质膜钙离子ATP酶

钙离子(Ca2+)是重要的第二信使,通过与效应蛋白的结合和解离,以及在不同细胞器之间的穿梭运动而精确调控细胞活动,参与多种重要生命过程。细胞内具有精确调节Ca2+时空分布的调控系统。在静息状态下,细胞内的游离Ca2+浓度约为100 nmol/L;而当细胞受到信号刺激后,胞内的Ca2+浓度可上升至1000 nmol/L甚至更高。细胞中存在多种跨膜运送Ca2+的膜蛋白,以精确调节Ca2+浓度的时空动态变化,其中,细胞质膜上的多种Ca2+通道(包括电压门控通道、受体门控通道、储存控制通道等),以及内质网/肌质网和线粒体等胞内"钙库"膜上的雷诺丁受体、三磷酸肌醇受体等膜蛋白复合物,均可提升胞内Ca2+浓度,而细胞质膜上的钠钙交换体、质膜Ca2+-ATP酶、"钙库"膜上的内质网Ca2+-ATP酶、线粒体Ca2+单向转运体等,可将Ca2+浓度降低至静息态水平。质膜钙ATP酶是向细胞外运送Ca2+的关键膜蛋白,本文将对其结构、功能及其酶活性的调控机制做一简要综述。     

Rapidly exchanging Ca2+ stores of non-muscle cells

The rapid and transient redistribution of calcium from intracellular stores is a key event of cell activation. The nature and molecular composition of intracellular Ca2+ stores of non-muscle cells are the object of intense investigation. In this paper, we review: (a) the experimental evidence in favor of the existence of intracellular, membrane-bound compartments specialized for uptake, storage and release of calcium, (b) the main protein components of rapidly exchanging Ca2+ stores, i.e. Ca2+ pump, intralumenal Ca2+ binding proteins (calsequestrin, calreticulin, etc.) and Ca2+ channels sensitive to either inositol 1,4,5-trisphosphate or Ca2+, caffeine and ryanodine, and (c) the relationship between Ca2+ stores and the endoplasmic reticulum.     

Quantification of Ca(2+) binding to melanin supports the hypothesis that melanosomes serve a functional role in regulating calcium homeostasis

Calcium regulation in melanocytes affects numerous biological pathways including protecting the redox balance in the cell and regulating the supply of substrate, l-tyrosine, for melanogenesis. The pigment contained in the melanocytes, melanin, has been implicated in maintaining calcium homeostasis in the cell and is known to be involved with calcium ion regulation in the inner ear. Herein, the association constant for Ca(2+) binding to Sepia melanin is determined by isothermal titration calorimetry to be 3.3 (+/-0.2) x 10(3)/M. This value is comparable with other well-established intracellular calcium-binding proteins that serve to buffer calcium concentrations, lending further support to the hypothesis that melanosomes serve as intracellular mediators of calcium homeostasis in melanocytes. Using this binding constant and the data from a fluorescent Ca(2+) displacement assay, the pK(a) of the carboxyl group coordinated to Ca(2+) is determined to be 3.1 +/- 0.1.     

钙调蛋白及其结合蛋白对神经递质释放的调控作用

钙离子（Ca2+）是调节突触前神经递质的胞吐释放的关键离子信号.作为胞内最普遍存在的钙离子感受器的钙调蛋白（CaM）被发现能通过与多种蛋白的相互作用,调控着突触小泡的生发、运输及再填充,从而传递胞内Ca2+浓度变化的信号,对神经递质的释放及突触电生理活动起到至关重要的调控作用.本文综述了CaM及其结合蛋白是如何参与对突触小泡的胞吐释放和胞吞恢复的调控,并探讨了其中可能的分子机制.     

An interaction-based analysis of calcium-induced conformational changes in Ca2+ sensor proteins.

Calcium sensor proteins translate transient increases in intracellular calcium levels into metabolic or mechanical responses, by undergoing dramatic conformational changes upon Ca2+ binding. A detailed analysis of the calcium binding-induced conformational changes in the representative calcium sensors calmodulin (CaM) and troponin C was performed to obtain insights into the underlying molecular basis for their response to the binding of calcium. Distance difference matrices, analysis of interresidue contacts, comparisons of interhelical angles, and inspection of structures using molecular graphics were used to make unbiased comparisons of the various structures. The calcium-induced conformational changes in these proteins are dominated by reorganization of the packing of the four helices within each domain. Comparison of the closed and open conformations confirms that calcium binding causes opening within each of the EF-hands. A secondary analysis of the conformation of the C-terminal domain of CaM (CaM-C) clearly shows that CaM-C occupies a closed conformation in the absence of calcium that is distinct from the semi-open conformation observed in the C-terminal EF-hand domains of myosin light chains. These studies provide insight into the structural basis for these changes and into the differential response to calcium binding of various members of the EF-hand calcium-binding protein family. Factors contributing to the stability of the Ca2+-loaded open conformation are discussed, including a new hypothesis that critical hydrophobic interactions stabilize the open conformation in Ca2+ sensors, but are absent in "non-sensor" proteins that remain closed upon Ca2+ binding. A role for methionine residues in stabilizing the open conformation is also proposed.     

