Chromobindin A, a Ca2+- and ATP-dependent chromaffin granule-binding protein, is found in a variety of tissues and in yeast

Chromobindin A is a ring shaped, multisubunit protein which exhibits Ca2+ and ATP-dependent binding to chromaffin granule membranes. Here we report biochemical and immunochemical evidence for the presence of chromobindin A in a surprisingly broad range of tissues: The protein is abundant in bovine skeletal muscle, pancreas, adrenal cortex and in brain gray and white matter; it is present in low quantities in the parotid, intestine and spleen and is undetectable in lung. Interestingly, chromobindin A was also detected in extracts of yeast. Electron micrographs of the yeast protein reveal a morphology virtually identical to the mammalian protein. These results suggest that chromobindin A is an important protein in a wide variety of cell types and that it has been highly conserved through evolution.     

Kinetic studies of Ca2+ binding and Ca2+ clearance in the cytosol of adrenal chromaffin cells.

The Ca2+ binding kinetics of fura-2, DM-nitrophen, and the endogenous Ca2+ buffer, which determine the time course of Ca2+ changes after photolysis of DM-nitrophen, were studied in bovine chromaffin cells. The in vivo Ca2+ association rate constants of fura-2, DM-nitrophen, and the endogenous Ca2+ buffer were measured to be 5.17 x 10(8) M-1 s-1, 3.5 x 10(7) M-1 s-1, and 1.07 x 10(8) M-1 s-1, respectively. The endogenous Ca2+ buffer appeared to have a low affinity for Ca2+ with a dissociation constant around 100 microM. A fast Ca2+ uptake mechanism was also found to play a dominant role in the clearance of Ca2+ after flashes at high intracellular free Ca2+ concentrations ([Ca2+]), causing a fast [Ca2+]i decay within seconds. This Ca2+ clearance was identified as mitochondrial Ca2+ uptake. Its uptake kinetics were studied by analyzing the Ca2+ decay at high [Ca2+]i after flash photolysis of DM-nitrophen. The capacity of the mitochondrial uptake corresponds to a total cytosolic Ca2+ load of approximately 1 mM.     

Ca2+-independent,protein-mediated fusion of chromaffin granule ghosts with liposomes

We have investigated the interaction between isolated membrane vesicles from chromaffin granules and large unilamellar phospholipid vesicles (liposomes). Mixing of membrane lipids has been monitored continuously, utilizing the fluorescence resonance energy transfer assay described by Struck et al. ((1982) Biochemistry 20, 4093–4099). To demonstrate coalescence of the internal vesicle volumes the transfer of colloidal gold from the liposomes to the interior of the granule membrane vesicles has been examined. Efficient fusion of the liposomes with the granule membranes was observed. Significant fusion occurred in the absence of Ca²⁺, although the extent of interaction was enhanced in its presence. The sensitivity of the interaction to pretreatment of the granule membranes with trypsin showed the fusion reaction to be a protein-mediated process.     

Two-dimensional determination of the cellular Ca2+ binding in bovine chromaffin cells.

