Phospholipid-dependent Ca2+ binding by the 36-kDa tyrosine kinase substrate (calpactin) and its 33-kDa core

A 36-kDa protein, which is a component of the membrane skeleton, has been shown to co-localize with spectrin in addition to serving as a major substrate for tyrosine-protein kinases. This protein, which will be referred to as calpactin (for calcium-dependent phospholipid and actin binding protein), was isolated from bovine intestine as the complex with a 10-kilodalton light chain and the Ca2+ binding was analyzed by equilibrium dialysis with 45Ca2+ in the presence or absence of phospholipid. Although Ca2+ binding by calpactin alone was negligible at micromolar free Ca2+, it was greatly enhanced by liposomes containing phosphatidylserine or phosphatidylinositol. A proteolytic derivative of calpactin, termed the "core," which has lost the site of association with the light chain in addition to the site of tyrosine phosphorylation by pp60src, was also found to contain this high affinity phospholipid enhanced Ca2+-binding activity. Scatchard plots reveal that each calpactin monomer or core polypeptide bound 2 Ca2+ ions with a Kd of 4.5 X 10(-6) M at 200 micrograms of phosphatidylserine/ml. Liposome binding experiments confirmed that calpactin as a complex with light chain as well as calpactin monomer or the 33-kDa core interact with phosphatidylserine liposomes in a Ca2+-dependent manner.     

"Fluctuograms" reveal the intermittent intra-protein communication in subtilisin Carlsberg and correlate mechanical coupling with co-evolution

The mechanism of intra-protein communication and allosteric coupling is key to understanding the structure-property relationship of protein function. For subtilisin Carlsberg, the Ca2+-binding loop is distal to substrate-binding and active sites, yet the serine protease function depends on Ca2+ binding. The atomic molecular dynamics (MD) simulations of apo and Ca2+-bound subtilisin show similar structures and there is no direct evidence that subtilisin has alternative conformations. To model the intra-protein communication due to Ca2+ binding, we transform the sequential segments of an atomic MD trajectory into separate elastic network models to represent anharmonicity and nonlinearity effectively as the temporal and spatial variation of the mechanical coupling network. In analogy to the spectrogram of sound waves, this transformation is termed the "fluctuogram" of protein dynamics. We illustrate that the Ca2+-bound and apo states of subtilisin have different fluctuograms and that intra-protein communication proceeds intermittently both in space and in time. We found that residues with large mechanical coupling variation due to Ca2+ binding correlate with the reported mutation sites selected by directed evolution for improving the stability of subtilisin and its activity in a non-aqueous environment. Furthermore, we utilize the fluctuograms calculated from MD to capture the highly correlated residues in a multiple sequence alignment. We show that in addition to the magnitude, the variance of coupling strength is also an indicative property for the sequence correlation observed in a statistical coupling analysis. The results of this work illustrate that the mechanical coupling networks calculated from atomic details can be used to correlate with functionally important mutation sites and co-evolution.     

Binding of calcium ions and SNAP-25 to the hexa EF-hand protein secretagogin

Secretagogin is a hexa EF-hand protein, which has been identified as a novel potential tumour marker. In the present study, we show that secretagogin binds four Ca2+ ions (log K1=7.1+/-0.4, log K2=4.7+/-0.6, log K3=3.6+/-0.7 and log K4=4.6+/-0.6 in physiological salt buffers) with a [Ca2+](0.5) of approx. 25 microM. The tertiary structure of secretagogin changes significantly upon Ca2+ binding, but not upon Mg2+ binding, and the amount of exposed hydrophobic surface in secretagogin increases upon Ca2+ binding, but not upon Mg2+ binding. These properties suggest that secretagogin belongs to the 'sensor' family of Ca2+-binding proteins. However, in contrast with the prototypical Ca2+ sensor calmodulin, which interacts with a very large number of proteins, secretagogin is significantly less promiscuous. Only one secretagogin-interacting protein was reproducibly identified from insulinoma cell lysates and from bovine and mouse brain homogenates. This protein was identified as SNAP-25 (25 kDa synaptosome-associated protein), a protein involved in Ca2+-induced exocytosis in neurons and in neuroendocrine cells. K(d) was determined to be 1.2x10(-7) M in the presence of Ca2+ and 1.5x10(-6) M in the absence of Ca2+. The comparatively low Ca2+ affinity for secretagogin and the fact that it undergoes Ca2+-induced conformational changes and interacts with SNAP-25 raise the possibility that secretagogin may link Ca2+ signalling to exocytotic processes.     

