Expression of Distinct α Subunits of GABAA Receptor Regulates Inhibitory Synaptic Strength

2004 ◽  
Vol 92 (3) ◽  
pp. 1718-1727 ◽  
Author(s):  
Pavel I. Ortinski ◽  
Congyi Lu ◽  
Kentaroh Takagaki ◽  
Zhanyan Fu ◽  
Stefano Vicini
Keyword(s):  
Primary Cultures ◽  
Molecular Assembly ◽  
Inhibitory Synapses ◽  
Critical Determinant ◽  
Pharmacological Agents ◽  
Α Subunit ◽  
Subunit Expression ◽  
Α Subunits ◽  
In Cells

Distinct α subunit subtypes in the molecular assembly of GABAA receptors are a critical determinant of the functional properties of inhibitory synapses and their modulation by a range of pharmacological agents. We investigated the contribution of these subunits to the developmental changes of inhibitory synapses in cerebellar granule neurons in primary cultures from wild-type and α1 subunit −/− mice. The decay time of miniature inhibitory postsynaptic currents (mIPSCs) halved between 6 days in vitro (DIV6) and DIV12. This was paralleled by the decrease of α2 and α3 subunits, the increase of α1 and α6 subunits expression at synapses, and changes in the action of selective α subunit modulators. A small but significant shortening of mIPSCs was observed with development in cells from −/− mice together with a decrease in the expression of α3 subunit. In contrast, the expression of α2 subunit at inhibitory synapses in −/− cells was significantly higher than in +/+ cells at DIV11-12. α5 subunit was not detected, and increased sensitivity to a selective α4/α6 subunit agonist suggests increased expression of extrasynaptic receptors in −/− mice. β2/β3 subunit expression and loreclezole sensitivity increased with development in +/+ but not in −/− cells, supporting the preferential association of the α1 with the β2 subunit. Synaptic charge transfer strongly decreased with development but was not different between cells in the +/+ and −/− groups until DIV11-12. Our results uncover a pattern of sequential expression of α subunits underlying the changes in functional efficacy of GABAergic networks with development.

scholarly journals Identification of an Essential Cytoplasmic Region of Interleukin-7 Receptor α Subunit in B-Cell Development

2018 ◽  
Vol 19 (9) ◽  
pp. 2522 ◽  
Author(s):  
Hirotake Kasai ◽  
Taku Kuwabara ◽  
Yukihide Matsui ◽  
Koichi Nakajima ◽  
Motonari Kondo
Keyword(s):  
B Cells ◽  
B Cell ◽  
Myeloid Cell ◽  
Cell Development ◽  
B Cell Development ◽  
Α Subunit ◽  
Interleukin 7 ◽  
In Cells

Interleukin-7 (IL-7) is essential for lymphocyte development. To identify the functional subdomains in the cytoplasmic tail of the IL-7 receptor (IL-7R) α chain, here, we constructed a series of IL-7Rα deletion mutants. We found that IL-7Rα-deficient hematopoietic progenitor cells (HPCs) gave rise to B cells both in vitro and in vivo when a wild-type (WT) IL-7Rα chain was introduced; however, no B cells were observed under the same conditions from IL-7Rα-deficient HPCs with introduction of the exogenous IL-7Rα subunit, which lacked the amino acid region at positions 414–441 (d414–441 mutant). Signal transducer and activator of transcription 5 (STAT5) was phosphorylated in cells with the d414–441 mutant, similar to that in WT cells, in response to IL-7 stimulation. In contrast, more truncated STAT5 (tSTAT5) was generated in cells with the d414–441 mutant than in WT cells. Additionally, the introduction of exogenous tSTAT5 blocked B lymphopoiesis but not myeloid cell development from WT HPCs in vivo. These results suggested that amino acids 414–441 in the IL-7Rα chain formed a critical subdomain necessary for the supportive roles of IL-7 in B-cell development.

