Faculty Opinions recommendation of The unique N-terminal sequence of the BKCa channel α-subunit determines its modulation by β-subunits.

Oxidative stress may alter the functions of many proteins including the Slo1 large conductance calcium-activated potassium channel (BKCa). Previous results demonstrated that in the virtual absence of Ca2+, the oxidant chloramine-T (Ch-T), without the involvement of cysteine oxidation, increases the open probability and slows the deactivation of BKCa channels formed by human Slo1 (hSlo1) α subunits alone. Because native BKCa channel complexes may include the auxiliary subunit β1, we investigated whether β1 influences the oxidative regulation of hSlo1. Oxidation by Ch-T with β1 present shifted the half-activation voltage much further in the hyperpolarizing direction (−75 mV) as compared with that with α alone (−30 mV). This shift was eliminated in the presence of high [Ca2+]i, but the increase in open probability in the virtual absence of Ca2+ remained significant at physiologically relevant voltages. Furthermore, the slowing of channel deactivation after oxidation was even more dramatic in the presence of β1. Oxidation of cysteine and methionine residues within β1 was not involved in these potentiated effects because expression of mutant β1 subunits lacking cysteine or methionine residues produced results similar to those with wild-type β1. Unlike the results with α alone, oxidation by Ch-T caused a significant acceleration of channel activation only when β1 was present. The β1 M177 mutation disrupted normal channel activation and prevented the Ch-T–induced acceleration of activation. Overall, the functional effects of oxidation of the hSlo1 pore-forming α subunit are greatly amplified by the presence of β1, which leads to the additional increase in channel open probability and the slowing of deactivation. Furthermore, M177 within β1 is a critical structural determinant of channel activation and oxidative sensitivity. Together, the oxidized BKCa channel complex with β1 has a considerable chance of being open within the physiological voltage range even at low [Ca2+]i.

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Contribution of BKCa channels to vascular tone regulation by PDE3 and PDE4 is lost in heart failure

Cardiovascular Research ◽

10.1093/cvr/cvy161 ◽

2018 ◽

Vol 115 (1) ◽

pp. 130-144 ◽

Cited By ~ 2

Author(s):

Sarah Idres ◽

Germain Perrin ◽

Valérie Domergue ◽

Florence Lefebvre ◽

Susana Gomez ◽

...

Keyword(s):

Heart Failure ◽

Coronary Arteries ◽

Vascular Tone ◽

Cyclic Adenosine Monophosphate ◽

Adenosine Monophosphate ◽

Bkca Channel ◽

Adrenergic Stimulation ◽

Bkca Channels ◽

Α Subunit ◽

Pde Inhibitors

Abstract Aims Regulation of vascular tone by 3′,5′-cyclic adenosine monophosphate (cAMP) involves many effectors including the large conductance, Ca2+-activated, K+ (BKCa) channels. In arteries, cAMP is mainly hydrolyzed by type 3 and 4 phosphodiesterases (PDE3, PDE4). Here, we examined the specific contribution of BKCa channels to tone regulation by these PDEs in rat coronary arteries, and how this is altered in heart failure (HF). Methods and results Concomitant application of PDE3 (cilostamide) and PDE4 (Ro-20-1724) inhibitors increased BKCa unitary channel activity in isolated myocytes from rat coronary arteries. Myography was conducted in isolated, U46619-contracted coronary arteries. Cilostamide (Cil) or Ro-20-1724 induced a vasorelaxation that was greatly reduced by iberiotoxin (IBTX), a BKCa channel blocker. Ro-20-1724 and Cil potentiated the relaxation induced by the β-adrenergic agonist isoprenaline (ISO) or the adenylyl cyclase activator L-858051 (L85). IBTX abolished the effect of PDE inhibitors on ISO but did not on L85. In coronary arteries from rats with HF induced by aortic stenosis, contractility and response to acetylcholine were dramatically reduced compared with arteries from sham rats, but relaxation to PDE inhibitors was retained. Interestingly, however, IBTX had no effect on Ro-20-1724- and Cil-induced vasorelaxations in HF. Expression of the BKCa channel α-subunit, of a 98 kDa PDE3A and of a 80 kDa PDE4D were lower in HF compared with sham coronary arteries, while that of a 70 kDa PDE4B was increased. Proximity ligation assays demonstrated that PDE3 and PDE4 were localized in the vicinity of the channel. Conclusion BKCa channels mediate the relaxation of coronary artery induced by PDE3 and PDE4 inhibition. This is achieved by co-localization of both PDEs with BKCa channels, enabling tight control of cAMP available for channel opening. Contribution of the channel is prominent at rest and on β-adrenergic stimulation. This coupling is lost in HF.