Ca2+ regulation of interactions between endoplasmic reticulum chaperones

Casade Blue (CB), a fluorescent dye, was used to investigate the dynamics of interactions between endoplasmic reticulum (ER) lumenal chaperones including calreticulin, protein disulfide isomerase (PDI), and ERp57. PDI and ERp57 were labeled with CB, and subsequently, we show that the fluorescence intensity of the CB-conjugated proteins changes upon exposure to microenvironments of a different polarity. CD analysis of the purified proteins revealed that changes in the fluorescence intensity of CB-ERp57 and CB-PDI correspond to conformational changes in the proteins. Using this technique we demonstrate that PDI interacts with calreticulin at low Ca2+ concentration (below 100 microM), whereas the protein complex dissociates at >400 microM Ca2+. These are the Ca2+ concentrations reminiscent of Ca2+ levels found in empty or full ER Ca2+ stores. The N-domain of calreticulin interacts with PDI, but Ca2+ binding to the C-domain of the protein is responsible for Ca2+ sensitivity of the interaction. ERp57 also interacts with calreticulin through the N-domain of the protein. Initial interaction between these proteins is Ca2+-independent, but it is modulated by Ca2+ binding to the C-domain of calreticulin. We conclude that changes in ER lumenal Ca2+ concentration may be responsible for the regulation of protein-protein interactions. Calreticulin may play a role of Ca2+ "sensor" for ER chaperones via regulation of Ca2+-dependent formation and maintenance of structural and functional complexes between different proteins involved in a variety of steps during protein synthesis, folding, and post-translational modification.     

Purification and characterization of a Ca2+-binding protein in Lumbricus terrestris.

A Ca2+-binding protein which is capable of activating mammalian Ca2+-activatable cyclic nucleotide phosphodiesterase has been purified from Lumbricus terrestris and characterized. This protein and the Ca2+-dependent protein modulator from bovine tissues have many similar properties. Both proteins have molecular weights of approximately 18,000, isoelectric points of about pH 4, similar and characteristic ultraviolet spectra, and similar amino acid compositions. Both proteins bind calcium ions with high affinity. However, the protein from Lumbricus terrestris binds 2 mol of calcium ions with equal affinity, Kdiss = 6 X 10(-6) M, whereas the Ca2+-dependent protein modulator from bovine tissues binds 4 mol of calcium ions with differing affinities. Although the Ca2+-binding protein of Lumbricus terrestris activates the Ca2+-activatable cyclic nucleotide phosphodiesterase from mammalian tissues, we have failed to detect the existence of a Ca2+-activatable phosphodiesterase activity in Lumbricus terrestris. The activation of phosphodiesterase by the Ca2+-binding protein from Lumbricus terrestris is inhibited by the recently discovered bovine brain modulator binding protein (Wang, J. H., and Desai, R. (1977) J. Biol. Chem. 252, 4175-4184). Since the modulator binding protein has been shown to associate with the mammalian protein modulator to result in phosphodiesterase inhibition, it can be concluded that the Lumbricus terrestris Ca2+-binding protein also associates with the bovine brain modulator binding protein. Attempts to demonstrate the existence of a similar modulator binding protein in Lumbricus terrestris have been unsuccessful.     

Isolation and characterization of calmodulin from an insulin-secreting tumour.