Modeling the interrelations between the calcium oscillations and ER membrane potential oscillations

A refined electrochemical model accounting for intracellular calcium oscillations and their interrelations with oscillations of the potential difference across the membrane of the endoplasmic reticulum (ER) or other intracellular calcium stores is established. The ATP dependent uptake of Ca2+ from the cytosol into the ER, the Ca2+ release from the ER through channels following a calcium-induced calcium release mechanism, and a potential-dependent Ca2+ leak flux out of the ER are included in the model and described by plausible rate laws. The binding of calcium to specific proteins such as calmodulin is taken into account. The quasi-electroneutrality condition allows us to express the transmembrane potential in terms of the concentrations of cytosolic calcium and free binding sites on proteins, which are the two independent variables of the model. We include monovalent ions in the model, because they make up a considerable portion in the balance of electroneutrality. As the permeability of the endoplasmic membrane for these ions is much higher than that for calcium ions, we assume the former to be in Nernst equilibrium. A stability analysis of the steady-state solutions (which are unique or multiple depending on parameter values) is carried out and the Hopf bifurcation leading from stable steady states to self-sustained oscillations is analysed with the help of appropriate mathematical techniques. The oscillations obtained by numerical integration exhibit the typical spike-like shape found in experiments and reasonable values of frequency and amplitude. The model describes the process of switching between stationary and pulsatile regimes as well as changes in oscillation frequency upon parameter changes. It turns out that calcium oscillations can arise without a permanent influx of calcium into the cell, when a calcium-buffering system such as calmodulin is included.     

A compact intermediate state of calmodulin in the process of target binding

Calmodulin undergoes characteristic conformational changes by binding Ca(2+), which allows it to bind to more than 300 target proteins and regulate numerous intracellular processes in all eukaryotic cells. We measured the conformational changes of calmodulin upon Ca(2+) and mastoparan binding using the time-resolved small-angle X-ray scattering technique combined with flash photolysis of caged calcium. This measurement system covers the time range of 0.5-180 ms. Within 10 ms of the stepwise increase in Ca(2+) concentration, we identified a distinct compact conformational state with a drastically different molecular dimension. This process is too fast to study with a conventional stopped-flow apparatus. The compact conformational state was also observed without mastoparan, indicating that the calmodulin forms a compact globular conformation by itself upon Ca(2+) binding. This new conformational state of calmodulin seems to regulate Ca(2+) binding and conformational changes in the N-terminal domain. On the basis of this finding, an allosteric mechanism, which may have implications in intracellular signal transduction, is proposed.     

Calcium distribution and exchange in the rat uterus

The calcium content and distribution of the rat uterus were determined employing flame photometry and Ca⁴⁵ determinations. The total uterine calcium concentration was found to be 2.25 millimoles (mmoles) per kilogram wet weight, 0.45 of which was inexchangeable. The exchangeable Ca could be divided into 0.8 mmole/kg wet weight extracellular and 1.0 mmole/kg wet weight intracellular. The concentration of ionic Ca in rat serum was obtained by equilibrium dialysis as 1.5 mM or 53 % of the total serum Ca. The observed Ca distribution required that its active transport be postulated, since the membrane was shown to be permeable to Ca and the internal Ca concentration was far below its electrochemical equilibrium value. Metabolic inhibition by iodoacetate or dinitrophenol caused a net Ca uptake, but cooling to 4°C and ouabain did not. Iodoacetate did not affect the Ca⁴⁵ efflux, but did increase the influx, suggesting that active Ca transport is accomplished by an exclusion mechanism. In experiments with varied external sodium concentrations, no evidence was obtained that sodium competes with calcium for inward transport. Cellular Ca binding was measured under conditions of prolonged metabolic inhibition, which abolished both active transport and the membrane potential. The association constants obtained were compatible with intracellular Ca binding to proteins, but insufficient to account for the low level of intracellular ionic Ca believed essential for relaxation. Hence metabolically dependent intracellular Ca binding was postulated. The Ca⁴⁵ efflux was slowed down by Ca-free efflux media. The presence of Sr or EDTA could completely prevent this decrease in efflux rate, and Ba could partly prevent it. Changes in Mg and Na concentration did not affect the rate of Ca⁴⁵ efflux. A model to explain Ca exchange across smooth muscle membranes has been proposed.     

