Synexin and GTP increase surfactant secretion in permeabilized alveolar type II cells

We have previously suggested that synexin (annexin VII), a Ca(2+)-dependent phospholipid binding protein, may have a role in surfactant secretion, since it promotes membrane fusion between isolated lamellar bodies (the surfactant-containing organelles) and plasma membranes. In this study, we investigated whether exogenous synexin can augment surfactant phosphatidylcholine (PC) secretion in synexin-deficient lung epithelial type II cells. Isolated rat type II cells were cultured for 20-22 h with [(3)H]choline to label cellular PC. The cells were then treated with beta-escin, which forms pores in the cell membrane and releases cytoplasmic proteins including synexin. These cells, however, retained lamellar bodies. The permeabilized type II cells were evaluated for PC secretion during a 30-min incubation. Compared with PC secretion under basal conditions, the presence of Ca(2+) (up to 10 microM) did not increase PC secretion. In the presence of 1 microM Ca(2+), synexin increased PC secretion in a concentration-dependent manner, which reached a maximum at approximately 5 microg/ml synexin. The secretagogue effect of synexin was abolished when synexin was inactivated by heat treatment (30 min at 65 degrees C) or by treatment with synexin antibodies. GTP or its nonhydrolyzable analog beta:gamma-imidoguanosine-5'-triphosphate also increased PC secretion in permeabilized type II cells. The PC secretion was further increased in an additive manner when a maximally effective concentration of synexin was added in the presence of 1 mM GTP, suggesting that GTP acts by a synexin-independent mechanism to increase membrane fusion. Thus our results support a direct role for synexin in surfactant secretion. Our study also suggests that membrane fusion during surfactant secretion may be mediated by two independent mechanisms.     

Cation charge and size selectivity of the C2 domain of cytosolic phospholipase A(2).

C2 domains regulate numerous eukaryotic signaling proteins by docking to target membranes upon binding Ca(2+). Effective activation of the C2 domain by intracellular Ca(2+) signals requires high Ca(2+) selectivity to exclude the prevalent physiological metal ions K(+), Na(+), and Mg(2+). The cooperative binding of two Ca(2+) ions to the C2 domain of cytosolic phospholipase A(2) (cPLA(2)-alpha) induces docking to phosphatidylcholine (PC) membranes. The ionic charge and size selectivities of this C2 domain were probed with representative mono-, di-, and trivalent spherical metal cations. Physiological concentrations of monovalent cations and Mg(2+) failed to bind to the domain and to induce docking to PC membranes. Superphysiological concentrations of Mg(2+) did bind but still failed to induce membrane docking. In contrast, Ca(2+), Sr(2+), and Ba(2+) bound to the domain in the low micromolar range, induced electrophoretic mobility shifts in native polyacrylamide gels, stabilized the domain against thermal denaturation, and induced docking to PC membranes. In the absence of membranes, the degree of apparent positive cooperativity in binding of Ca(2+), Sr(2+), and Ba(2+) decreased with increasing cation size, suggesting that the C2 domain binds two Ca(2+) or Sr(2+) ions, but only one Ba(2+) ion. These stoichiometries were correlated with the abilities of the ions to drive membrane docking, such that micromolar concentrations of Ca(2+) and Sr(2+) triggered docking while even millimolar concentrations of Ba(2+) yielded poor docking efficiency. The simplest explanation is that two bound divalent cations are required for stable membrane association. The physiological Ca(2+) ion triggered membrane docking at 20-fold lower concentrations than Sr(2+), due to both the higher Ca(2+) affinity of the free domain and the higher affinity of the Ca(2+)-loaded domain for membranes. Kinetic studies indicated that Ca(2+) ions bound to the free domain are retained at least 5-fold longer than Sr(2+) ions. Moreover, the Ca(2+)-loaded domain remained bound to membranes 2-fold longer than the Sr(2+)-loaded domain. For both Ca(2+) and Sr(2+), the two bound metal ions dissociate from the protein-membrane complex in two kinetically resolvable steps. Finally, representative trivalent lanthanide ions bound to the domain with high affinity and positive cooperativity, and induced docking to PC membranes. Overall, the results demonstrate that both cation charge and size constraints contribute to the high Ca(2+) selectivity of the C2 domain and suggest that formation of a cPLA(2)-alpha C2 domain-membrane complex requires two bound multivalent metal ions. These features are proposed to stem from the unique structural features of the metal ion-binding site in the C2 domain.     