A major protein constituent of a rat islet cell tumour that exhibited Ca2+-dependent changes in electrophoretic mobility has been purified to homogeneity and compared in its physicochemical and biological properties with bovine brain and rat brain calmodulin (synonymous with phosphodiesterase activator protein, calcium-dependent regulator, troponin C-like protein and modulator protein). The protein, like these calmodulins, contained trimethyl-lysine, exhibited a blocked N-terminus and had an identical amino-acid composition and molecular weight on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Peptide "maps' prepared after digestion of the three proteins with trypsin, papain or Staphylococcus V-8 proteinase were virtually superimposable. Ca2+ altered the electrophoretic mobilities the enhanced the native protein fluorescence in an equivalent manner with all three proteins. Equilibrium dialysis experiments demonstrated in each case the binding of 4g-atoms of calcium/mol of protein; the binding sites were equivalent and showed Kd 0.8 microM. Tumour and brain proteins were equipotent as Ca2+-dependent activators of partially purified rat brain cyclic nucleotide phosphodiesterase, and in this action were inhibited in an identical manner by trifluoperazine. The proteins also exhibited the common property of Ca2+-dependent binding to troponin I, histone H2B and myelin basic protein. The estimated tumour content of calmodulin was 450 mg/kg fresh wt., a value similar to that reported in islets of Langerhans. These results further document the validity of the islet cell tumour as an experimental model of Ca2+-mediated molecular events associated with insulin secretion. They also suggest that brain calmodulin may be substituted for endogenous calmodulin in experimental investigations into the mechanism of insulin secretion.     

Dansylaziridine-labeled troponin C. A new fluorescent "physiological" probe for calcium regulation by sarcoplasmic reticulum

Dansylaziridine-labeled troponin C (TnCDANZ) undergoes a greater than 2-fold fluorescence enhancement with Ca2+ binding to the Ca2+-specific regulatory sites of troponin C. Hence, TnCDANZ serves as a very good indicator of Ca2+ in the range of 3.0 to 70 micron. Ca2+ uptake and release by sarcoplasmic reticulum can be easily and accurately monitored by the fluorescence changes in TnCDANZ. In this manner, we can monitor directly the removal of Ca2+ from troponin C by the sarcoplasmic reticulum. Thus, this reaction represents a useful analogue ot the Ca2+ "on-off" process in muscle.     

The binding of calcium to a salivary phosphoprotein, protein C, and comparison with calcium binding to protein A, a related salivary phosphoprotein.

The binding of Ca2+ to a salivary phosphoprotein, protein C, was studied by equilibrium dialysis. In 5mM-Tris/HCl buffer, pH 7.5, protein C bound 190 nmol of Ca2+/mg of protein. The apparent dissociation constant, K, was determined to be 1.9 x 10(-4)M and the binding of Ca2+ to the protein was non-co-operative. The binding of Ca2+ to protein C apparently depends on groups which ionize above pH 5.0. Ca2+ binding decreased with increased concentration of the dialysis buffer and on addition of SrCL2, MgCl2 and MnCl2 to the dialysis buffer. Digestion of protein C with trypsin or collagenase or heating of the protein to 60 degrees or 100 degrees C had little or no effect on the Ca2+ binding. Digestion of protein C with alkaline phosphatase caused a decrease in the amount of protein-bound Ca2+. This was also found for another salivary phosphoprotein, protein A. In the absence of Ca2+ the S020,w for protein C was 1.29 S and in the presence of Ca2+ it was 1.46S. Ca2+ may cause a conformational change in the protein or an aggregation of the protein molecules. No conformational changes of protein C in the presence of Ca2+ could be detected by circular dichroism or nuclear magnetic resonance.     

