Influence of red light and acetylcholine on 45Ca2+ uptake by oat coleoptile cells

Influence of red light and acetylcholine on ⁴⁵Ca²⁺ uptake by oat coleoptile cells was examined. It was found that the uptake is passive in darkness, while short, 10–15 min. exposure of coleoptile sections to red light or treatment with acetylcholine solution increases the rate of ⁴⁵Ca²⁺ uptake from the medium. Calcium channel blockers, La³⁺ and Verapamil, hinder ⁴⁵Ca²⁺ uptake in darkness and neutralize the stimulative influence of red light and acetylcholine.     

Attempts to alter 45Ca2+ binding to brain tissue with pulse-modulated microwave energy

Rat brain tissue, loaded with ⁴⁵Ca²⁺ by intraventricular injection was exposed in vitro to pulsemodulated 1-GHz (SAR of 0.29 or 2.9 W/kg) or 2.45-GHz radiation (SAR = 0.3 W/kg), and in vivo to 2.06-GHz radiation (SAR of 0.12 to 2.4 W/kg). There were no significant differences in efflux of ⁴⁵Ca²⁺ between the microwave- and sham-irradiated groups.     

Binding of Ca 2+ to normal and dicoumarol-induced prothrombin

The Ca²⁺ binding properties of normal bovine prothrombin have been studied and compared with those of an abnormal bovine prothrombin induced by dicoumarol. The normal prothrombin binds up to 10–12 Ca²⁺ per mole of protein. The three first Ca²⁺ were bound to sites which exhibited positive cooperativity. A Ca²⁺ dependent conformational change was demonstrated during the binding of the first three Ca²⁺. In contrast with normal prothrombin, the dicoumarol-induced prothrombin had only one high affinity binding site. No ligand-induced conformational change was detected in this prothrombin.     

Ca2+ Binding to Site I of the Cardiac Ca2+ Pump Is Sufficient to Dissociate Phospholamban

Phospholamban (PLB) inhibits the activity of SERCA2a, the Ca²⁺-ATPase in cardiac sarcoplasmic reticulum, by decreasing the apparent affinity of the enzyme for Ca²⁺. Recent cross-linking studies have suggested that PLB binding and Ca²⁺ binding to SERCA2a are mutually exclusive. PLB binds to the E2 conformation of the Ca²⁺-ATPase, preventing formation of E1, the conformation that binds two Ca²⁺ (at sites I and II) with high affinity and is required for ATP hydrolysis. Here we determined whether Ca²⁺ binding to site I, site II, or both sites is sufficient to dissociate PLB from the Ca²⁺ pump. Seven SERCA2a mutants with amino acid substitutions at Ca²⁺-binding site I (E770Q, T798A, and E907Q), site II (E309Q and N795A), or both sites (D799N and E309Q/E770Q) were made, and the effects of Ca²⁺ on N30C-PLB cross-linking to Lys³²⁸ of SERCA2a were measured. In agreement with earlier reports with the skeletal muscle Ca²⁺-ATPase, none of the SERCA2a mutants (except E907Q) hydrolyzed ATP in the presence of Ca²⁺; however, all were phosphorylatable by P_i to form E2P. Ca²⁺ inhibition of E2P formation was observed only in SERCA2a mutants retaining site I. In cross-linking assays, strong cross-linking between N30C-PLB and each Ca²⁺-ATPase mutant was observed in the absence of Ca²⁺. Importantly, however, micromolar Ca²⁺ inhibited PLB cross-linking only to mutants retaining a functional Ca²⁺-binding site I. The dynamic equilibrium between Ca²⁺ pumps and N30C-PLB was retained by all mutants, demonstrating normal regulation of cross-linking by ATP, thapsigargin, and anti-PLB antibody. From these results we conclude that site I is the key Ca²⁺-binding site regulating the physical association between PLB and SERCA2a.     

Binding of Ca2+ to glycoprotein from bovine heart mitochondria

It is shown that glycoprotein from bovine heart mitochondria which forms Ca2+-selective conductance channels in a bilayer lipid membrane possesses Ca2+-binding activity. Ca2+-binding sites of two kinds were revealed in the glycoprotein molecule: high affinity sites with Kd = 2.8 X 10(-6) M and low affinity sites with Kd 1.1 X 10(-5) M. Ca2+-binding by the high affinity sites occurs co-operatively. The Hill coefficient is about 2.     

