Effect of transmembrane Ca2+ gradient on the coupling of β-adrenergic receptors and adenylyl cyclase

In order to investigate the effect of transmembrane Ca²⁺ gradient on G_s mediated coupling of -AR and adenylyl cyclase, -AR from duck erythrocytes and G_s and adenylyl cyclase from bovine brain cortices were co-reconstituted into asolectin liposomes with different transmembrane Ca²⁺ gradient. These proteoliposomes were proven to be impermeable to water-soluble substances. The results obtained indicate that a physiological transmembrane Ca^2– gradient (1000-fold) is essential for higher stimulation of adenylyl cyclase by hormone-activated -AR via coupling to G_s and can be further enhanced by the decrease of such Ca²⁺ gradient within certain range (100 fold) following Ca²⁺ influx into cells during signal transduction. Fluorescence polarization of DPH revealed that transmembrane Ca²⁺ gradient modulates adenylyl cyclase and its stimulation by hormones through mediating a change in lipid fluidity. Correspondent conformational changes of -AR were also detected from the fluorescence spectra and quenching of Acrylodan-labelled -AR in those proteoliposomes. It is suggested that a proper transmembrane Ca²⁺ gradient is essential for the optimal fluidity of the phospholipid bilayer in the proteoliposomes, which favors the formation of a suitable conformation of the reconstituted -AR and thus promotes the stimulation of adenylyl cyclase activities by hormone-activated -AR via Gs.Abbreviations ATP adenosine triphosphate - -AR -adrenergic receptors - AC adenylyl cyclase - DHA dihydroalprenolol - DPH diphenylhexatriene - [Ca²⁺]_i Ca²⁺ concentration inside proteoliposomes - [Ca²⁺]_o Ca²⁺ concentration outside proteoliposomes - cAMP cyclic adenosine monophosphate - DTT Dithiothreitol - FS fluorescein sulfonate - G_s Stimulatory GTP-binding protein - GTP guanosine triphosphate - GTPS guanosine 5-O-(3-thiotriphosphate) - kDa kilodalton - SDS sodium dodecyl sulfate - Tris N-tris(hydroxymethyl)aminomethane     

Characterization of the Effect of the Mitochondrial Protein Hint2 on?Intracellular Ca2+ dynamics

Hint2, one of the five members of the superfamily of the histidine triad AMP-lysine hydrolase proteins, is expressed in mitochondria of various cell types. In human adrenocarcinoma cells, Hint2 modulates Ca²⁺ handling by mitochondria. As Hint2 is highly expressed in hepatocytes, we investigated if this protein affects Ca²⁺ dynamics in this cell type. We found that in hepatocytes isolated from Hint2^−/− mice, the frequency of Ca²⁺ oscillations induced by 1 μM noradrenaline was 150% higher than in the wild-type. Using spectrophotometry, we analyzed the rates of Ca²⁺ pumping in suspensions of mitochondria prepared from hepatocytes of either wild-type or Hint2^−/− mice; we found that Hint2 accelerates Ca²⁺ pumping into mitochondria. We then resorted to computational modeling to elucidate the possible molecular target of Hint2 that could explain both observations. On the basis of a detailed model for mitochondrial metabolism proposed in another study, we identified the respiratory chain as the most probable target of Hint2. We then used the model to predict that the absence of Hint2 leads to a premature opening of the mitochondrial permeability transition pore in response to repetitive additions of Ca²⁺ in suspensions of mitochondria. This prediction was then confirmed experimentally.     

Impact of Ca2+ on structure of soybean CDPKβ and accessibility of the Tyr-24 autophosphorylation site

Several plant CDPKs were recently shown to be dual specificity kinases rather than Ser/Thr kinases as traditionally classified by sequence analysis. In the present study we confirm the autophosphorylation of recombinant soybean His₆-GmCDPKβ at the Tyr-24 site using sequence- and modification- specific antibodies. Homology modeling of soybean CDPKβ based on recent structures determined for several apicomplexan CDPKs suggested that phosphotyrosine-24 may be inaccessible to phosphatases. However, we report that dephosphorylation of CDPKβ by the protein tyrosine phosphatase 1B, PTP1B, was not restricted in the presence of calcium. Thus, despite conformational changes likely associated with calcium binding to the CDPKs, phosphotyrosine sites remain fully accessible to dephosphorylation suggesting the possibility of conformational breathing and flexing.     