Strength of Ca(2+) binding to retinal lipid membranes: consequences for lipid organization

There is evidence that membranes of rod outer segment (ROS) disks are a high-affinity Ca(2+) binding site. We were interested to see if the high occurrence of sixfold unsaturated docosahexaenoic acid in ROS lipids influences Ca(2+)-membrane interaction. Ca(2+) binding to polyunsaturated model membranes that mimic the lipid composition of ROS was studied by microelectrophoresis and (2)H NMR. Ca(2+) association constants of polyunsaturated membranes were found to be a factor of approximately 2 smaller than constants of monounsaturated membranes. Furthermore, strength of Ca(2+) binding to monounsaturated membranes increased with the addition of cholesterol, while binding to polyunsaturated lipids was unaffected. The data suggest that the lipid phosphate groups of phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylserine (PS) in PC/PE/PS (4:4:1, mol/mol) are primary targets for Ca(2+). Negatively charged serine in PS controls Ca (2+) binding by lowering the electric surface potential and elevating cation concentration at the membrane/water interface. The influence of hydrocarbon chain unsaturation on Ca(2+) binding is secondary compared to membrane PS content. Order parameter analysis of individual lipids in the mixture revealed that Ca(2+) ions did not trigger lateral phase separation of lipid species as long as all lipids remained liquid-crystalline. However, depending on temperature and hydrocarbon chain unsaturation, the lipid with the highest chain melting temperature converted to the gel state, as observed for the monounsaturated phosphatidylethanolamine (PE) in PC/PE/PS (4:4:1, mol/mol) at 25 degrees C.     

Homeostatic control of slow vacuolar channels by luminal cations and evaluation of the channel-mediated tonoplast Ca2+ fluxes in situ

Ca(2+), Mg(2+), and K(+) activities in red beet (Beta vulgaris L.) vacuoles were evaluated using conventional ion-selective microelectrodes and, in the case of Ca(2+), by non-invasive ion flux measurements (MIFE) as well. The mean vacuolar Ca(2+) activity was approximately 0.2 mM. Modulation of the slow vacuolar (SV) channel voltage dependence by Ca(2+) in the absence and presence of other cations at their physiological concentrations was studied by patch-clamp in excised tonoplast patches. Lowering pH at the vacuolar side from 7.5 to 5.5 (at zero vacuolar Ca(2+)) did not affect the channel voltage dependence, but abolished sensitivity to luminal Ca(2+) within a physiological range of concentrations (0.1-1.0 mM). Aggregation of the physiological vacuolar Na(+) (60 mM) and Mg(2+) (8 mM) concentrations also results in the SV channel becoming almost insensitive to vacuolar Ca(2+) variation in a range from nanomoles to 0.1 mM. At physiological cation concentrations at the vacuolar side, cytosolic Ca(2+) activates the SV channel in a voltage-independent manner with K(d)=0.7-1.5 microM. Comparison of the vacuolar Ca(2+) fluxes measured by both the MIFE technique and from estimating the SV channel activity in attached patches, suggests that, at resting membrane potentials, even at elevated (20 microM) cytosolic Ca(2+), only 0.5% of SV channels are open. This mediates a Ca(2+) release of only a few pA per vacuole (approximately 0.1 pA per single SV channel). Overall, our data suggest that the release of Ca(2+) through SV channels makes little contribution to a global cytosolic Ca(2+) signal.     

Ca(2+) and membrane binding to annexin 3 modulate the structure and dynamics of its N terminus and domain III