The affinity of a major Ca2+ binding site on GRP78 is differentially enhanced by ADP and ATP

GRP78 is a major protein regulated by the mammalian endoplasmic reticulum stress response, and up-regulation has been shown to be important in protecting cells from challenge with cytotoxic agents. GRP78 has ATPase activity, acts as a chaperone, and interacts specifically with other proteins, such as caspases, as part of a mechanism regulating apoptosis. GRP78 is also reported to have a possible role as a Ca2+ storage protein. In order to understand the potential biological effects of Ca2+ and ATP/ADP binding on the biology of GRP78, we have determined its ligand binding properties. We show here for the first time that GRP78 can bind Ca2+, ATP, and ADP, each with a 1:1 stoichiometry, and that the binding of cation and nucleotide is cooperative. These observations do not support the hypothesis that GRP78 is a dynamic Ca2+ storage protein. Furthermore, we demonstrate that whereas Mg2+ enhances GRP78 binding to ADP and ATP to the same extent, Ca2+ shows a differential enhancement. In the presence of Ca2+, the KD for ATP is lowered approximately 11-fold, and the KD for ADP is lowered around 930-fold. The KD for Ca2+ is lowered approximately 40-fold in the presence of ATP and around 880-fold with ADP. These findings may explain the biological requirement for a nucleotide exchange factor to remove ADP from GRP78. Taken together, our data suggest that the Ca2+-binding property of GRP78 may be part of a signal transduction pathway that modulates complex interactions between GRP78, ATP/ADP, secretory proteins, and caspases, and this ultimately has important consequences for cell viability.     

Comparison of properties of Ca2+ release channels between rabbit and frog skeletal muscles

Biochemical investigation of Ca2+ release channel proteins has been carried out mainly with rabbit skeletal muscles, while frog skeletal muscles have been preferentially used for physiological investigation of Ca2+ release. In this review, we compared the properties of ryanodine receptors (RyR), Ca2+ release channel protein, in skeletal muscles between rabbit and frog. While the Ryr1 isoform is the main RyR of rabbit skeletal muscles, two isoforms, - and -RyR which are homologous to Ryr1 and Ryr3 isoforms in mammals, respectively, coexist as a homotetramer in a similar amount in frog skeletal muscles. The two isoforms in an isotonic medium show very similar property in [3H]ryanodine binding activity which is parallel to Ca2+-induced Ca2+ release (CICR) activity, and make independent contributions to the activities of the sarcoplasmic reticulum. CICR and [3H]ryanodine binding activities of rabbit and frog are qualitatively similar in stimulation by Ca2+, adenine nucleotide and caffeine, however, they showed the following quantitative differences. First, rabbit RyR showed higher Ca2+ affinity than the frog. Second, rabbit RyR showed higher activity in the presence of Ca2+ alone with less stimulation by adenine nucleotide than the frog. Third, rabbit RyR displayed less enhancement of [3H]ryanodine binding by caffeine in spite of having a similar magnitude of Ca2+ sensitization than the frog, which may explain the occasional difficulty by researchers to demonstrate caffeine contracture with mammalian skeletal muscles. Finally, but not least, rabbit RyR still showed marked inhibition of [3H]ryanodine binding in the presence of high Ca2+ concentrations in the 1 M NaCl medium, while frog RyR showed disinhibition. Other matters relevant to Ca2+ release were also discussed.     

Omega-conotoxin GVIA blocks a Ca2+ current in bovine chromaffin cells that is not of the "classic" N type.

Previous studies have identified two components of whole-cell Ca2+ current in bovine chromaffin cells. The "standard" component was activated by single depolarizations, while "facilitation" could be activated by large prepulses or repetitive depolarizations. Neither current component was sensitive to changes in holding potential between -100 and -50 mV; thus neither appeared to be carried by N-type Ca2+ channels. We now report that the facilitation Ca2+ current is insensitive to omega-conotoxin GVIA (omega-CgTx), but that the toxin blocks approximately 50% of the standard Ca2+ current. In some cells the toxin blocks all of the standard Ca2+ current, in others about half of the current, while in others it has no effect. Kinetic differences in current activation are observed after toxin application. These results suggest that the standard component of chromaffin cell Ca2+ current is composed of two pharmacologically distinct channels-one is omega-CgTx sensitive and the other is not. Two kinetically distinct types of 14 pS Ca2+ channels that may correspond to the omega-CgTx-sensitive and -insensitive components were observed in single-channel experiments. Because omega-CgTx blocked Ca2+ channels that were not inactivated by a depolarized holding potential, the commonly used Ca2+ channel categorization scheme may be inadequate to describe the Ca2+ channels found in chromaffin cells.     