Effect of Chlorpromazine on the Binding of Oestradiol-17β by Human Uterine Cytosol

Abstract

Chlorpromazine increases the binding of oestradiol-17β by human uterine cytosol in vitro. This effect is due to an increase in the number of receptor sites, and the dissociation constant for oestradiol-17β is unaffected by chlorpromazine.     

Interaction of Phospholipase A of the E. coli Outer Membrane with the Inhibitors of Eucaryotic Phospholipases A2 and Their Effect on the Ca2+-Induced Permeabilization of the Bacterial Membrane

Phospholipase A of the bacterial outer membrane (OMPLA) is a β-barrel membrane protein which is activated under various stress conditions. The current study examines interaction of inhibitors of eucaryotic phospholipases A₂—palmitoyl trifluoromethyl ketone (PACOCF₃) and aristolochic acid (AA)—with OMPLA and considers a possible involvement of the enzyme in the Ca²⁺-dependent permeabilization of the outer membrane of Escherichia coli. Using the method of molecular docking, it has been predicted that PACOCF₃ and AA bind to OMPLA at the same site and with the same affinity as the OMPLA inhibitors, hexadecanesulfonylfluoride and bromophenacyl bromide, and the substrate of the enzyme palmitoyl oleoyl phosphatidylethanolamine. It has also been shown that PACOCF₃, AA, and bromophenacyl bromide inhibit the Ca²⁺-induced temperature-dependent changes in the permeability of the bacterial membrane for the fluorescent probe propidium iodide and suppressed the transformation of E. coli cells with plasmid DNA induced by Ca²⁺ and heat shock. The cell viability was not affected by the eucaryotic phospholipases A₂ inhibitors. The study discusses a possible involvement of OMPLA in the mechanisms of bacterial transmembrane transport based on the permeabilization of the bacterial outer membrane.     

Effect of Quinoline-Type Antimalarial Drugs on the Binding of Oestradiol-17β and Progesterone by Rabbit and Human Uterine Cytosols

Abstract

Rabbit and human uterine cytosol, prepared and tested in phosphate buffer, bound less oestradiol-17β or progesterone than cytosol from the same source prepared and tested in Tris-HCl buffer. Dissociation constants were the same in both buffer systems, and the difference in binding was due to a difference in the number of binding sites. Three quinoline-type antimalarial drugs, chloroquine, quinine and mefloquine, and the quinoline derivative, 4-(4'-hydroxy-l'-methylbutylamino)-7-chloroquinoline, increased the steroid binding capacity of phosphate-buffered cytosol to that of Tris-buffered cytosol, the optimal concentration of quinoline derivative being 1.4–1.6 mM. Tris (50 mM) increased the binding capacity of phosphate-buffered cytosol to that of Tris-buffered cytosol. The effects of Tris and quinoline derivatives were not additive. By gel chromatography and sucrose density gradient centrifugation it was shown that the molecular size and sedimentation behaviour of the oestradiol and progesterone receptors were not affected by the quinoline derivatives. Two types of binding site are proposed, one requiring the presence of low molecular weight, basic compounds. The uterine levels of chloroquine attained by normal pharmacological doses of the drug are potentially capable of influencing the binding of oestradiol-17β and progesterone in the uterine cytosol.     

Effect of the Disease-Causing R266K Mutation on the Heme and PLP Environments of Human Cystathionine β-Synthase

Cystathionine β-synthase (CBS) is an essential pyridoxal 5'-phosphate (PLP)-dependent enzyme of the transsulfuration pathway that condenses serine with homocysteine to form cystathionine; intriguingly, human CBS also contains a heme b cofactor of unknown function. Herein we describe the enzymatic and spectroscopic properties of a disease-associated R266K hCBS variant, which has an altered hydrogen-bonding environment. The R266K hCBS contains a low-spin, six-coordinate Fe(III) heme bearing a His/Cys ligation motif, like that of WT hCBS; however, there is a geometric distortion that exists at the R266K heme. Using rR spectroscopy, we show that the Fe(III)-Cys(thiolate) bond is longer and weaker in R266K, as evidenced by an 8 cm(-1) downshift in the ν(Fe-S) resonance. Presence of this longer and weaker Fe(III)-Cys(thiolate) bond is correlated with alteration of the fluorescence spectrum of the active PLP ketoenamine tautomer. Activity data demonstrate that, relative to WT, the R266K variant is more impaired in the alternative cysteine-synthesis reaction than in the canonical cystathionine-synthesis reaction. This diminished cysteine synthesis activity and a greater sensitivity to exogenous PLP correlate with the change in PLP environment. Fe-S(Cys) bond weakening causes a nearly 300-fold increase in the rate of ligand switching upon reduction of the R266K heme. Combined, these data demonstrate cross talk between the heme and PLP active sites, consistent with previous proposals, revealing that alteration of the Arg(266)-Cys(52) interaction affects PLP-dependent activity and dramatically destabilizes the ferrous thiolate-ligated heme complex, underscoring the importance of this hydrogen-bonding residue pair.     