Binding of Ca2+ to phosphoinositols, phosphatidylserines and gangliosides

The effect of K+, Mg2+ and serotonin on the interaction between Ca2+ and different phospholipids as well as glycosphingolipids (gangliosides) was studied by equilibrium dialysis using 45Ca as tracer. The highly polar phosphatidylinositol-4,5-bisphosphate (TPI) was found to bind more Ca2+ per lipid molecule than all other lipids tested and Ca2+ could not be released as easily as in the other lipids by K+, Mg2+ and serotonin. Ca2+ is released from all lipid-Ca2+ complexes most effectively by Mg2+, serotonin is less effective but enhances K+ in its capacity to displace Ca2+ from the respective binding sites. A remarkable dissociating influence of serotonin on ganglioside-Ca2+ and phosphatidylserine-Ca2+ complexes is observed. This effect is less pronounced with phosphatidylinositol-Ca2+ complexes under comparable comparable conditions. The possible functional role of phospholipids and gangliosides in vivo is discussed with regard to the specific Ca2+-binding properties of these lipids.     

Binding of Ca2+ to the calcium adenosinetriphosphatase of sarcoplasmic reticulum

The binding of Ca2+ and the resulting change in catalytic specificity that allows phosphorylation of the calcium ATPase of sarcoplasmic reticulum by ATP were examined by measuring the amount of phosphoenzyme formation from [32P]ATP, or 45Ca incorporation into vesicles, after the simultaneous addition of ATP and EGTA at different times after mixing enzyme and Ca2+ (25 degrees C, pH 7.0, 5 mM MgSO4, 0.1 M KCl). A "burst" of calcium binding in the presence of high [Ca2+] gives approximately 12% phosphorylation and internalization of two Ca2+ at very short times after the addition of Ca2+ with this assay. This shows that calcium binding sites are available on the cytoplasmic-facing side of the free enzyme. Calcium binding to these sites induces the formation of cE.Ca2, the stable high-affinity form of the enzyme, with k = 40 s-1 at saturating [Ca2+] and a half-maximal rate at approximately 20 microM Ca2+ (from Kdiss = 7.4 X 10(-7) M for Ca.EGTA). The formation of cE.Ca2 through a "high-affinity" pathway can be described by the scheme E 1 in equilibrium cE.Ca1 2 in equilibrium cE.Ca2, with k1 = 3 X 10(6) M-1 s-1, k2 = 4.3 X 10(7) M-1 s-1, k-1 = 30 s-1, k-2 = 60 s-1, K1 = 9 X 10(-6) M, and K2 = 1.4 X 10(-6) M. The approach to equilibrium from E and 3.2 microM Ca2+ follows kobsd = kf + kr = 18 s-1 and gives kf = kr = 9 s-1. The rate of exchange of 45Ca into the inner position of cE.Ca2 shows an induction period and is not faster than the approach to equilibrium starting with E and 45Ca. The dissociation of 45Ca from the inner position of cE.45Ca.Ca in the presence of 3.2 microM Ca2+ occurs with a rate constant of 7 s-1. These results are inconsistent with a slow conformational change of free E to give cE, followed by rapid binding-dissociation of Ca2+.     

Raising the cytosolic Ca2+ concentration increases the membrane capacitance of maize coleoptile protoplasts: Evidence for Ca2+-stimulated exocytosis

Enhanced elongation of coleoptile cells has been proposed to be related to a rise in secretory activity. Therefore, to obtain a direct measurement of exocytotic events in maize (Zea mays L.) coleoptile protoplasts we used the patch-clamp method to record changes in membrane capacitance (Cm) as a parameter proportional to fluctuations of the membrane surface area. The secretory activity of protoplasts was correlated with the cytosolic free Ca²⁺ concentration ([Ca²⁺]_cyt): dialyzing protoplasts with 1 M [Ca²⁺]_cyt caused a steady rise in Cm of 3.3 ± pF·s^–1. In contrast, dialysis with a solution containing <20 nM Ca²⁺ produced a small and persistent decrease in Cm. This demonstrates that secretory activity in coleoptile cells can be controlled by factors which modulate [Ca²⁺]_cyt.Abbreviation Cm membrane capacitance This work was made possible by a visiting grant from the Research Council of Slovenia and financial support of the Deutsche Forschungsgemeinschaft to G.T. We are grateful to Dr. W. Diekmann (University of Göttingen) for teaching us the preparation of coleoptile protoplasts.     