Effects of crude and highly purified bovine inhibin (Mr 32 000 form) on gonadotrophin production by ovine pituitary cells in vitro: inhibin enhances gonadotrophin-releasing hormone-induced release of LH

1990 ◽  
Vol 127 (1) ◽  
pp. 149-159 ◽  
Author(s):  
S. Muttukrishna ◽  
P. G. Knight
Keyword(s):  
Primary Cultures ◽  
Suppressive Effect ◽  
Pituitary Cells ◽  
Dependent Manner ◽  
Α Subunit ◽  
Releasing Hormone ◽  
Lh Release ◽  
Gonadotrophin Releasing Hormone

ABSTRACT Primary cultures of ovine pituitary cells (from adult ewes) were used to investigate the actions of steroid-free bovine follicular fluid (bFF) and highly-purified Mr 32 000 bovine inhibin on basal and gonadotrophin-releasing hormone (GnRH)-induced release of FSH and LH. Residual cellular contents of each hormone were also determined allowing total gonadotrophin content/well to be calculated. As in rats, both crude and highly purified inhibin preparations promoted a dose (P < 0·001)- and time (P < 0·001)-dependent suppression of basal and GnRH-induced release of FSH as well as an inhibition of FSH synthesis, reflected by a fall in total FSH content/well. However, while neither inhibin preparation affected basal release of LH or total LH content/well, GnRH-induced LH release was significantly (P< 0·001) increased by the presence of either bFF (+ 75%) or highly-purified inhibin (+ 64%) in a dose- and time-dependent manner. This unexpected action of bFF on GnRH-induced LH release was abolished in the presence of 5 μl specific anti-inhibin serum, confirming that the response was indeed mediated by inhibin. Furthermore, neither oestradiol-17β (1 pmol/l–10 nmol/l) nor monomeric α-subunit of bovine inhibin (2·5–40 ng/ml) significantly affected basal or GnRH-induced release of LH. These in-vitro findings for the ewe lend support to a number of recent in-vivo observations and indicate that, in addition to its well-documented suppressive effect on the synthesis and secretion of FSH, inhibin may actually facilitate LH release in this species, in marked contrast to its action in the rat. Journal of Endocrinology (1990) 127, 149–159

Estrogen regulates β1-subunit expression in Ca2+-activated K+ channels in arteries from reproductive tissues

2005 ◽  
Vol 289 (4) ◽  
pp. H1417-H1427 ◽  
Author(s):  
Deepa Nagar ◽  
Xiao-tie Liu ◽  
Charles R. Rosenfeld
Keyword(s):  
Vascular Tone ◽  
Immunoblot Analysis ◽  
Uterine Blood Flow ◽  
Α Subunit ◽  
Uterine Arteries ◽  
Subunit Mrna ◽  
Reverse Transcription Pcr ◽  
Subunit Expression ◽  
Estradiol 17Β ◽  
Α Subunits

Daily estradiol-17β (E2β) increases basal uterine blood flow (UBF) and enhances acute E2β-mediated increases in UBF in ovariectomized nonpregnant ewes. The acute E2β-mediated rise in UBF involves vascular smooth muscle (VSM) large-conductance Ca2+-activated K+ channels (BKCa). BKCa consist of pore-forming α-subunits and regulatory β1-subunits that modulate channel function and E2β responsiveness. It is unclear whether E2β also alters subunit expression and thus channel density and/or function, thereby contributing to the rise in basal UBF and enhanced UBF responses that follow daily E2β. Therefore, we examined BKCa subunit expression by using reverse transcription-PCR and immunoblot analysis of arterial VSM from reproductive and nonreproductive tissues and myometrium from ovariectomized nonpregnant ewes after daily E2β (1 μg/kg iv) or vehicle without or with acute E2β (1 μg/kg). Tissue distribution was determined by immunohistochemistry. Acute E2β did not alter α- or β1-subunit expression in any tissue ( P > 0.1). Daily E2β also did not affect α-subunit mRNA or protein in any tissue ( P > 0.1) or mesenteric arterial VSM β1-subunit. However, daily E2β increased uterine and mammary arterial VSM β1-subunit mRNA by 32% and 83% ( P < 0.05), uterine VSM protein by 30%, and myometrial β1-subunit mRNA and protein by 74% ( P ≤ 0.005). Immunostaining of uterine arteries, myometrium, and intramyometrial arteries paralleled immunoblot analyses for both subunits. Although BKCa density is unaffected by daily and acute E2β, daily E2β increases β1-subunit in proximal and distal uterine arterial VSM. Thus prolonged E2β exposure may alter BKCa function, estrogen responsiveness, and basal vascular tone and reactivity in reproductive arteries by modifying α:β1 stoichiometry.

scholarly journals Role of the RNA Polymerase α Subunits in MetR-Dependent Activation of metE and metH: Important Residues in the C-Terminal Domain and Orientation Requirements within RNA Polymerase