The spatiotemporal profile of intracellular calcium signals is determined by the flux of calcium ions across different biological membranes as well as by the diffusional mobility of calcium and different calcium buffers in the cell. To arrive at a quantitative understanding of the determinants of these signals, one needs to dissociate the flux contribution from the redistribution and buffering of calcium. Since the cytosol can be heterogeneous with respect to its calcium buffering property, it is essential to assess this property in a spatially resolved manner. In this paper we report on two different methods to estimate the cellular calcium binding of bovine adrenal chromaffin cells. In the first method, we use voltage-dependent calcium channels as a source to generate calcium gradients in the cytosol. Using imaging techniques, we monitor the dissipation of these gradients to estimate local apparent calcium diffusion coefficients and, from these, local calcium binding ratios. This approach requires a very high signal-to-noise ratio of the calcium measurement and can be used when well-defined calcium gradients can be generated throughout the cell. In the second method, we overcome these problems by using calcium-loaded DM-nitrophen as a light-dependent calcium source to homogeneously and quantitatively release calcium in the cytosol. By measuring [Ca2+] directly before and after the photorelease process and knowing the total amount of calcium being released photolytically, we get an estimate of the fraction of calcium ions which does not appear as free calcium and hence must be bound to either the indicator dye or the endogenous calcium buffer. This finally results in a two-dimensional map of the distribution of the immobile endogenous calcium buffer. We did not observe significant variations of the cellular calcium binding at a spatial resolution of approximately 2 micron. Furthermore, the calcium binding is not reduced by increasing the resting [Ca2+] to levels as high as 1.1 microM. This is indicative of a low calcium affinity of the corresponding buffers and is in agreement with a recent report on the affinity of these buffers (Xu, T., M. Naraghi, H. Kang, and E. Neher. 1997. Biophys. J. 73:532-545). In contrast to the homogeneous distribution of the calcium buffers, the apparant calcium diffusion coefficient did show inhomogeneities, which can be attributed to restricted diffusion at the nuclear envelope and to rim effects at the cell membrane.     

Ca2+ and proton transport in chromaffin granule membranes: a proton NMR study

High-resolution proton NMR spectroscopy has been used to monitor the internal pH of chromaffin granule ghosts during Ca2+ influx through the membrane. For this purpose, ghosts were prepared by lysing and resealing chromaffin granules in a medium containing the disodium-ethylenediaminetetraacetic acid complex (Na2.EDTA). Uncomplexed EDTA and Ca.EDTA give rise to distinct sets of methylene peaks in the proton NMR spectrum. Free EDTA titrates with a pK near 6.6 in deuterated media; the chemical shifts that accompany titration have been used to monitor intravesicular pH changes which occur inside chromaffin granule ghosts as a result of ATPase activity and deprotonation of EDTA during Ca2+ influx and complex formation. ATPase activity results in an NMR-detectable proton gradient which is dissipated by nigericin. Experiments monitoring Ca2+ uptake showed that protons which are liberated inside ghosts as a result of Ca.EDTA complex formation are not extruded from the ghosts via a process coupled to Ca2+ entry. This suggests that the Ca2+ transport system of the chromaffin granule membrane occurs without concurrent proton antiport and is not directly coupled energetically to the transmembrane pH gradient.     

The Ca2+ antagonists binding cytosolic protein has properties of the Ca2+ channel.

In our previous work (Krizanová et al. 1989) we have described a protein from rabbit skeletal muscle cytosolic fraction, which is able to bind dihydropyridines and phenothiazines. In the present work conclusive evidence is provided for the ability of the phospholipid-reconstituted cytosolic protein to transport calcium. The calcium transport was stimulated by BAY K 8644 and inhibited in the presence of PN 200-110. Our observations were confirmed also by electrophysiological measurements on planar lipid bilayers. The possibility that the cytosolic fraction was contaminated with membranes could be definitely ruled out. Nevertheless, the nature of the protein under study is still in the frame of guess.     

Ca2+ regulated modulator protein interacting agents: inhibition of Ca2+-Mg2+-ATPase of human erythrocyte ghost.

Agents such as N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), and its derivatives, chlorpromazine and amitriptyline that interact with calcium-regulated modulator protein were found to inhibit not only Ca²⁺ dependent cyclic nucleotide phosphodiesterase but also Ca²⁺-Mg²⁺-ATPase of human erythrocyte ghosts. I₅₀ values of modulator interacting agents for testis modulator-activated, brain modulator-activated and erythrocyte modulator-activated-ATPase are indistinguishable. However, I₅₀ of W-7 for troponin C-activated-ATPase is lower than that for modulator-activated ATPase. The specificity of these agents toward modulator-related enzyme reaction is also shown by the negative effect on modulator-unrelated enzyme system such as erythrocyte ghost protein kinase and Mg²⁺-ATPase. These agents provide a useful tool for elucidating the physiological role of modulator.     