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Human leucocyte glycosylasparaginase is an α/β-heterodimer of 19 kDa α-subunit and 17 and 18 kDa β-subunit

Biochemical Journal ◽

10.1042/bj3000541 ◽

1994 ◽

Vol 300 (2) ◽

pp. 541-544 ◽

Cited By ~ 1

Author(s):

O K Tollersrud ◽

T Heiskanen ◽

L Peltonen

Keyword(s):

Molecular Mass ◽

Reversed Phase ◽

Alpha Subunit ◽

Cross Linking ◽

Terminal Sequence ◽

Α Subunit ◽

Sds Page ◽

Specific Antisera ◽

Purification Scheme ◽

Chemical Cross Linking

Human lysosomal glycosylasparaginase (AGA; EC 3.5.1.26) consists of two glycosylated subunits, alpha and beta. Treatment with 3% SDS at 45 degrees C as part of a new purification scheme did not affect enzyme activity, but the alpha-subunit migrated an apparent 19 kDa peptide on SDS/PAGE instead of as a 24 kDa peptide, as observed without this SDS treatment. The N-terminal sequence was similar to that of the 24 kDa form, and, after reversed-phase h.p.l.c., the 19 kDa form was transformed to an apparent 24 kDa peptide on SDS/PAGE, indicating that their primary structures were identical. As the molecular mass of the alpha-subunit deduced from its cDNA was 19.5 kDa, the variation might be due to incomplete SDS coating of the 24 kDa form. This was confirmed by the tendency of the 24 kDa variant to polymerize even in the presence of SDS. The molecular mass of the beta-subunit was 17 and 18 kDa in accordance with previous reports. Chemical cross-linking with 1-ethyl-3-(3-dimethylaminopropyl)carbodi-imide resulted in the appearance of a 38 kDa peptide on SDS/PAGE which reacted with both the subunit-specific antisera on Western-blot analysis. On SDS/PAGE at pH 10.2 the active enzyme migrated as an apparent 43 kDa peptide. These results indicate that native human glycosylasparaginase is a heterodimer.

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A C-terminal sequence of soybean β-conglycinin α′ subunit acts as a vacuolar sorting determinant in seed cells

The Plant Journal ◽

10.1046/j.1365-313x.2003.01754.x ◽

2003 ◽

Vol 34 (5) ◽

pp. 647-659 ◽

Cited By ~ 53

Author(s):

Keito Nishizawa ◽

Nobuyuki Maruyama ◽

Ryohei Satoh ◽

Yoshihiro Fuchikami ◽

Takahiko Higasa ◽

...

Keyword(s):

Terminal Sequence ◽

Α Subunit ◽

Vacuolar Sorting ◽

Seed Cells

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Crotalus adamanteus phospholipase A2-α: Subunit structure, NH2-terminal sequence, and homology with other phospholipases

Archives of Biochemistry and Biophysics ◽

10.1016/0003-9861(75)90515-9 ◽

1975 ◽

Vol 167 (2) ◽

pp. 706-717 ◽

Cited By ~ 23

Author(s):

Francis H.C. Tsao ◽

Pamela S. Keim ◽

Robert L. Heinrikson

Keyword(s):

Phospholipase A2 ◽

Subunit Structure ◽

Terminal Sequence ◽

Α Subunit

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INHIBITION OF FSH-STIMULATED GRANULOSA CELL FUNCTION BY A SYNTHETIC FRAGMENT OF THE PORCINE INHIBIN α-SUBUNIT: EVIDENCE FOR INVOLVEMENT OF GnRH RECEPTORS

Journal of Endocrinology ◽

10.1677/joe.0.113r003 ◽

1987 ◽

Vol 113 (2) ◽

pp. R3-R5 ◽

Cited By ~ 10

Author(s):

S.G. Hillier ◽

C.G. Tsonis ◽

E.J. Wickings ◽

K.A. Eidne

Keyword(s):

Granulosa Cell ◽

Culture Medium ◽

Cell Function ◽

Specific Binding ◽

Female Rat ◽

Terminal Sequence ◽

Α Subunit ◽

Gnrh Receptors ◽

Synthetic Peptide Fragment ◽

Dose Dependent

ABSTRACT The bioactivity of a synthetic peptide fragment which mimics the N-terminal sequence of the 134-amino-acid porcine Inhibin α-subunit (pl- α1-26-Gly27Tyr28-OH) was tested and compared with the bioactivity of GnRH in rat granulosa cell cultures. Granulosa cells from immature female rat ovaries were cultured with hFSH and testosterone to stimulate the production of cyclic AMP, progesterone and oestradiol. Addition of pl- α1-26-Gly27Tyr28-OH to the culture medium caused a dose-dependent suppression of all three parameters (ID50 700-1,000 nmol/l). GnRH caused similar but higher-potency inhibition (ID50 2-4 nmol/l). Suppression of granulosa cell function by both peptides was fully reversible by a synthetic GnRH antagonist. Moreover, specific binding of the porcine inhibin fragment to ovarian GnRH receptors was demonstrated by radioreceptor assay. This is evidence that the porcine inhibin α-subunit fragment suppresses FSH-induced rat granulosa cell function via a mechanism of action similar to that of GnRH.