2000 ◽  
Vol 182 (19) ◽  
pp. 5539-5550 ◽  
Author(s):  
Paula S. Fritsch ◽  
Mark L. Urbanowski ◽  
George V. Stauffer
Keyword(s):  
Rna Polymerase ◽  
Random Mutagenesis ◽  
Complex Surface ◽  
Α Subunit ◽  
C Terminus ◽  
Distinct Cluster ◽  
In Vitro Reconstitution ◽  
The Difference ◽  
Α Subunits

ABSTRACT Many transcription factors activate by directly interacting with RNA polymerase (RNAP). The C terminus of the RNAP α subunit (αCTD) is a common target of activators. We used both random mutagenesis and alanine scanning to identify αCTD residues that are crucial for MetR-dependent activation of metE and metH. We found that these residues localize to two distinct faces of the αCTD. The first is a complex surface consisting of residues important for α-DNA interactions, activation of both genes (residues 263, 293, and 320), and activation of either metE only (residues 260, 276, 302, 306, 309, and 322) or metH only (residues 258, 264, 290, 294, and 295). The second is a distinct cluster of residues important for metE activation only (residues 285, 289, 313, and 314). We propose that a difference in the location of the MetR binding site for activation at these two promoters accounts for the differences in the residues of α required for MetR-dependent activation. We have designed an in vitro reconstitution-purification protocol that allows us to specifically orient wild-type or mutant α subunits to either the β-associated or the β′-associated position within RNAP (comprising α2, β, β′, and ς subunits). In vitro transcriptions using oriented α RNAP indicate that a single αCTD on either the β- or the β′-associated α subunit is sufficient for MetR activation of metE, while MetR interacts preferentially with the αCTD on the β-associated α subunit at metH. We propose that the different αCTD requirements at these two promoters are due to a combination of the difference in the location of the activation site and limits on the rotational flexibility of the αCTD.

scholarly journals Crystallographic evidence for oxidative Met→Asp conversion in human hemoglobin

2014 ◽  
Vol 70 (a1) ◽  
pp. C310-C310
Author(s):  
Jayashree Soman ◽  
Michael Strader ◽  
Wayne Hicks ◽  
Tigist Kassa ◽  
Eileen Singleton ◽  
...  
Keyword(s):  
Electron Density ◽  
Dose Dependence ◽  
Side Chain ◽  
Β Subunit ◽  
Α Subunit ◽  
In The Wild ◽  
Ferryl Species ◽  
Coordinated Water ◽  
Α Subunits

The mutants HbA Bristol-Alesha (βV(E11)67M) and HbF Toms River (γV(E11)67M) [1,2] are examples of a `silent' posttranslational modification in which the side chain of the substituted amino acid is chemically modified (Met→Asp) resulting in a disparity between the DNA and protein sequences. In both cases the patients' hemolysate contained both V67M and V67D isoforms. But in the analogous α subunit mutant, Hb Evans αV(E11)62M, the conversion to Asp was not identified and DNA sequencing confirmed the Met replacement [3]. Our crystal structures of the three (ferrous) CO-bound recombinant V(E11)M mutants show the MetE11 side chain in similar conformations. But the air-oxidized β mutant crystals clearly showed a `bifurcated' and smaller electron density pattern for the E11 side chain, indicating the appearance of Asp. Also, the ligand electron-density at the iron atom in the oxidized β subunit appears to be an oxoferryl Fe4+=O rather than a Fe3+OH2 ferric complex. In contrast, there was little change in the electron density for αMetE11 in oxidized αV62M crystals. The ligand in the ferric α subunit is clearly a coordinated water molecule. But again, a ferryl Fe4+=O complex appears to occur in the wild-type β subunit. This strongly suggest that β subunits have a greater propensity to form highly reactive ferryl species, and that the ferryl species play a role in the Met→Asp conversion. Our autoxidation and proteomics studies showed that although all three recombinant VE11M mutants had similar, high rates of autooxidation and a strong H2O2 dose dependence on sulfoxide and sulfone formation, no Asp formation was detected in α subunits whereas MetE11 is converted to Asp to levels as high as 15% in vitro in β and γ subunits. We propose that the Met→Asp conversion specifically involves H2O2 mediated oxidation of the ferrous heme to an oxoferryl state, and because the transient ferryl intermediates are much less stable in the α subunits, there is no oxidative conversion.