Interaction of ATP sensor, cAMP sensor, Ca2+ sensor, and voltage-dependent Ca2+ channel in insulin granule exocytosis

ATP, cAMP, and Ca(2+) are the major signals in the regulation of insulin granule exocytosis in pancreatic beta cells. The sensors and regulators of these signals have been characterized individually. The ATP-sensitive K(+) channel, acting as the ATP sensor, couples cell metabolism to membrane potential. cAMP-GEFII, acting as a cAMP sensor, mediates cAMP-dependent, protein kinase A-independent exocytosis, which requires interaction with both Piccolo as a Ca(2+) sensor and Rim2 as a Rab3 effector. l-type voltage-dependent Ca(2+) channels (VDCCs) regulate Ca(2+) influx. In the present study, we demonstrate interactions of these molecules. Sulfonylurea receptor 1, a subunit of ATP-sensitive K(+) channels, interacts specifically with cAMP-GEFII through nucleotide-binding fold 1, and the interaction is decreased by a high concentration of cAMP. Localization of cAMP-GEFII overlaps with that of Rim2 in plasma membrane of insulin-secreting MIN6 cells. Localization of Rab3 co-incides with that of Rim2. Rim2 mutant lacking the Rab3 binding region, when overexpressed in MIN6 cells, is localized exclusively in cytoplasm, and impairs cAMP-dependent exocytosis in MIN6 cells. In addition, Rim2 and Piccolo bind directly to the alpha(1)1.2-subunit of VDCC. These results indicate that ATP sensor, cAMP sensor, Ca(2+) sensor, and VDCC interact with each other, which further suggests that ATP, cAMP, and Ca(2+) signals in insulin granule exocytosis are integrated in a specialized domain of pancreatic beta cells to facilitate stimulus-secretion coupling.     

ATP and Ca binding by the Ca pump protein of sarcoplasmic reticulum

The binding of ATP and Ca²⁺ by the Ca²⁺ pump protein of sarcoplasmic reticulum from rabbit skeletal muscle has been studied and correlated with the formation of a phoshorylated intermediate. The Ca²⁺ pump protein has been found to contain one specific ATP and two specific Ca²⁺ binding sites per phosphorylation site. ATP binding is dependent on Mg²⁺ and is severely decreased when a phosphorylated intermediate is formed by the addition of Ca²⁺. In the presence of Mg²⁺ and the absence of Ca²⁺, ATP and ADP bind completely to the membrane. Pre-incubation with N-ethylmaleimide results in inhibition of ATP binding and decrease of Ca²⁺ binding. In the absence of ATP, Ca²⁺ binding is noncooperative at pH 6–7 and negatively cooperative at pH 8. Mg²⁺, Sr²⁺ and La³⁺, in that order, decrease Ca²⁺ binding by the Ca²⁺ pump protein. The affinity of the Ca²⁺ pump protein for both ATP and Ca²⁺ increases when the pH is raised from 6 to 8. At the infection point (pH ≈ 7.3) the binding constants of the Ca²⁺ pump protein-MgATP²⁻ and Ca²⁺ pump protein-calcium complexes are approx. 0.25 and 0.5 μM⁻¹, respectively. The unphosphorylated Ca²⁺ pump protein does not contain a Mg²⁺ binding site with an affinity comparable to those of the ATP and Ca²⁺ binding sites.The affinity of the Ca²⁺ pump protein for Ca²⁺ is not appreciably changed by the addition of ATP. The ratio of phosphorylated intermediate formed to bound Ca²⁺ is close to 2 over a 5-fold range of phosphoenzyme concentration. The equilibrium constant for phosphoenzyme formation is less than one at saturating levels of Ca²⁺. The phosphoenzyme is thus a “high-energy” intermediate, whose energy may then be used for the translocation of the two Ca²⁺.A reaction scheme is discussed showing that phosphorylation of sarcoplasmic reticulum proceeds via an enzyme-Ca₂²⁺-MgATP²⁻ complex. This complex is then converted to a phosphoenzyme intermediate which binds two Ca²⁺ and probably Mg²⁺.     