The Local Control of Cytosolic Ca2+ as a Propagator of CNS Communication—Integration of Mitochondrial Transport Mechanisms and Cellular Responses

Ca²⁺ signals propagate in wave form along individual cells of the central nervous system(CNS) and through networks of connected cells of neuronal and multiple glial cell types. Inorder for wave fronts to convey information, signaling mechanisms are required that allowwaves to propagate reproducibly and without decrement in signal strength over long distances.CNS Ca²⁺ waves are under specific integrated local control, made possible by interactions atlocal subcellular microdomains between endoplasmic reticulum and mitochondria. Activemitochondria located near the mouth of inositol trisphosphate receptor (InsP₃R) channel clustersin glia take up Ca²⁺, which may prevent a buildup of Ca²⁺ around the InsP₃R channel, therebydecreasing the rate of Ca²⁺-induced receptor inactivation, and prolonging channel open time.Mitochondria may amplify InsP;i3-dependent Ca2;pl signals by a transient permeability transitionin response to Ca²⁺ uptake into the mitochondrion. Other evidence suggests privileged accessinto mitochondria for Ca²⁺ entering neurons by glutamatergic receptor channels. This enablesspecific signal modulation as the Ca²⁺ wave is propagated into neurons, such that mitochondrialocated close to glutamate channels can prolong the neuronal cytosolic response time bysuccessive uptake and release of Ca²⁺. Disruption of mitochondrial function deregulates theability of CNS-derived cells to undergo normal Ca²⁺ signaling and wave propagation.     

Effect of the α-agonist noradrenaline on total and 45Ca2+ movements in mitochondria of rat liver cells

Ca²⁺ movements triggered by noradrenaline were determined in isolated cells and mitochondria from rat livers. It has been shown that these depend on experimental conditions. In cells incubated in 1.8mm-Ca²⁺, results suggest that noradrenaline mobilizes Ca²⁺ from reticulum before releasing Ca²⁺ from mitochondria.     

Effect of micromolar Ca2+ on NADH inhibition of bovine kidney α-ketoglutarate dehydrogenase complex and possible role of Ca2+ in signal amplification

Summary NADH inhibition of bovine kidney -ketoglutarate dehydrogenase complex was compared at 10 m free Ca²⁺ or in the absence of Ca²⁺ (i.e., < 1.0 nM free Ca²⁺). In the presence of Ca^2–, NADH inhibition was appreciably decreased for a wide range of NADH : NAD⁺ ratios. A half-maximal decrease in NADH inhibition occurred at slightly less than 1 m free Ca²⁺ (as determined with EGTA-Ca buffers). Of necessity this was observed on top of an effect of Ca²⁺ on the S_0.5 for -ketoglutarate which was decreased by Ca²⁺ with a half-maximal effect at a similar concentration. The effect of Ca²⁺ on NADH inhibition was not observed in assays of the dihydrolipoyl dehydrogenase component (using dihydrolipoamide as a substrate) or in assays of bovine kidney pyruvate dehydrogenase complex. This indicates that the overall reaction catalyzed by the -ketoglutarate dehydrogenase complex is required to elicit the effect of Ca²⁺ on NADH inhibition.At a fixed -ketoglutarate concentration (50 m), removal of Ca² reduced the activity of the -ketoglutarate dehydrogenase complex by 8,5-fold (due to an increase in S_0.5 for -ketoglutarate) and, in the presence of different NADH : NAD⁺ ratios, decreased the activity of the complex by 50 to 100-fold. Effects of the phosphate potential (ATP/ADPxP_i) or a combination of the phosphate potential and NADH :NAD⁺ ratio are also described. The possibility that the level of intramitochondrial free Ca²⁺ serves as a signal amplifier normally coupled to the energy state of mitochondria is discussed.     

Effects of the H6R and D7H Mutations on the Heparin-Dependent Modulation of Zinc-Induced Aggregation of Amyloid β

Molecular Biology - Zinc ions and glycosaminoglycans (GAGs) are found in amyloid deposits and are known to modulate the β-amyloid peptide (Аβ) aggregation, which is thought to be a...     