N-Myristoylation is essential for protein phosphatases PPM1A and PPM1B to dephosphorylate their physiological substrates in cells

2013 ◽  
Vol 449 (3) ◽  
pp. 741-749 ◽  
Author(s):  
Toko Chida ◽  
Masakatsu Ando ◽  
Tasuku Matsuki ◽  
Yutaro Masu ◽  
Yuko Nagaura ◽  
...  
Keyword(s):  
Protein Phosphatase ◽  
Specific Activity ◽  
Wild Type ◽  
Α Subunit ◽  
Nitrophenyl Phosphate ◽  
Dependent Protein ◽  
Amp Activated Protein Kinase ◽  
Physiological Substrates ◽  
In Cells

PPM [metal-dependent protein phosphatase, formerly called PP2C (protein phosphatase 2C)] family members play essential roles in regulating a variety of signalling pathways. While searching for protein phosphatase(s) that act on AMPK (AMP-activated protein kinase), we found that PPM1A and PPM1B are N-myristoylated and that this modification is essential for their ability to dephosphorylate the α subunit of AMPK (AMPKα) in cells. N-Myristoylation was also required for two other functions of PPM1A and PPM1B in cells. Although a non-myristoylated mutation (G2A) of PPM1A and PPM1B prevented membrane association, this relocalization did not likely cause the decreased activity towards AMPKα. In in vitro experiments, the G2A mutants exhibited reduced activities towards AMPKα, but much higher specific activity against an artificial substrate, PNPP (p-nitrophenyl phosphate), compared with the wild-type counterparts. Taken together, the results of the present study suggest that N-myristoylation of PPM1A and PPM1B plays a key role in recognition of their physiological substrates in cells.

Isolation and characterization of distinct bioactive forms of LH from male buffalo pituitaries: differences localized to their α subunits

1994 ◽  
Vol 143 (2) ◽  
pp. 313-323 ◽  
Author(s):  
M R Sairam ◽  
A A H Zaky ◽  
A A Hassan
Keyword(s):  
Receptor Binding ◽  
Binding Activity ◽  
Α Subunit ◽  
Basic Fraction ◽  
Isolation And Characterization ◽  
Receptor Binding Activity ◽  
Acidic Fraction ◽  
Α Subunits

Abstract The isolation of highly purified forms of pituitary LH from Egyptian male (Nile) buffaloes is described. The total LH content (receptor binding activity) which was approximately 30 to 50 fold higher than FSH in the pituitary could be divided into three pools based upon fractionation patterns on a cation exchanger. The acidic fraction which also contained FSH was not purified to homogeneity. A basic fraction (bu-LH-2; 300 mg/kg anterior pituitary) and a very basic fraction (bu-LH-3; 80 mg/kg) were both highly purified and free of FSH activity as tested by specific FSH receptor and immunoassays. The basic buffalo LH fraction, bu-LH-2, was as active as highly purified ovine LH (oLH). The most basic form of buffalo LH, bu-LH-3, was, however, about twice as active as highly purified oLH in the in vitro bioassay using mouse Leydig tumour (MA-10) cells. In a receptor binding assay employing 125I-labelled buffalo LH (bu-LH-3) and porcine testicular membranes, the affinity of bu-LH-3 was about five times higher than purified oLH. The Mr of both forms of purified buffalo LH and subunits was similar to that of oLH. Amino acid composition of buffalo LH was also very similar to oLH except for small differences. Fractionation by fast protein liquid chromatography on Mono-Q columns revealed further evidence of microheterogeneity in each of the pools of buffalo LH with bu-LH-3 exhibiting a predominant single component. By reverse-phase high-pressure liquid chromotography analysis we have localized differences in the two purified isoforms of male buffalo LH to the α subunit. It is suggested that differences in biological potencies could be due to variations in terminal glycosylation and/or differences in branching of this subunit which is known to be important for signal transduction. Journal of Endocrinology (1994) 143, 313–323

scholarly journals Adrenoceptor-Mediated Elevation of Ambient GABA Levels Activates Presynaptic GABAB Receptors in Rat Sensorimotor Cortex

1997 ◽  
Vol 78 (1) ◽  
pp. 561-566 ◽  
Author(s):  
Ben D. Bennett ◽  
John R. Huguenard ◽  
David A. Prince
Keyword(s):  
Sensorimotor Cortex ◽  
Gaba Release ◽  
Gabab Receptors ◽  
Inhibitory Synapses ◽  
Pharmacological Agents ◽  
Physiological Conditions ◽  
Spontaneous Action Potential ◽  
Ambient Gaba ◽  
Spontaneous Action