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Mutation of PKC phosphorylation site 1075T on the BKCa channel α subunit modulates BKCa channel activity

The FASEB Journal ◽

10.1096/fasebj.20.5.a1113-c ◽

2006 ◽

Vol 20 (5) ◽

Author(s):

Shu Zhu ◽

Richard E. White ◽

Darren Browning ◽

Yali Hou ◽

Mary L. Meadows ◽

...

Keyword(s):

Phosphorylation Site ◽

Bkca Channel ◽

Channel Activity ◽

Α Subunit

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Molecular Cloning, Nucleotide Sequence, and Expression of Genes Encoding a Polycyclic Aromatic Ring Dioxygenase from Mycobacterium sp. Strain PYR-1

Applied and Environmental Microbiology ◽

10.1128/aem.67.8.3577-3585.2001 ◽

2001 ◽

Vol 67 (8) ◽

pp. 3577-3585 ◽

Cited By ~ 156

Author(s):

Ashraf A. Khan ◽

Rong-Fu Wang ◽

Wei-Wen Cao ◽

Daniel R. Doerge ◽

David Wennerstrom ◽

...

Keyword(s):

Genomic Library ◽

Terminal Sequence ◽

Α Subunit ◽

Pah Degradation ◽

Expression Of Genes ◽

Genes Encoding ◽

Polycyclic Aromatic ◽

2D Gel ◽

Α Subunits ◽

Induced Proteins

ABSTRACT Mycobacterium sp. strain PYR-1 degrades high-molecular-weight polycyclic hydrocarbons (PAHs) primarily through the introduction of both atoms of molecular oxygen by a dioxygenase. To clone the dioxygenase genes involved in PAH degradation, two-dimensional (2D) gel electrophoresis of PAH-induced proteins from cultures of Mycobacterium sp. strain PYR-1 was used to detect proteins that increased after phenanthrene, dibenzothiophene, and pyrene exposure. Comparison of proteins from induced and uninduced cultures on 2D gels indicated that at least six major proteins were expressed (105, 81, 52, 50, 43, and 13 kDa). The N-terminal sequence of the 50-kDa protein was similar to those of other dioxygenases. A digoxigenin-labeled oligonucleotide probe designed from this protein sequence was used to screen dioxygenase-positive clones from a genomic library of Mycobacterium sp. strain PYR-1. Three clones, each containing a 5,288-bp DNA insert with three genes of the dioxygenase system, were obtained. The genes in the DNA insert, from the 5′ to the 3′ direction, were a dehydrogenase, the dioxygenase small (β)-subunit, and the dioxygenase large (α)-subunit genes, arranged in a sequence different from those of genes encoding other bacterial dioxygenase systems. Phylogenetic analysis showed that the large α subunit did not cluster with most of the known α-subunit sequences but rather with three newly described α subunits of dioxygenases from Rhodococcus spp. andNocardioides spp. The genes fromMycobacterium sp. strain PYR-1 were subcloned and overexpressed in Escherichia coli with the pBAD/ThioFusion system. The functionality of the genes for PAH degradation was confirmed in a phagemid clone containing all three genes, as well as in plasmid subclones containing the two genes encoding the dioxygenase subunits.

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Heat-Shock-Induced Proteins fromMyxococcus xanthus

Journal of Bacteriology ◽

10.1128/jb.183.21.6282-6287.2001 ◽

2001 ◽

Vol 183 (21) ◽

pp. 6282-6287 ◽

Cited By ~ 28

Author(s):

Mieko Otani ◽

Junko Tabata ◽

Toshiyuki Ueki ◽

Keiji Sano ◽

Sumiko Inouye

Keyword(s):

Heat Shock ◽

Ribosomal Protein ◽

Atp Synthase ◽

Terminal Sequence ◽

Protein Patterns ◽

Two Dimensional Gel Electrophoresis ◽

Α Subunit ◽

Ribosomal Protein S6 ◽

Alkyl Hydroperoxide ◽

Induced Proteins

ABSTRACT Optimal conditions for two-dimensional gel electrophoresis of total cellular proteins from Myxococcus xanthus were established. Using these conditions, we analyzed protein patterns of heat-shockedM. xanthus cells. Eighteen major spots and 15 minor spots were found to be induced by heat shock. From N-terminal sequences of 15 major spots, DnaK, GroEL, GroES, alkyl hydroperoxide reductase, aldehyde dehydrogenase, succinyl coenzyme A (CoA) synthetase, 30S ribosomal protein S6, and ATP synthase α subunit were identified. Three of the 18 major spots had an identical N-terminal sequence, indicating that they may be different forms of the same protein. Although a DnaK homologue, SglK, has been identified in M. xanthus (R. M. Weimer, C. Creghton, A. Stassinopoulos, P. Youderian, and P. L. Hartzell, J. Bacteriol. 180:5357–5368, 1998; Z. Yang, Y. Geng, and W. Shi, J. Bacteriol. 180:218–224, 1998), SglK was not induced by heat shock. In addition, there were seven substitutions within the N-terminal 30-residue sequence of the newly identified DnaK. This is the first report to demonstrate that succinyl CoA synthetase, 30S ribosomal protein S6, and ATP synthase α subunit are heat shock inducible.

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