Mapping the BKCa channel's "Ca2+ bowl": side-chains essential for Ca2+ sensing

There is controversy over whether Ca(2+) binds to the BK(Ca) channel's intracellular domain or its integral-membrane domain and over whether or not mutations that reduce the channel's Ca(2+) sensitivity act at the point of Ca(2+) coordination. One region in the intracellular domain that has been implicated in Ca(2+) sensing is the "Ca(2+) bowl". This region contains many acidic residues, and large Ca(2+)-bowl mutations eliminate Ca(2+) sensing through what appears to be one type of high-affinity Ca(2+)-binding site. Here, through site-directed mutagenesis we have mapped the residues in the Ca(2+) bowl that are most important for Ca(2+) sensing. We find acidic residues, D898 and D900, to be essential, and we find them essential as well for Ca(2+) binding to a fusion protein that contains a portion of the BK(Ca) channel's intracellular domain. Thus, much of our data supports the conclusion that Ca(2+) binds to the BK(Ca) channel's intracellular domain, and they define the Ca(2+) bowl's essential Ca(2+)-sensing motif. Overall, however, we have found that the relationship between mutations that disrupt Ca(2+) sensing and those that disrupt Ca(2+) binding is not as strong as we had expected, a result that raises the possibility that, when examined by gel-overlay, the Ca(2+) bowl may be in a nonnative conformation.     

Different conformation changes induced by calcium and terbium of the porcine intestinal calcium-binding protein

The fluorescence of 1-anilino-8-naphthalenesulfonate (ANS) is progressively enhanced with increasing concentration of it, showing a proportionate blue shift of the emission maximum, by the interaction with the porcine intestinal Ca2+-binding protein (CaBP) in the absence of Ca2+. The apo-CaBP has a single binding site for ANS as determined by the fluorescence change, the apparent dissociation constant (Kd) estimated at 49.1 microM. Addition of Ca2+ or Tb3+ to the ANS-apo-CaBP system is capable of enhancing its fluorescence up to about 2- or 5-fold, respectively, causing further blue shift of the emission maximum. These metal ions do not affect the capacity of ANS binding, but Ca2+ slightly increases the Kd value. Increase of the fluorescence of the ANS-CaBP complex by increasing binding of Ca2+ to it was monophasic, while that with Tb3+ was biphasic, both saturated at the same molar ratio, 2, of added cations to the complex. Biphasic change of response has also been observed in UV absorption of the CaBP with increasing concentration of Tb3+. With a half-saturating concentration of Tb3+, Ca2+ can induce a much higher enhancement of the ANS fluorescence than excess Ca2+ alone. All these results indicate that the CaBP molecule contains a single ANS binding site and the conformation and/or microenvironment surrounding bound ANS of the protein is altered reversibly with binding of Ca2+ or Tb3+ to it and that there are differences between Ca2+- and Tb3+-induced conformation changes around the ANS-binding site and the tyrosine residue of it.     

Innocuous character of [ethylenebis(oxyethylenenitrilo)]tetraacetic acid and EDTA as metal-ion buffers in studying Ca2+ binding by alpha-lactalbumin