VAMP2 and synaptotagmin mobility in chromaffin granule membranes: implications for regulated exocytosis

Granule-plasma membrane docking and fusion can only occur when proteins that enable these reactions are present at the granule-plasma membrane contact. Thus, the mobility of granule membrane proteins may influence docking and membrane fusion. We measured the mobility of vesicle associated membrane protein 2 (VAMP2), synaptotagmin 1 (Syt1), and synaptotagmin 7 (Syt7) in chromaffin granule membranes in living chromaffin cells. We used a method that is not limited by standard optical resolution. A bright flash of strongly decaying evanescent field produced by total internal reflection was used to photobleach GFP-labeled proteins in the granule membrane. Fluorescence recovery occurs as unbleached protein in the granule membrane distal from the glass interface diffuses into the more bleached proximal regions, enabling the measurement of diffusion coefficients. We found that VAMP2-EGFP and Syt7-EGFP are mobile with a diffusion coefficient of ∼3 × 10⁻¹⁰ cm²/s. Syt1-EGFP mobility was below the detection limit. Utilizing these diffusion parameters, we estimated the time required for these proteins to arrive at docking and nascent fusion sites to be many tens of milliseconds. Our analyses raise the possibility that the diffusion characteristics of VAMP2 and Syt proteins could be a factor that influences the rate of exocytosis.     

Calcyclin-like protein from Ehrlich ascites tumour cells. Ca2+ and Zn2+ binding, distribution and target protein

The calcyclin-like protein from Ehrlich ascites tumour cells is a 10.5 kDa heat stable protein, which binds two Ca2+ ions each with different affinity. Upon Ca2+ binding, the protein changes its conformation exposing hydrophobic regions. In this conformation it is able to interact with fluphenazine and with a 36 kDa protein immunologically similar to mammalian calpactin. Calcyclin-like protein binds Zn2+ and forms dimers like other members of the S-100 protein family. The calcyclin-like protein is present in several mouse tissues such as stomach, skeletal muscle, heart, spleen, lung and kidney, but seems to be absent from brain, intestine and liver as well as from some tumorigenic cells lines.     

Characterization of the chromobindins. Soluble proteins that bind to the chromaffin granule membrane in the presence of Ca2+

A group of proteins that bind to the chromaffin granule membrane in the presence of Ca2+ has been isolated by affinity chromatography of bovine adrenal medullary cytosol on granule membranes coupled to Sepharose 4B. Twenty-two of these proteins were resolved into classes depending upon the Ca2+ concentration at which they were eluted from the affinity column (40 or 0.1 microM), upon their affinities for native granule membranes or for liposomes prepared from extracted granule lipids, and upon the requirement of seven of the proteins for ATP in the cytosol fraction and column buffers to promote binding. The molecular weights and isoelectric points of these proteins were determined by two-dimensional electrophoresis. Two of the granule-binding proteins were identified: synexin and calmodulin. Calmodulin was found to bind to seven specific granule membrane proteins after diffusion of 125I-labeled calmodulin into an acrylamide gel of membrane proteins separated by electrophoresis in the presence of sodium dodecyl sulfate. A phospholipid-activated protein kinase activity, possibly due to protein kinase C, was present in the granule-binding fraction. Two major granule-binding proteins were found to present a pattern in two-dimensional electrophoresis that was very similar to but shifted slightly toward the basic end of the gel from the pattern generated by light chains associated with clathrin in adrenal medullary coated vesicles. In the chromaffin cell, these proteins, by associating with the granule membrane in the presence of an increased cytosolic Ca2+ concentration, might play a variety of roles in the process of exocytosis.     

ATP synthesis by Ca2+ + Mg2+-ATPase in detergent solution at constant Ca2+ levels.