Annexin 3 (ANX A3) represents approximately 1% of the total protein of human neutrophils and promotes tight contact between membranes of isolated specific granules in vitro leading to their aggregation. Like for other annexins, the primary molecular events of the action of this protein is likely its binding to negatively charged phospholipid membranes in a Ca(2+)-dependent manner, via Ca(2+)-binding sites located on the convex side of the highly conserved core of the molecule. The conformation and dynamics of domain III can be affected by this process, as it was shown for other members of the family. The 20 amino-acid, N-terminal segment of the protein also could be affected and also might play a role in the modulation of its binding to the membranes. The structure and dynamics of these two regions were investigated by fluorescence of the two tryptophan residues of the protein (respectively, W190 in domain III and W5 in the N-terminal segment) in the wild type and in single-tryptophan mutants. By contrast to ANX A5, which shows a closed conformation and a buried W187 residue in the absence of Ca(2+), domain III of ANX A3 exhibits an open conformation and a widely solvent-accessible W190 residue in the same conditions. This is in agreement with the three-dimensional structure of the ANX A3-E231A mutant lacking the bidentate Ca(2+) ligand in domain III. Ca(2+) in the millimolar concentration range provokes nevertheless a large mobility increase of the W190 residue, while interaction with the membranes reduces it slightly. In the N-terminal region, the W5 residue, inserted in the central pore of the protein, is weakly accessible to the solvent and less mobile than W190. Its amplitude of rotation increases upon binding of Ca(2+) and returns to its original value when interacting with membranes. Ca(2+) concentration for half binding of the W5A mutant to negatively charged membranes is approximately 0.5 mM while it increases to approximately 1 mM for the ANX A3 wild type and to approximately 3 mM for the W190 ANX A3 mutant. In addition to the expected perturbation of the W190 environment at the contact surface between the protein and the membrane bilayer, binding of the protein to Ca(2+) and to membranes modulates the flexibility of the ANX A3 hinge region at the opposite of this interface and might affect its membrane permeabilizing properties.     

Characterization of Na/Ca exchange in plasmalemmal vesicles from zona fasciculata cells of the bovine adrenal gland

The presence of an Na/Ca exchange system in fasciculata cells of the bovine adrenal gland was tested using isolated plasmalemmal vesicles. In the presence of an outwardly Na(+) gradient, Ca(2+) uptake was about 2-fold higher than in K(+) condition. Li(+) did not substitute for Na(+) and 5 mM Ni(2+) inhibited Ca(2+) uptake. Ca(2+) efflux from Ca(2+)-loaded vesicles was Na(+)-stimulated and Ni(2+)-inhibited. The saturable part of Na(+)-dependent Ca(2+) uptake displayed Michaelis-Menten kinetics. The relationship of Na(+)-dependent Ca(2+) uptake versus intravesicular Na(+) concentration was sigmoid (apparent K(0.5) approximately 24 mM; Hill number approximately 3) and Na(+) acted on V(max) without significant effect on K(m). Na(+)-stimulated Ca(2+) uptake was temperature-dependent (apparent Q(10) approximately 2.2). The inhibition properties of several divalent cations (Cd(2+), Sr(2+), Ni(2+), Ba(2+), Mn(2+), Mg(2+)) were tested and were similar to those observed in kidney basolateral membrane. The above results indicate the presence of an Na/Ca exchanger located on plasma membrane of zona fasciculata cells of bovine adrenal gland. This exchanger displays similarities with that of renal basolateral cell membrane.     

fMLP-induced arachidonic acid release in db-cAMP-differentiated HL-60 cells is independent of phosphatidylinositol-4, 5-bisphosphate-specific phospholipase C activation and cytosolic phospholipase A(2) activation

In inflammatory cells, agonist-stimulated arachidonic acid (AA) release is thought to be induced by activation of group IV Ca(2+)-dependent cytosolic phospholipase A(2) (cPLA(2)) through mitogen-activated protein kinase (MAP kinase)- and/or protein kinase C (PKC)-mediated phosphorylation and Ca(2+)-dependent translocation of the enzyme to the membrane. Here we investigated the role of phospholipases in N-formylmethionyl-l-leucyl-l-phenylalanine (fMLP; 1 nM-10 microM)-induced AA release from neutrophil-like db-cAMP-differentiated HL-60 cells. U 73122 (1 microM), an inhibitor of phosphatidyl-inositol-4,5-biphosphate-specific phospholipase C, or the membrane-permeant Ca(2+)-chelator 1, 2-bis?2-aminophenoxy?thane-N,N,N',N'-tetraacetic acid (10 microM) abolished fMLP-mediated Ca(2+) signaling, but had no effect on fMLP-induced AA release. The protein kinase C-inhibitor Ro 318220 (5 microM) or the inhibitor of cPLA(2) arachidonyl trifluoromethyl ketone (AACOCF(3); 10-30 microM) did not inhibit fMLP-induced AA release. In contrast, AA release was stimulated by the Ca(2+) ionophore A23187 (10 microM) plus the PKC activator phorbol myristate acetate (PMA) (0.2 microM). This effect was inhibited by either Ro 318220 or AACOCF(3). Accordingly, a translocation of cPLA(2) from the cytosol to the membrane fraction was observed with A23187 + PMA, but not with fMLP. fMLP-mediated AA release therefore appeared to be independent of Ca(2+) signaling and PKC and MAP kinase activation. However, fMLP-mediated AA release was reduced by approximately 45% by Clostridium difficile toxin B (10 ng/ml) or by 1-butanol; both block phospholipase D (PLD) activity. The inhibitor of phosphatidylcholine-specific phospholipase C (PC-PLC), D609 (100 microM), decreased fMLP-mediated AA release by approximately 35%. The effect of D609 + 1-butanol on fMLP-induced AA release was additive and of a magnitude similar to that of propranolol (0.2 mM), an inhibitor of phosphatidic acid phosphohydrolase. This suggests that the bulk of AA generated by fMLP stimulation of db-cAMP-differentiated HL-60 cells is independent of the cPLA(2) pathway, but may originate from activation of PC-PLC and PLD.     