The binding of EGTA and EDTA to alpha-lactalbumin, first demonstrated by Kronman and Bratcher (Kronman, M. J., and Bratcher, S. C. (1983) J. Biol. Chem. 258, 5707-5709) and afterwards regarded as a significant source of error in estimating the binding constant of Ca2+ to the protein, has been investigated by comparison of the thermal unfolding curves of the protein in the absence and presence of the chelators and also by measuring the NMR spectra of the protein, the chelators, and the protein-chelator mixtures. The unfolding curve in the presence of a large excess of each chelator has been found to be identical to that in the absence of chelator, indicating that there is essentially no interaction between the chelators and alpha-lactalbumin. The NMR results have also supported this conclusion, and the innocuous character of these chelators as metal-ion buffers in studying the Ca2+-binding properties of alpha-lactalbumin is demonstrated. In order to re-examine the binding constant for Ca2+ of alpha-lactalbumin without the aid of chelating metal-ion buffers, the thermal unfolding curve of the protein in the presence of 0.1 mM excess Ca2+ but without chelators has been compared with the unfolding curve in the absence of Ca2+ at a constant concentration of Na+ (0.010 or 0.10 M) at pH 7.0. The binding constant of alpha-lactalbumin can be calculated from the increment of melting temperature caused by the presence of Ca2+ and from the enthalpy and heat capacity changes in the unfolding. Because Ca2+ binding to the unfolded protein can be neglected under the conditions employed, the binding constant evaluated corresponds to the binding constant to protein that has native structure. The constant obtained is 3-5 X 10(9) M-1 after corrections for binding of Na+ to the protein and for ionic strength, and this shows excellent agreement with the corresponding value previously estimated (2.9 +/- 1.0 X 10(9) M-1), although the latter value was obtained in the presence of EDTA. The apparent Ca2+-binding constant that has been discussed in most previous studies, without taking account of the folding-unfolding equilibrium associated with the binding process, also depends on the concentration of monovalent cations such as Na+, and the present results lead to values of 1.5 X 10(8) and 8.7 X 10(6) M-1 at 0.01 and 0.1 M Na+, respectively.     

Inhibition of calcium flux and calcium channel antagonist binding in the PC12 neural cell line by phorbol esters and protein kinase C

Ca2+- and phospholipid-dependent protein kinase (protein kinase C) has been shown to modify receptor-mediated Ca2+ responses in a variety of cells. To assess its possible role in modulating voltage-dependent Ca2+ responses, we examined the effect of tumor-promoting phorbol esters, which activate protein kinase C, on Ca2+ channel function in the PC12 neural cell line. Phorbol 12-myristate 13-acetate reduced K+-depolarization-evoked 45Ca uptake and decreased binding of the Ca2+ channel antagonist [3H] (+)PN200-110 to intact cells. Inhibition of binding was markedly reduced in PC12 membranes, but was restored by reconstituting membranes with protein kinase C activity. Protein kinase C may therefore participate in endogenous regulation of voltage-dependent Ca2+ channels in mammalian neural cells.     

Ca2+ binding to bovine lactoferrin enhances protein stability and influences the release of bacterial lipopolysaccharide.

Bovine lactoferrin (bLf) is known to damage the outer membrane of Gram-negative bacteria by binding to bacterial lipopolysaccharide (LPS). We report that LPS is released from bacterial outer membranes also when apo- or metal-saturated Lf is separated from bacterial cells by a dialysis membrane. This process occurs in phosphate-buffered saline with no added Ca2+ and Mg2+ and is hindered by addition of these cations. The effect of bLf is similar to that induced by EDTA and has been ascribed to chelation of Ca2+. In fact, it may be envisaged that Ca2+-binding sites on LPS have different affinities and that bLf can remove those ions that are more weakly bound. Ca2+ binding does not alter Lf iron-binding properties significantly or its UV and CD spectral features but brings about changes in the FT-IR bands due to carboxylate residues. Ca2+ binding is characterized by an apparent dissociation constant of 6 microM and a stoichiometry of 1.55 Ca2+ per Lf molecule; it enhances bLf stability towards chemical and thermal denaturation. The increase in stability takes place in both the apo- and iron-saturated forms but not in the desialilated protein, indicating that the carboxylate groups of the sialic acid residues present on two of the glycan chains are involved in Ca2+ binding.     

Calcineurin is a calcium ion-dependent, calmodulin-stimulated protein phosphatase

A Ca2+-dependent, calmodulin-stimulated protein phosphatase (EC 3.1.3.16) is known to be associated with calcineurin, a major calmodulin binding protein in brain. The protein phosphatase activity has now been shown to be retained by a substrate affinity column (thiophosphorylated myosin P-light chain Sepharose) in the presence of Ca2+, and to be eluted specifically with EGTA. Calcineurin behaved identically. This establishes that calcineurin is the Ca2+-dependent protein phosphatase, and that interaction of Ca2+ with the B-subunit is essential for substrate binding.     