The EF-Hand Ca2+ Binding Protein MICU Choreographs Mitochondrial Ca2+ Dynamics in Arabidopsis

Plant organelle function must constantly adjust to environmental conditions, which requires dynamic coordination. Ca²⁺ signaling may play a central role in this process. Free Ca²⁺ dynamics are tightly regulated and differ markedly between the cytosol, plastid stroma, and mitochondrial matrix. The mechanistic basis of compartment-specific Ca²⁺ dynamics is poorly understood. Here, we studied the function of At-MICU, an EF-hand protein of Arabidopsis thaliana with homology to constituents of the mitochondrial Ca²⁺ uniporter machinery in mammals. MICU binds Ca²⁺ and localizes to the mitochondria in Arabidopsis. In vivo imaging of roots expressing a genetically encoded Ca²⁺ sensor in the mitochondrial matrix revealed that lack of MICU increased resting concentrations of free Ca²⁺ in the matrix. Furthermore, Ca²⁺ elevations triggered by auxin and extracellular ATP occurred more rapidly and reached higher maximal concentrations in the mitochondria of micu mutants, whereas cytosolic Ca²⁺ signatures remained unchanged. These findings support the idea that a conserved uniporter system, with composition and regulation distinct from the mammalian machinery, mediates mitochondrial Ca²⁺ uptake in plants under in vivo conditions. They further suggest that MICU acts as a throttle that controls Ca²⁺ uptake by moderating influx, thereby shaping Ca²⁺ signatures in the matrix and preserving mitochondrial homeostasis. Our results open the door to genetic dissection of mitochondrial Ca²⁺ signaling in plants.     

Crystal Structures of Progressive Ca2+ Binding States of the Ca2+ Sensor Ca2+ Binding Domain 1 (CBD1) from the CALX Na+/Ca2+ Exchanger Reveal Incremental Conformational Transitions

Na⁺/Ca²⁺ exchangers (NCX) constitute a major Ca²⁺ export system that facilitates the re-establishment of cytosolic Ca²⁺ levels in many tissues. Ca²⁺ interactions at its Ca²⁺ binding domains (CBD1 and CBD2) are essential for the allosteric regulation of Na⁺/Ca²⁺ exchange activity. The structure of the Ca²⁺-bound form of CBD1, the primary Ca²⁺ sensor from canine NCX1, but not the Ca²⁺-free form, has been reported, although the molecular mechanism of Ca²⁺ regulation remains unclear. Here, we report crystal structures for three distinct Ca²⁺ binding states of CBD1 from CALX, a Na⁺/Ca²⁺ exchanger found in Drosophila sensory neurons. The fully Ca²⁺-bound CALX-CBD1 structure shows that four Ca²⁺ atoms bind at identical Ca²⁺ binding sites as those found in NCX1 and that the partial Ca²⁺ occupancy and apoform structures exhibit progressive conformational transitions, indicating incremental regulation of CALX exchange by successive Ca²⁺ binding at CBD1. The structures also predict that the primary Ca²⁺ pair plays the main role in triggering functional conformational changes. Confirming this prediction, mutagenesis of Glu⁴⁵⁵, which coordinates the primary Ca²⁺ pair, produces dramatic reductions of the regulatory Ca²⁺ affinity for exchange current, whereas mutagenesis of Glu⁵²⁰, which coordinates the secondary Ca²⁺ pair, has much smaller effects. Furthermore, our structures indicate that Ca²⁺ binding only enhances the stability of the Ca²⁺ binding site of CBD1 near the hinge region while the overall structure of CBD1 remains largely unaffected, implying that the Ca²⁺ regulatory function of CBD1, and possibly that for the entire NCX family, is mediated through domain interactions between CBD1 and the adjacent CBD2 at this hinge.     

Effect of Quin-2 on 45Ca2+ uptake mediated by Na+i/Ca2+o exchange and 45Ca2+ efflux in rat brain synaptosomes: a requirement for [Ca2+]i

The Na+/Ca2+ exchanger of squid axons, barnacle muscle and sarcolemma requires micromolar intracellular calcium for activation in the Na+i/Ca2+o exchange mode ('reverse' Na+/Ca2+ exchange). The requirement for [Ca2+]i has been demonstrated with the use of intracellular calcium buffers, such as Quin-2, to inhibit Na+i/Ca2+o exchange. However, the inhibition of Na+i/Ca2+o exchange in mammalian nerve terminals loaded with Quin-2 has not been observed [7], suggesting a lower sensitivity to low [Ca2+]i for this system. In contrast, the results reported herein indicate that 45Ca2+ uptake in synaptosomes through Na+i/Ca2+o exchange is inhibited by Quin-2 much in the same way as it is in the squid, provided that synaptosomes are preincubated in low Ca2+ medium to avoid saturation of Quin-2. Under these conditions, 45Ca2+ efflux via Ca2+i/Ca2+o exchange is also inhibited. Our results indicate that the Na+i/Ca2+o and Ca2+i/Ca2+o modes of the Na+/Ca2+ exchanger from rat brain synaptosomes require intracellular calcium for activation. However, because no clear relationship between the observed [Ca2+]i values and the inhibition of Na+i/Ca2+o exchange has been found, it is suggested that localised submembrane calcium concentrations not detected by the [Ca2+]i probe might regulate the exchanger.     