Iron-induced oxidative damage of corn root plasma membrane H(+)-ATPase

The effect of iron on the activity of the plasma membrane H(+)-ATPase (PMA) from corn root microsomal fraction (CRMF) was investigated. In the presence of either Fe(2+) or Fe(3+) (100-200 microM of FeSO(4) or FeCl(3), respectively), 80-90% inhibition of ATP hydrolysis by PMA was observed. Half-maximal inhibition was attained at 25 microM and 50 microM for Fe(2+) and Fe(3+), respectively. Inhibition of the ATPase activity was prevented in the presence of metal ion chelators such as EDTA, deferoxamine or o-phenanthroline in the incubation medium. However, preincubation of CRMF in the presence of 100 microM Fe(2+), but not with 100 microM Fe(3+), rendered the ATPase activity (measured in the presence of excess EDTA) irreversibly inhibited. Inhibition was also observed using a preparation further enriched in plasma membranes by gradient centrifugation. Addition of 0.5 mM ATP to the preincubation medium, either in the presence or in the absence of 5 mM MgCl(2), reduced the extent of irreversible inhibition of the H(+)-ATPase. Addition of 40 microM butylated hydroxytoluene and/or 5 mM dithiothreitol, or deoxygenation of the incubation medium by bubbling a stream of argon in the solution, also caused significant protection of the ATPase activity against irreversible inhibition by iron. Western blots of CRMF probed with a polyclonal antiserum against the yeast plasma membrane H(+)-ATPase showed a 100 kDa cross-reactive band, which disappeared in samples previously exposed to 500 microM Fe(2+). Interestingly, preservation of the 100 kDa band was observed when CRMF were exposed to Fe(2+) in the presence of either 5 mM dithiothreitol or 40 microM butylated hydroxytoluene. These results indicate that iron causes irreversible inhibition of the corn root plasma membrane H(+)-ATPase by oxidation of sulfhydryl groups of the enzyme following lipid peroxidation.     

Induction of 5-lipoxygenase activation in polymorphonuclear leukocytes by 1-oleoyl-2-acetylglycerol

1,2-Diacylglycerols (DAGs) can prime polymorphonuclear leukocytes (PMNL) for enhanced release of arachidonic acid (AA) and generation of 5-lipoxygenase (5-LO) products upon subsequent agonist stimulation. Here, we demonstrate that in isolated human PMNL, 1-oleoyl-2-acetylglycerol (OAG) functions as a direct agonist stimulating 5-LO product formation (up to 42-fold). OAG caused no release of endogenous AA, but in the presence of exogenous AA, the magnitude of 5-LO product synthesis induced by OAG was comparable to that obtained with the Ca(2+)-ionophore A23187. Interestingly, OAG-induced 5-LO product synthesis was not connected with increased 5-LO nuclear membrane association. Examination of diverse glycerides revealed that the sn-2-acetyl-group is important, thus, also 1-O-hexadecyl-2-acetylglycerol (EAG) stimulated 5-LO product formation (up to 8-fold).Treatment of PMNL with OAG did not alter the mobilization of Ca(2+) but removal of intracellular Ca(2+) abolished the upregulatory OAG effects. Notably, the PKC activator phorbol-myristate-acetate hardly increased 5-LO product synthesis and PKC inhibitors failed to suppress the effects of OAG. Although OAG rapidly activated p38 MAPK and p42/44(MAPK), which can stimulate 5-LO for product synthesis, specific inhibitors of these kinases could not prevent 5-LO activation by OAG. Together, OAG acts as a direct agonist for 5-LO product synthesis in PMNL stimulating 5-LO by novel undefined mechanisms.     