A Cytosolic Relay of Heat Shock Proteins HSP70-1A and HSP90β Monitors the Folding Trajectory of the Serotonin Transporter

Mutations in the C terminus of the serotonin transporter (SERT) disrupt folding and export from the endoplasmic reticulum. Here we examined the hypothesis that a cytosolic heat shock protein relay was recruited to the C terminus to assist folding of SERT. This conjecture was verified by the following observations. (i) The proximal portion of the SERT C terminus conforms to a canonical binding site for DnaK/heat shock protein of 70 kDa (HSP70). A peptide covering this segment stimulated ATPase activity of purified HSP70-1A. (ii) A GST fusion protein comprising the C terminus of SERT pulled down HSP70-1A. The interaction between HSP70-1A and SERT was visualized in live cells by Förster resonance energy transfer: it was restricted to endoplasmic reticulum-resident transporters and enhanced by an inhibitor that traps HSP70-1A in its closed state. (iv) Co-immunoprecipitation confirmed complex formation of SERT with HSP70-1A and HSP90β. Consistent with an HSP relay, co-chaperones (e.g. HSC70-HSP90-organizing protein) were co-immunoprecipitated with the stalled mutants SERT-R607A/I608A and SERT-P601A/G602A. (v) Depletion of HSP90β by siRNA or its inhibition increased the cell surface expression of wild type SERT and SERT-F604Q. In contrast, SERT-R607A/I608A and SERT-P601A/G602A were only rendered susceptible to inhibition of HSP70 and HSP90 by concomitant pharmacochaperoning with noribogaine. (vi) In JAR cells, inhibition of HSP90 also increased the levels of SERT, indicating that endogenously expressed transporter was also susceptible to control by HSP90β. These findings support the concept that the folding trajectory of SERT is sampled by a cytoplasmic chaperone relay.     

Differential Effect of High Extracellular Ca2+ on K+ and Cl− Conductances in Murine Osteoclasts

Effects of the extracellular Ca²⁺ concentration ([Ca²⁺] _o ) on whole cell membrane currents were examined in mouse osteoclastic cells generated from bone marrow/stromal cell coculture. The major resting conductance in the presence of 1 mm Ca²⁺ was mediated by a Ba²⁺-sensitive, inwardly rectifying K⁺ (IR_K) current. A rise in [Ca²⁺] _o (5–40 mm) inhibited the IR_K current and activated an 4,4′-diisothiocyano-2,2′-stilbenedisulfonate (DIDS)-sensitive, outwardly rectifying Cl⁻ (OR_Cl) current. The activation of the OR_Cl current developed slowly and needed higher [Ca²⁺] _o than that required to inhibit the IR_K current. The inhibition of the IR_K current consisted of two components, initial and subsequent late phases. The initial inhibition was not affected by intracellular application of guanosine 5′-O-(3-thiotriphosphate) (GTPγS) or guanosine 5′-O-(2-thiodiphosphate) (GDPβS). The late inhibition, however, was enhanced by GTPγS and attenuated by GDPβS, suggesting that GTP-binding proteins mediate this inhibition. The activation of the OR_Cl current was suppressed by pretreatment with pertussis toxin, but not potentiated by GTPγS. An increase in intracellular Ca²⁺ level neither reduced the IR_K current nor activated the OR_Cl current. Staurosporine, an inhibitor for protein kinase C, did not modulate the [Ca²⁺] _o -induced changes in the IR_K and OR_Cl conductances. These results suggest that high [Ca²⁺] _o had a dual action on the membrane conductance of osteoclasts, an inhibition of an IR_K conductance and an activation of an OR_Cl conductance. The two conductances modulated by [Ca²⁺] _o may be involved in different phases of bone resorption because they differed in Ca²⁺ sensitivity, temporal patterns of changes and regulatory mechanisms. Received: 28 May 1996/Revised: 28 January 1997     

Inverse Regulation of the Cytosolic Ca2+ Buffer Parvalbumin and Mitochondrial Volume in Muscle Cells via SIRT1/PGC-1α Axis