Ca2+ dependence of stimulated 45Ca efflux in skinned muscle fibers

Stimulation of sarcoplasmic reticulum Ca release by Mg reduction of caffeine was studied in situ, to characterize further the Ca2+ dependence observed previously with stimulation by Cl ion. 45Ca efflux and isometric force were measured simultaneously at 19 degrees C in frog skeletal muscle fibers skinned by microdissection; EGTA was added to chelate myofilament space Ca either before or after the stimulus. Both Mg2+ reduction (20 or 110 microM to 4 microM) and caffeine (5 mM) induced large force responses and 45Ca release, which were inhibited by pretreatment with 5 mM EGTA. In the case of Mg reduction, residual efflux stimulation was undetectable, and 45Ca efflux in EGTA at 4 microM Mg2+ was not significantly increased. Residual caffeine stimulation at 20 microM Mg2+ was substantial and was reduced further in increased EGTA (10 mM); at 600 microM Mg2+, residual stimulation in 5 mM EGTA was undetectable. Caffeine appears to initiate a small Ca2+-insensitive efflux that produces a large Ca2+-dependent efflux. Additional experiments suggested that caffeine also inhibited influx. The results suggest that stimulated efflux is mediated mainly or entirely by a channel controlled by an intrinsic Ca2+ receptor, which responds to local [Ca2+] in or near the channel. Receptor affinity for Ca2+ probably is influenced by Mg2+, but inhibition is weak unless local [Ca2+] is very low.     

Binding of Zn2+ to a Ca2+ loop allosterically attenuates the activity of factor VIIa and reduces its affinity for tissue factor

The protease domain of coagulation factor VIIa (FVIIa) is homologous to trypsin with a similar active site architecture. The catalytic function of FVIIa is regulated by allosteric modulations induced by binding of divalent metal ions and the cofactor tissue factor (TF). To further elucidate the mechanisms behind these transformations, the effects of Zn2+ binding to FVIIa in the free form and in complex with TF were investigated. Equilibrium dialysis suggested that two Zn2+ bind with high affinity to FVIIa outside the N-terminal gamma-carboxyglutamic acid (Gla) domain. Binding of Zn2+ to FVIIa, which was influenced by the presence of Ca2+, resulted in decreased amidolytic activity and slightly reduced affinity for TF. After binding to TF, FVIIa was less susceptible to zinc inhibition. Alanine substitutions for either of two histidine residues unique for FVIIa, His216, and His257, produced FVIIa variants with decreased sensitivity to Zn2+ inhibition. A search for putative Zn2+ binding sites in the crystal structure of the FVIIa protease domain was performed by Grid calculations. We identified a pair of Zn2+ binding sites in the Glu210-Glu220 Ca2+ binding loop adjacent to the so-called activation domain canonical to serine proteases. Based on our results, we propose a model that describes the conformational changes underlying the Zn2+-mediated allosteric down-regulation of FVIIa's activity.     

Binding of hydrophobic drugs to lipid bilayers and to the (Ca2+ + Mg2+)-ATPase

Microelectrophoretic studies of the binding of a number of commonly used hydrophobic amine drugs to liposomes demonstrated the existence of relatively large surface potentials associated with binding of the protonated forms of the drugs. A theoretical treatment based on Langmuir adsorption isotherms and the Gouy-Chapman theory of the diffuse double layer allows estimation of drug-binding constants from electrophoretic mobility data. Such constants allow calculation of the charge effects arising from drug binding in more complex membrane systems, and it is shown that shifts in the apparent Ca+ affinity of the (Ca2+ + Mg2+)-ATPase of sarcoplasmic reticulum in the presence of hydrophobic amine drugs are readily explicable in terms of the electrostatic effects of drug binding.     

Structures of EF-hand Ca 2+-binding proteins: Diversity in the organization, packing and response to Ca 2+ Binding

The growing database of three-dimensional structures of EF-hand calcium-binding proteins is revealing a previously unrecognized variability in the coformations and organizations of EF-hand binding motifs. The structures of twelve different EF-hand proteins for which coordinates are publicly available are discussed and related to their respective biological and biophysical properties. The classical picture of calcium sensors and calcium signal modulators is presented, along with variants on the basic theme and new structural paradigms.© Kluwer Academic Publishers