Synaptotagmins form a hierarchy of exocytotic Ca(2+) sensors with distinct Ca(2+) affinities

Synaptotagmins constitute a large family of membrane proteins implicated in Ca(2+)-triggered exocytosis. Structurally similar synaptotagmins are differentially localized either to secretory vesicles or to plasma membranes, suggesting distinct functions. Using measurements of the Ca(2+) affinities of synaptotagmin C2-domains in a complex with phospholipids, we now show that different synaptotagmins exhibit distinct Ca(2+) affinities, with plasma membrane synaptotagmins binding Ca(2+) with a 5- to 10-fold higher affinity than vesicular synaptotagmins. To test whether these differences in Ca(2+) affinities are functionally important, we examined the effects of synaptotagmin C2-domains on Ca(2+)-triggered exocytosis in permeabilized PC12 cells. A precise correlation was observed between the apparent Ca(2+) affinities of synaptotagmins in the presence of phospholipids and their action in PC12 cell exocytosis. This was extended to PC12 cell exocytosis triggered by Sr(2+), which was also selectively affected by high-affinity C2-domains of synaptotagmins. Together, our results suggest that Ca(2+) triggering of exocytosis involves tandem Ca(2+) sensors provided by distinct plasma membrane and vesicular synaptotagmins. According to this hypothesis, plasma membrane synaptotagmins represent high-affinity Ca(2+) sensors involved in slow Ca(2+)-dependent exocytosis, whereas vesicular synaptotagmins function as low-affinity Ca(2+) sensors specialized for fast Ca(2+)-dependent exocytosis.     

Store-operated Ca2+ entry in porcine airway smooth muscle

Ca(2+) influx triggered by depletion of sarcoplasmic reticulum (SR) Ca(2+) stores [mediated via store-operated Ca(2+) channels (SOCC)] was characterized in enzymatically dissociated porcine airway smooth muscle (ASM) cells. When SR Ca(2+) was depleted by either 5 microM cyclopiazonic acid or 5 mM caffeine in the absence of extracellular Ca(2+), subsequent introduction of extracellular Ca(2+) further elevated [Ca(2+)](i). SOCC was insensitive to 1 microM nifedipine- or KCl-induced changes in membrane potential. However, preexposure of cells to 100 nM-1 mM La(3+) or Ni(2+) inhibited SOCC. Exposure to ACh increased Ca(2+) influx both in the presence and absence of a depleted SR. Inhibition of inositol 1,4,5-trisphosphate (IP)-induced SR Ca(2+) release by 20 microM xestospongin D inhibited SOCC, whereas ACh-induced IP(3) production by 5 microM U-73122 had no effect. Inhibition of Ca(2+) release through ryanodine receptors (RyR) by 100 microM ryanodine also prevented Ca(2+) influx via SOCC. Qualitatively similar characteristics of SOCC-mediated Ca(2+) influx were observed with cyclopiazonic acid- vs. caffeine-induced SR Ca(2+) depletion. These data demonstrate that a Ni(2+)/La(3+)-sensitive Ca(2+) influx via SOCC in porcine ASM cells involves SR Ca(2+) release through both IP(3) and RyR channels. Additional regulation of Ca(2+) influx by agonist may be related to a receptor-operated, noncapacitative mechanism.     