ATP has been synthesized by the purified Ca2+ + Mg2+-dependent ATPase from sarcoplasmic reticulum (SR) solubilized in nonionic detergent dodecyloctaoxyethylenglycol-monoether in a solution containing inorganic phosphate and glycerol by changing pH upon addition of ADP. The Ca2+ concentration is kept constant during the experiment. Optimum synthesis is found at CaCl2 = 0.6 mM and the delta pH = 2.9 +/- 0.2. The enzyme has been digested by trypsin for 1 and 20 min, and it is found that synthesis of ATP is correlated with the Ca2+-uptake into SR. The data indicate that the enzyme alone is responsible for active transport of Ca2+ in SR. The driving force for the ATP synthesis of the process may be due to various ion-protein interactions. H+ cannot substitute for Ca2+ in the synthesis of ATP but acts probably through a modification of the Ca2+ binding sites. The data give support that the integrity of the enzyme molecule between its hydrolytic site and the Ca2+-binding sites is essential for the overall Ca2+ transport.     

Matrix free Ca2+ in isolated chromaffin vesicles

Isolated secretory vesicles from bovine adrenal medulla contain 80 nmol of Ca2+ and 25 nmol of Mg2+ per milligram of protein. As determined with a Ca2+-selective electrode, a further accumulation of about 160 nmol of Ca2+/mg of protein can be attained upon addition of the Ca2+ ionophore A23187. During this process protons are released from the vesicles, in exchange for Ca2+ ions, as indicated by the decrease of the pH in the incubation medium or the release of 9-aminoacridine previously taken up by the vesicles. Intravesicular Mg2+ is not released from the vesicles by A23187, as determined by atomic emission spectroscopy. In the presence of NH4Cl, which causes the collapse of the secretory vesicle transmembrane proton gradient (delta pH), Ca2+ uptake decreases. Under these conditions A23187-mediated influx of Ca2+ and efflux of H+ cease at Ca2+ concentrations of about 4 microM. Below this concentration Ca2+ is even released from the vesicles. At the Ca2+ concentration at which no net flux of ions occurs the intravesicular matrix free Ca2+ equals the extravesicular free Ca2+. In the absence of NH4Cl we determined an intravesicular pH of 6.2. Under these conditions the Ca2+ influx ceases around 0.15 microM. From this value and the known pH across the vesicular membrane an intravesicular matrix free Ca2+ concentration of about 24 microM was calculated. This is within the same order of magnitude as the concentration of free Ca2+ in the vesicles determined in the presence of NH4Cl.(ABSTRACT TRUNCATED AT 250 WORDS)     

A brain-specific Ca2+/calmodulin-dependent protein kinase (CaM kinase-Gr) is regulated by autophosphorylation. Relevance to neuronal Ca2+ signaling.

A neuronal Ca2+/calmodulin-dependent protein kinase (CaM kinase-Gr) undergoes autophosphorylation on a serine residue(s) in response to Ca2+ and calmodulin. Phosphate incorporation leads to the formation of a Ca(2+)-independent (autonomous) activity state, as well as potentiation of the Ca2+/calmodulin-dependent response. The autonomous enzyme activity of the phosphorylated enzyme approximately equals the Ca2+/calmodulin-stimulated activity of the unphosphorylated enzyme, but displays diminished affinity toward ATP and the synthetic substrate, syntide-2. The Km(app) for ATP and syntide-2 increased 4.3- and 1.7-fold, respectively. Further activation of the autonomous enzyme by Ca2+/calmodulin yields a marked increase in the affinity for ATP and peptide substrate such that the Km(app) for ATP and syntide-2 decreased by 14- and 8-fold, respectively. Both autophosphorylation and the addition of Ca2+/calmodulin are required to produce the maximum level of enzyme activation and to increase substrate affinity. Unlike Ca2+/calmodulin-dependent protein kinase type II that is dephosphorylated by the Mg(2+)-independent phosphoprotein phosphatases 1 and 2A, CaM kinase-Gr is dephosphorylated by a Mg(2+)-dependent phosphoprotein phosphatase that may be related to the type 2C enzyme. Dephosphorylation of CaM kinase-Gr reverses the effects of autophosphorylation on enzyme activity. A comparison between the autophosphorylation and dephosphorylation reactions of CaM kinase-Gr and Ca2+/calmodulin-dependent protein kinase type II provides useful insights into the operation of Ca(2+)-sensitive molecular switches.