Bennett, Ben D., John R. Huguenard, and David A. Prince. Adrenoceptor-mediated elevation of ambient GABA levels activates presynaptic GABAB receptors in rat sensorimotor cortex. J. Neurophysiol. 78: 561–566, 1997. At inhibitory synapses in the mature neocortex and hippocampus in vitro, spontaneous action-potential-dependent and -independent release of γ-aminobutyric acid (GABA) activates postsynaptic GABAA receptors but not pre- or postsynaptic GABAB receptors. Elevation of synaptic GABA levels with pharmacological agents or electrical stimulation can cause activation of GABAB receptors, but the physiological conditions under which such activation occurs need further elucidation. In rodent sensorimotor cortex, epinephrine produced a depression in the amplitude of evoked monosynaptic inhibitory postsynaptic currents (IPSCs) and a concomitant, adrenoceptor-mediated increase in the frequency of spontaneous IPSCs. Blockade of GABAB receptors prevented the depression of evoked IPSC amplitude by epinephrine but did not affect the increase in spontaneous IPSC frequency. These data show that adrenoceptor-mediated increases in spontaneous IPSCs can cause activation of presynaptic GABAB receptors and indirectly modulate impulse-related GABA release, presumably through elevation of synaptic GABA levels.

scholarly journals Human Type 3 3α-Hydroxysteroid Dehydrogenase (Aldo-Keto Reductase 1C2) and Androgen Metabolism in Prostate Cells

Endocrinology ◽  
2003 ◽  
Vol 144 (7) ◽  
pp. 2922-2932 ◽  
Author(s):  
Tea Lanišnik Rižner ◽  
Hsueh K. Lin ◽  
Donna M. Peehl ◽  
Stephan Steckelbroeck ◽  
David R. Bauman ◽  
...  
Keyword(s):  
Primary Cultures ◽  
Hydroxysteroid Dehydrogenase ◽  
Product Inhibition ◽  
High Rate ◽  
Retinol Dehydrogenase ◽  
Normal Prostate ◽  
Androgen Metabolism ◽  
Prostate Cells ◽  
In Cells

Abstract Human aldo-keto reductases (AKRs) of the AKR1C subfamily function in vitro as 3-keto-, 17-keto-, and 20-ketosteroid reductases or as 3α-, 17β-, and 20α-hydroxysteroid oxidases. These AKRs can convert potent sex hormones (androgens, estrogens, and progestins) into their cognate inactive metabolites or vice versa. By controlling local ligand concentration AKRs may regulate steroid hormone action at the prereceptor level. AKR1C2 is expressed in prostate, and in vitro it will catalyze the nicotinamide adenine dinucleotide (NAD+)-dependent oxidation of 3α-androstanediol (3α-diol) to 5α-dihydrotestosterone (5α-DHT). This reaction is potently inhibited by reduced NAD phosphate (NADPH), indicating that the NAD+: NADPH ratio in cells will determine whether AKR1C2 makes 5α-DHT. In transient COS-1-AKR1C2 and in stable PC-3-AKR1C2 transfectants, 5α-DHT was reduced by AKR1C2. However, the transfected AKR1C2 oxidase activity was insufficient to surmount the endogenous 17β-hydroxysteroid dehydrogenase (17β-HSD) activity, which eliminated 3α-diol as androsterone. PC-3 cells expressed retinol dehydrogenase/3α-HSD and 11-cis-retinol dehydrogenase, but these endogenous enzymes did not oxidize 3α-diol to 5α-DHT. In stable LNCaP-AKR1C2 transfectants, AKR1C2 did not alter androgen metabolism due to a high rate of glucuronidation. In primary cultures of epithelial cells, high levels of AKR1C2 transcripts were detected in prostate cancer, but not in cells from normal prostate. Thus, in prostate cells AKR1C2 acts as a 3-ketosteroid reductase to eliminate 5α-DHT and prevents activation of the androgen receptor. AKR1C2 does not act as an oxidase due to either potent product inhibition by NADPH or because it cannot surmount the oxidative 17β-HSD present. Neither AKR1C2, retinol dehydrogenase/3α-HSD nor 11-cis-retinol dehydrogenase is a source of 5α-DHT in PC-3 cells.

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