Al(3+)-mediated changes in membrane physical properties participate in the inhibition of polyphosphoinositide hydrolysis

We investigated the possible involvement of Al(3+)-induced alterations in membrane physical properties in Al(3+)-mediated inhibition of polyphosphoinositide (PPI) hydrolysis by the enzyme phosphatidylinositol-specific phospholipase C (PI-PLC). Liposomes composed of brain phosphatidylcholine (PC) or of PC and a mixture of brain PPI (PC:PPI) were incubated in the presence of Al(3+) (1-100 microM). We evaluated: (1) the amount of membrane-bound Al(3+), (2) the effects of Al(3+) on key membrane physical properties (surface potential, lipid fluidity, and lipid arrangement), and (3) the hydrolysis of PPI. Al(3+) binding to PC:PPI (60:40 mol/mol) liposomes was 1.3 times higher than to PC:PPI (90:10 mol/mol) liposomes and did not change after treatment with Triton X-100. Al(3+) increased membrane surface potential, promoted the loss of membrane fluidity, and caused lateral phase separation in PC:PPI liposomes. Phosphatidylinositol and phosphatidylinositol monophosphate hydrolysis in the presence of PI-PLC was not affected by Al(3+), but a significant and concentration-dependent inhibition of PIP(2) hydrolysis was observed, an effect that was prevented by previous bilayer disruption with Triton X-100. The obtained results support the hypothesis that Al(3+) binding to liposomes promotes the formation of rigid clusters enriched in PPI, restricting the accessibility of the enzyme to the substrate and subsequently inhibiting PIP(2) hydrolysis by PI-PLC.     

Characterization of tescalcin, a novel EF-hand protein with a single Ca2+-binding site: metal-binding properties, localization in tissues and cells, and effect on calcineurin

The tescalcin gene is preferentially expressed during mouse testis differentiation. Here, we demonstrate that this gene encodes a 24 kDa Ca(2+)- and Mg(2+)-binding protein with one consensus EF-hand and three additional domains with EF-hand homology. Equilibrium dialysis with (45)Ca(2+) revealed that recombinant tescalcin binds approximately one Ca(2+) ion at physiological concentrations (pCa 4.5). The intrinsic tryptophan fluorescence of tescalcin was significantly reduced by Ca(2+), indicative of a conformational change. The apparent K(d) for Ca(2+) was 0.8 microM. A point mutation in the consensus EF-hand (D123A) abolished (45)Ca(2+) binding and prevented the fluorescence quenching, demonstrating that the consensus EF-hand alone mediates the Ca(2+)-induced conformational change. Tescalcin also binds Mg(2+) (K(d) 73 microM), resulting in a much smaller fluorescence decrease. In the presence of 1 mM Mg(2+), tescalcin's Ca(2+) affinity is shifted to 3.5 microM. These results illustrate that tescalcin should bind Mg(2+) constitutively in a quiescent cell, replacing it with Ca(2+) during stimulation. We also show that tescalcin is most abundant in adult mouse heart, brain, and stomach, as well as in HeLa and HL-60 cells. Immunofluorescence microscopy revealed that tescalcin is present in the cytoplasm and nucleus, with concentration in membrane ruffles and lamellipodia in the presence of serum, where it colocalizes with the small guanosine triphosphatase Rac-1. Tescalcin shares sequence and functional homology with calcineurin-B homologous protein (CHP), and we found that tescalcin, like CHP, can inhibit the phosphatase activity of calcineurin A. Hence, tescalcin is a novel calcineurin B-like protein that binds a single Ca(2+) ion.     

Ferrous iron-dependent volatilization of mercury by the plasma membrane of Thiobacillus ferrooxidans

Of 100 strains of iron-oxidizing bacteria isolated, Thiobacillus ferrooxidans SUG 2-2 was the most resistant to mercury toxicity and could grow in an Fe(2+) medium (pH 2.5) supplemented with 6 microM Hg(2+). In contrast, T. ferrooxidans AP19-3, a mercury-sensitive T. ferrooxidans strain, could not grow with 0.7 microM Hg(2+). When incubated for 3 h in a salt solution (pH 2.5) with 0.7 microM Hg(2+), resting cells of resistant and sensitive strains volatilized approximately 20 and 1.7%, respectively, of the total mercury added. The amount of mercury volatilized by resistant cells, but not by sensitive cells, increased to 62% when Fe(2+) was added. The optimum pH and temperature for mercury volatilization activity were 2.3 and 30 degrees C, respectively. Sodium cyanide, sodium molybdate, sodium tungstate, and silver nitrate strongly inhibited the Fe(2+)-dependent mercury volatilization activity of T. ferrooxidans. When incubated in a salt solution (pH 3.8) with 0.7 microM Hg(2+) and 1 mM Fe(2+), plasma membranes prepared from resistant cells volatilized 48% of the total mercury added after 5 days of incubation. However, the membrane did not have mercury reductase activity with NADPH as an electron donor. Fe(2+)-dependent mercury volatilization activity was not observed with plasma membranes pretreated with 2 mM sodium cyanide. Rusticyanin from resistant cells activated iron oxidation activity of the plasma membrane and activated the Fe(2+)-dependent mercury volatilization activity of the plasma membrane.     

