamster solitary nucleus

A brainstem slice preparation was used to investigate GABA-inducedresponses in the gustatory region of the nucleus of the solitarytract (NST) of the hamster. The baseline activities of 91 cellsin the rostral NST were examined extracellularly; 59 cells werelocated in the rostral central (RC), 21 in the rostral lateral(RL), six in the ventral (V) and five in the medial (M) subdivisionof the NST. Of the 80 cells in the gustatory region of the NST(RC and RL subdivisions), application of GABA produced dose-dependentinhibition in 55 (69%), excitation in 9 (11%) and no effectin 16 cells (22%). In contrast, only nine cells were responsiveto baclofen, a GABA^B agonist. In all subdivision of the rostralNST, 57 cells were inhibited by GABA and the responses of 48of these were blocked by the specific GABA^A antagonist, bicucullinemethiodide (BICM). Application of BICM alone often yielded anexcitatory burst of impulses; this effect was eliminated whensynaptic release was blocked by perfusion with a high magnesiumphysiological saline solution (PSS/Mg⁺⁺). The GABA^A-responsivecells were distributed predominantly within the RC subdivision,whereas the GABA^B-responsive neurons were mostly in the RL subdivisionof the NST. The influences of GABA on the membrane properties of cells withinthe gustatory region (RC and RL subdivisions) of the NST wererecorded using conventional intracellular (16 cells) or whole-cellpatch (17 cells) recording methods. Intracellular recordingrevealed that GABA produced hyperpolarisation of the membrane,decreased the firing frequency, and increased the membrane conductance.In the patch-clamp experiments, the application of GABA evokedboth inward and outward currents, and an increase in membraneconductance. The reversal potential produced by GABA was closeto the Cl– equilibrium potential. The effects of GABAwere blocked by BICM. These results suggest that (i) GABA hasa strong inhibitory influence on rostral NST neurons, whichin the majority of cells is mediated through GABA^A, receptors;and (ii) the gustatory region of the NST may contain a tonicallyactive GABAergic netw     

Circadian rhythms and glial cells of the central nervous system

Glial cells are the most abundant cells in the central nervous system and play crucial roles in neural development, homeostasis, immunity, and conductivity. Over the past few decades, glial cell activity in mammals has been linked to circadian rhythms, the 24-h chronobiological clocks that regulate many physiological processes. Indeed, glial cells rhythmically express clock genes that cell-autonomously regulate glial function. In addition, recent findings in rodents have revealed that disruption of the glial molecular clock could impact the entire organism. In this review, we discuss the impact of circadian rhythms on the function of the three major glial cell types – astrocytes, microglia, and oligodendrocytes – across different locations within the central nervous system. We also review recent evidence uncovering the impact of glial cells on the body's circadian rhythm. Together, this sheds new light on the involvement of glial clock machinery in various diseases.     

Determination of amide hydrogen exchange by mass spectrometry: a new tool for protein structure elucidation.

A new method based on protein fragmentation and directly coupled microbore high-performance liquid chromatography-fast atom bombardment mass spectrometry (HPLC-FABMS) is described for determining the rates at which peptide amide hydrogens in proteins undergo isotopic exchange. Horse heart cytochrome c was incubated in D2O as a function of time and temperature to effect isotopic exchange, transferred into slow exchange conditions (pH 2-3, 0 degrees C), and fragmented with pepsin. The number of peptide amide deuterons present in the proteolytic peptides was deduced from their molecular weights, which were determined following analysis of the digest by HPLC-FABMS. The present results demonstrate that the exchange rates of amide hydrogens in cytochrome c range from very rapid (k > 140 h-1) to very slow (k < 0.002 h-1). The deuterium content of specific segments of the protein was determined as a function of incubation temperature and used to indicate participation of these segments in conformational changes associated with heating of cytochrome c. For the present HPLC-FABMS system, approximately 5 nmol of protein were used for each determination. Results of this investigation indicate that the combination of protein fragmentation and HPLC-FABMS is relatively free of constraints associated with other analytical methods used for this purpose and may be a general method for determining hydrogen exchange rates in specific segments of proteins.     

Organization of the genes necessary for hydrogenase expression in Rhodobacter capsulatus. Sequence analysis and identification of two hyp regulatory mutants

A 25kbp DNA fragment from the chromosome of Rhodobacter capsulatus B10 carrying hydrogenase (hup) determinants was completely sequenced. Coding regions corresponding to 20 open reading frames were identified. The R. capsulatus hydrogenase-specific gene (hup and hyp) products bear significant structural identity to hydrogenase gene products from Escherichia coli (13), from Rhizobium liguminosarum (16), from Azotobacter vinelandii (10) and from Alcaligenes eutrophus (11). The sequential arrangement of the R. capsulatus genes is: hupR2-hupU-hypF -hupS-hupL-hupM-hupD -hupF -hupG -hupH -huoJ -hupK -hypA-hypB-hupR1-hypC -hypD -hypE -ORF19 -ORF20 , all contiguous and transcribed from the same DNA strand. The last two potential genes do not encode products that are related to identified hydrogenase-specific gene products in other species. The sequence of the 12 R. capsulatus genes underlined above is presented. The mutation site in two of the Hup^? mutants used in this study, RS13 and RCC12, was identified in the hypF gene (deletion of one G) and in the hypD qene (deletion of 54 bp), respectively. The hypF gene product shares 45% identity with the product of hydA from E. coli and the product of hypF from R. leguminosarum. Those products present at their N-terminus a Cys arrangement typical of zinc-finger proteins. The G deletion in the C-terminal region of hypF in the RS13 mutant