Fluorescence studies of the calcium binding to whiting (Gadus merlangus) parvalbumin

The calcium binding by parvalbumin of whiting (Gadus merlangus) has been studied using tryptophanyl fluorescence characteristics. Titration of Ca2+-free parvalbumin with Ca2+ leads to a very pronounced blue shift, narrowing and intensification of the fluorescence spectrum. These spectral changs proceed in two stages reflecting the existence of at least three forms which can be interpreted as (a) the protein without Ca2+, (b) with one Ca2+ and (c) with two bound Ca2+ ions/molecule. The fluorescence of these forms has been identified and the fluorescence spectra measured at varied Ca2+ concentrations were resolved into three components corresponding to these spectral forms. The dependence of the relative concentration of the three fomrs on Ca2+ concentrations agree well with the two-step binding of Ca2+ to parvalbumin: Protein + Ca in equilibrium K1 protein x Ca; Protein x Ca + Ca in equilibrium K2 Ca x protein x Ca. The equilibrium binding constants K1 and K2 obtained by the computer fit are approximately 5 X 10(8) M-1 and 6 X 10(6) M-1. This scheme and the K1 and K2 value are in a good agreement with the independent experimental data resulting from EGTA titration of Ca2+-saturated parvalbumin and pH titratin of parvalbumin in the presence of EGTA and CA2+.     

Membrane composition affects the reversibility of annexin A2t binding to solid supported membranes: a QCM study

By means of the quartz crystal microbalance (QCM) technique, we investigated the interaction of porcine heterotetrametric annexin A2t with solid supported lipid membranes. Dissociation and rate constants of annexin A2t binding to various lipid mixtures were determined as a function of Ca2+ concentrations in solution. In contrast to what has been observed for annexin A1, the binding affinity and kinetics of annexin A2t binding are not influenced by cholesterol. In the experimental setup chosen, the annexin A2t binding is strictly Ca2+-dependent and only affected by the amount of phosphatidylserine (PS) in the membrane and the Ca2+ concentration in solution. By Ca2+-titration experiments at constant annexin A2t concentration, we investigated the reversibility of annexin A2t adsorption and desorption. Surprisingly, Ca2+-titration curves display a significant hysteresis. Protein desorption curves starting from annexin A2t bound to the membrane at 1 mM CaCl2 exhibit high cooperativity with half-maximum Ca2+ concentrations in the submicromolar range. However, protein adsorption curves starting from an EGTA-containing solution with soluble annexin A2t always show two inflection points upon addition of Ca2+ ions. These two inflection points may be indicative of two protein populations differently bound to the solid-supported membrane. The ratio of these two annexin A2t populations depends on the amount of PS molecules and cholesterol in the membrane as well as on the Ca2+ concentration. We propose a model discussing the results obtained in terms of two binding sites differing in their affinity due to lipid rearrangement.     

Calcium signals mediated by STIM and Orai proteins--a new paradigm in inter-organelle communication

In all cells Ca2+ signals are key to controlling a spectrum of cellular responses. Ca2+ signals activated by phospholipase C-coupled receptors have two components-rapid Ca2+ release from ER stores followed by slower Ca2+ entry from outside the cell. The coupling process between ER and PM to mediate this "store-operated" Ca2+ entry process has remained a molecular and mechanistic mystery. Through a combination of high throughput screening and molecular physiological approaches, the machinery and mechanism of this process have been elucidated. Two proteins are key to the coupling process. STIM1, a single spanning membrane protein with an unpaired Ca2+ binding EF-hand functions as the sensor of ER luminal Ca2+ and through redistribution in the ER transduces information directly to the PM. Orai1, a tetra-spanning PM protein, functions as the highly Ca2+ selective channel in the PM that is gated through interactions with the store-activated ER Ca2+ sensor. This molecular pas-de-deux between ER and PM components represents not only a crucial signaling pathway, but also a new paradigm in inter-organelle communication.