Changes in phospholipid composition and calcium flux in LLC-PK cells cultured at low magnesium concentrations

Monolayers of porcine kidney cells (LLC-PK) were grown in a series of Nu-Serum-supplemented media containing different Mg(2+) concentrations (480, 250, 25, 6.3 or 2.6 microM) to study the effect of Mg(2+) depletion on cellular phospholipid changes and the consequent effect on the membrane permeability to Ca(2+). Cells grown on 6.3 or 2.6 microM Mg(2+) showed a decrease in PE, PS, Sph, PI and an increase of PC. These changes were attributed mainly to the decreased rate of Sph synthesis through the transfer of phosphocholine from PC to ceramide, or due to the increase of PE N-methylation as found in Mg(2+)-deficient cells. The (45)Ca uptake was increased in cells grown on 25.0 microM Mg(2+), while it was decreased in cells grown on 6.3 or 2.6 microM Mg(2+). These changes in Ca(2+) uptake were related to changes of cellular phospholipids and fatty acids which affect adenylate cyclase activity in the membrane, as well as the membrane fluidity.     

Mg2+ enhances voltage sensor/gate coupling in BK channels

BK (Slo1) potassium channels are activated by millimolar intracellular Mg(2+) as well as micromolar Ca(2+) and membrane depolarization. Mg(2+) and Ca(2+) act in an approximately additive manner at different binding sites to shift the conductance-voltage (G(K)-V) relation, suggesting that these ligands might work through functionally similar but independent mechanisms. However, we find that the mechanism of Mg(2+) action is highly dependent on voltage sensor activation and therefore differs fundamentally from that of Ca(2+). Evidence that Ca(2+) acts independently of voltage sensor activation includes an ability to increase open probability (P(O)) at extreme negative voltages where voltage sensors are in the resting state; 2 microM Ca(2+) increases P(O) more than 15-fold at -120 mV. However 10 mM Mg(2+), which has an effect on the G(K)-V relation similar to 2 microM Ca(2+), has no detectable effect on P(O) when voltage sensors are in the resting state. Gating currents are only slightly altered by Mg(2+) when channels are closed, indicating that Mg(2+) does not act merely to promote voltage sensor activation. Indeed, channel opening is facilitated in a voltage-independent manner by Mg(2+) in a mutant (R210C) whose voltage sensors are constitutively activated. Thus, 10 mM Mg(2+) increases P(O) only when voltage sensors are activated, effectively strengthening the allosteric coupling of voltage sensor activation to channel opening. Increasing Mg(2+) from 10 to 100 mM, to occupy very low affinity binding sites, has additional effects on gating that more closely resemble those of Ca(2+). The effects of Mg(2+) on steady-state activation and I(K) kinetics are discussed in terms of an allosteric gating scheme and the state-dependent interactions between Mg(2+) and voltage sensor that may underlie this mechanism.     

Purification and characterization of S-modulin, a calcium-dependent regulator on cGMP phosphodiesterase in frog rod photoreceptors.

S-modulin is a 26 kDa protein that regulates light sensitivity of cGMP phosphodiesterase in a Ca(2+)-dependent manner in frog rod outer segments (ROSs). In the present study, we purified S-modulin by taking advantage of a hydrophobic interaction between Phenyl Sepharose and S-modulin at high Ca2+ concentrations. The yield was greater than 90%. 45Ca(2+)-binding experiment showed that S-modulin is a Ca(2+)-binding protein. At high Ca2+ concentrations, S-modulin binds to ROS membranes. The binding target of the Ca2+/S-modulin complex is possibly a ROS membrane lipid(s), but it was difficult to identify. The binding was observed mainly at greater than 1 microM Ca2+. The amino acid sequence deduced from proteolytic fragments of S-modulin was approximately 80% and 60% identical to those of recovering and visinin, respectively.     