Skeletal muscles show a high plasticity to cope with various physiological demands. Different muscle types can be distinguished by the force, endurance, contraction/relaxation kinetics (fast-twitch vs. slow-twitch muscles), oxidative/glycolytic capacity, and also with respect to Ca²⁺-signaling components. Changes in Ca²⁺ signaling and associated Ca²⁺-dependent processes are thought to underlie the high adaptive capacity of muscle fibers. Here we investigated the consequences and the involved mechanisms caused by the ectopic expression of the Ca²⁺-binding protein parvalbumin (PV) in C2C12 myotubes in vitro, and conversely, the effects caused by its absence in in fast-twitch muscles of parvalbumin null-mutant (PV−/−) mice in vivo. The absence of PV in fast-twitch muscle tibialis anterior (TA) resulted in an increase in the peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) and of its positive regulator, the deacetylase sirtuin 1 (SIRT1). TA muscles from PV−/− mice also have an increased mitochondrial volume. Mild ionophore treatment of control (PV-devoid) C2C12 myotubes causing a moderate elevation in [Ca²⁺]_c resulted in an increase in mitochondrial volume, together with elevated PGC-1α and SIRT1 expression levels, whilst it increased PV expression levels in myotubes stably transfected with PV. In PV-expressing myotubes the mitochondrial volume, PGC-1α and SIRT1 were significantly lower than in control C2C12 myotubes already at basal conditions and application of ionophore had no effect on either one. SIRT1 activation causes a down-regulation of PV in transfected myotubes, whilst SIRT1 inhibition has the opposite effect. We conclude that PV expression and mitochondrial volume in muscle cells are inversely regulated via a SIRT1/PGC-1α signaling axis.     

New Molecular Determinant for Inactivation of the Human L-Type α1C Ca2+ Channel

Molecular cloning of the human fibroblast Ca²⁺ channel pore-forming α_1C subunit revealed (Soldatov, 1992. Proc. Natl. Acad. Sci. USA 89:4628-4632) a naturally occurring mutation g²²⁵⁴→ a that causes the replacement of the conservative alanine for threonine at the position 752 at the cytoplasmic end of transmembrane segment IIS6. Using stably transfected HEK293 cell lines, we have compared electrophysiological properties of the conventional α_1C,77 human recombinant L-type Ca²⁺ channel with those of its mutated isoform α_1C,94 containing the A752T replacement. Comparative quantification of steady-state availability of the current carried by α_1C,94 and α_1C,77 showed that A752T mutation prevented a large (≈25%) fraction of the current carried by Ca²⁺ or Ba²⁺ from fully inactivating. This mutation, however, did not appear to alter significantly the Ca²⁺-dependence and kinetics of decay of the inactivating fraction of the current or its voltage-dependence. The data suggests that Ala752 at the cytoplasmic end of IIS6 might serve as a molecular determinant of the Ca²⁺ channel inactivation, possibly regulating the voltage-dependence of its availability. Received: 14 January 2000/Revised: 20 June 2000     

Investigation of properties of the Ca2+ influx and of the Ca2+-activated K+ efflux (Gárdos effect) in vanadate-treated and ATP-depleted human red blood cells

In this study the properties of the 45Ca2+ influx in human red blood cells (RBC) induced by NaVO3 or ATP-depletion were compared. Both NaVO3-induced and ATP-depletion-induced 45Ca2+ influxes were in the range 10(-6)-10(-5) mol Ca2+ x l(-1)cells x h(-1). The saturatability of ATP-depletion-induced 45Ca2+ influx with Ca2+ was much less pronounced than that of NaVO3-induced 45Ca2+ influx. The NaVO3-induced Ca2+ influx was sensitive to nifedipine (IC50 = 50 micromol/l) and Cu2+ (IC50 = 9 micromol/l) but these inhibitors had only a marginal effect when ATP-depletion was used as the Ca2+ influx inducer. On the other hand, polymyxin B (PXB) (1-5 mg/ml) strongly stimulated the ATP-depletion-induced 45Ca2+ influx whereas its effect on the NaVO3-induced Ca2+ influx was biphasic, with about 10% stimulation at lower PXB concentrations and an inhibition of 40% at higher concentrations. SDS-PAGE revealed that both NaVO3 and PXB induced changes in the protein phosphorylation pattern in the presence of Ca2+. NaVO3 stimulated the phosphorylation of several proteins and this effect was counteracted by PXB. The comparison of the kinetics and temperature dependencies of the Gárdos effect induced by NaVO3 and the ATP-depletion showed marked differences. The ability of NaVO3 to induce the Gárdos effect dramatically increased in ATP-depleted cells. These findings indicate that the 45Ca2+ influxes preceding the activation of the Ca2+-activated K+ efflux (Gárdos effect) stimulated by NaVO3 and by ATP-depletion, are mediated by different transport pathways. In addition, obtained results demonstrate that ATP-depletion and NaVO3-treatment exert additive action in triggering the Gárdos effect.