Roles of factor Va heavy and light chains in protein and lipid rearrangements associated with the formation of a bovine factor Va-membrane complex.

Factor Va is an essential protein cofactor of the enzyme factor Xa, which activates prothrombin to thrombin during blood coagulation. Peptides with an apparent Mr of approximately 94,000 (heavy chain; HC) and approximately 74,000 or 72,000 (light chain; LC) interact in the presence of Ca2+ to form active Va. The two forms of Va-LC differ in their carboxyl-terminal C2 domain. Using Va reconstituted with either LC form, we examined the effects of the two LC species on membrane binding and on the activity of membrane-bound Va. We found that 1) Va composed of the 72,000 LC bound only slightly more tightly to membranes composed of a mixture of neutral and acidic lipids, the Kd being reduced by a factor of approximately 3 at 5 mM and by a factor of 6 at 2 mM Ca2+. 2) The two forms of Va seemed to undergo different conformational changes when bound to a membrane. 3) The activity of bovine Va varied somewhat with LC species, the difference being greatest at limiting Xa concentration. We have also addressed the role of the two Va peptides in membrane lipid rearrangements and binding: 1) Va binding increased lateral packing density in mixed neutral/acidic lipid membranes. In the solid phase, Va-HC had no effect, whereas Va-LC and whole Va had similar but small effects. In the fluid phase, Va-HC and whole Va both altered membrane packing, with Va-HC having the largest effect. 2) Va-HC bound reversibly and in a Ca2+-independent fashion to membranes composed of neutral phospholipid (Kd, approximately 0.3 microM; stoichiometry approximately 91). High ionic strength had little effect on binding. 3) The substantial effect of Va on packing within neutral phospholipid membranes was mimicked by Va-HC. 4) Based on measurements of membrane phase behavior, binding of Va or its peptide components did not induce thermodynamically discernible lateral membrane domains. These results suggest that the membrane association of factor Va is a complex process involving both chains of Va, changes in lipid packing, and changes in protein structure.     

Kinetics and thermodynamics of annexin A1 binding to solid-supported membranes: a QCM study

By means of the quartz crystal microbalance (QCM) technique, the interaction of annexin A1 with lipid membranes was quantified using solid-supported bilayers immobilized on gold electrodes deposited on 5 MHz quartz plates. Solid-supported lipid bilayers were composed of a first octanethiol monolayer chemisorbed on gold and a physisorbed phospholipid monolayer obtained from vesicle fusion. This experimental setup enabled us to determine for the first time rate constants and affinity constants of annexin A1 binding to phosphatidylserine-containing layers as a function of the calcium ion concentration in solution and the cholesterol content within the outer leaflet of the solid-supported bilayer. The results reveal that a decrease in Ca(2+) concentration from 1 mM to 100 microM significantly increases the rate of annexin A1 binding to the membrane independent of the cholesterol content. However, the presence of cholesterol in the membrane altered the affinity constants considerably. While the association constant decreases with decreasing Ca(2+) concentration in the case of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine (POPS) membranes lacking cholesterol, it remains high in the presence of cholesterol.     

Arachidonic acid increases intracellular calcium in erythrocytes

Recently, we have measured in erythrocytes a voltage-modulated and dihydropyridine-inhibited calcium influx. Since arachidonic acid and other polyunsaturated fatty acids influence the activities of most ion channels, we studied their effects on the erythrocyte Ca(2+) influx. It was measured on fresh erythrocytes, isolated from healthy donors, using the fluorescent dye Fura 2 as indicator of [Ca(2+)](i). AA (5-50 microM) and EPA (20-30 microM) stimulated a concentration-dependent increase in [Ca(2+)](i), deriving from extracellular calcium (1 mM), without affecting the intra- and extracellular pH and membrane voltage. The Ca(2+) influx rate varied from 0.5 to 3 nM Ca(2+)/s in the presence of AA and from 0.9 to 1.7 nM Ca(2+)/s with EPA. The Ca(2+) influx elicited by AA and EPA was not inhibited by dihydropyridines, while cyclooxygenase inhibitors were effective and PGE1 or PGE2 did not produce any effect. We conclude that AA could activate an erythrocyte voltage-independent Ca(2+) transport via an intermediate product of cyclooxygenase pathway; however, a direct interaction with the membrane lipid-protein cannot be excluded.