Calcium binding sequences in calmyrin regulates interaction with presenilin-2

Calmyrin is a myristoylated calcium binding protein that contains four putative EF-hands. Calmyrin interacts with a number of proteins, including presenilin-2 (PS2). However, the biophysical properties of calmyrin, and the molecular mechanisms that regulate its binding to different partners, are not well understood. By site-directed mutagenesis and Ca2+ binding studies, we found that calmyrin binds two Ca2+ ions with a dissociation constant of approximately 53 microM, and that the two C-terminal EF-hands 3 and 4 bind calcium. Using ultraviolet spectroscopy, circular dichroism (CD), and NMR, we found that Ca(2+)-free and -bound calmyrin have substantially different protein conformations. By yeast two-hybrid assays, we found that both EF-hands 3 and 4 of calmyrin must be intact for calmyrin to interact with PS2-loop sequences. Pulse-chase studies of HeLa cells transfected with calmyrin expression constructs indicated that wild-type (Wt) calmyrin has a half-life of approximately 75 min, whereas a mutant defective in myristoylation turns over more rapidly (half-life of 35 min). By contrast, the half-lives of calmyrin mutants with a disrupted EF-hand 3 or EF-hand 4 were 52 and 170 min, respectively. Using immunofluorescence staining of HeLa cells transfected with Wt and mutant calmyrin cDNAs, we found that both calcium binding and myristoylation are important for dynamic intracellular targeting of calmyrin. Double immunofluorescence microscopy indicated that Wt and myristoylation-defective calmyrin proteins colocalize efficiently and to the same extent with PS2, whereas calmyrin mutants defective in calcium binding display less colocalization with PS2. Our results suggest that calmyrin functions as a calcium sensor and that calcium binding sequences in calmyrin are important for interaction with the PS2 loop.     

Ca2+ binding by Myxicola neurofilament proteins

Titrimetric, 45Ca dialysis, and autoradiographic methods were used to examine how axoplasmic proteins from the giant neuron of the marine annelid Myxicola infundibulum bind calcium. Following the autoradiographic method of Maruyama et al., the 150-160 kD neurofilament subunits were identified as prominent intracellular Ca-binding peptides. Using equilibrium dialysis, extracts of axoplasmic proteins (greater than 50% neurofilament subunits) were examined in 300 mM KCl at different concentrations of free Ca and Mg, and at different pH. Axoplasmic proteins showed a high affinity Ca binding site (K1/2 3-6 microM, capacity 3-7 mumole g-1 protein) at pH 6.8 or pH 7.5. Changing the Mg concentration from 0 to 5 mM had no effect on the Ca binding. Elevating the dialysis pH from 7.0 to 9.0 reduced the apparent number of binding sites for Ca. Using microelectrodes to record the free Ca, microtitrations of axoplasmic proteins were completed by adding small amounts of CaCl2 to 100 microliters volumes of protein solutions. In a medium containing ionic constituents closely resembling those of the Myxicola axon, a Ca binding capacity of 5.0 mumole g-1 protein and a K1/2 of approximately 1 microM were measured.     

A buffering model for calcium-dependent neurotransmitter release

A simple model is proposed, whereby a single buffering system for intracellular calcium accounts for the steep external Ca dependence of neurotransmitter release during depolarization of the presynaptic nerve terminal. Ca entry and buffering in the nerve terminal are assumed to be saturable; release is assumed to be proportional to intracellular Ca. The novel feature of this model is that it explains the apparent cooperative relationship between transmitter release and extracellular calcium, without invoking cooperative Ca binding.     

Dynamic and structural properties of the calcium binding site of bovine serine proteases and their zymogens. A multinuclear nuclear magnetic resonance and stopped-flow study

The combined use of 43Ca and 113Cd nuclear magnetic resonance (n.m.r.) has provided information on the structural and dynamic properties of the calcium binding site located in homologous positions in bovine beta-trypsin, alpha-chymotrypsin and their zymogens. The 43Ca and 113Cd n.m.r. chemical shifts are consistent with an octahedral symmetry of the binding site and with the substitution of one of the two carboxylate ligands present in trypsin(ogen) with a neutral ligand in chymotrypsin(ogen). The constancy of the 113Cd n.m.r. chemical shifts upon binding of the pancreatic trypsin inhibitor and/or the dipeptide Ile-Val to trypsinogen confirms that structural changes in the activation domain do not affect the calcium binding site. The exchange between bound and "free" (solvated) Ca2+ is slow on the 43Ca n.m.r. time-scale for trypsin(ogen), but falls in the intermediate exchange region for chymotrypsin(ogen). In trypsin, the Ca2+ off-rate was measured by stopped-flow making use of the calcium indicator 1,2-bis(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid and was found to be 3(+/- 1) s-1. In chymotrypsin(ogen) the off-rates calculated from the 43Ca n.m.r. data are 70 s-1 and 350 s-1, respectively. The dynamic properties of the calcium binding site of serine (pro)enzymes have been related to the flexibility of the binding site itself and have been compared to those of other extracellular and intracellular calcium binding proteins.     

Multiple calcium binding sites make calmodulin multifunctional

Protein-protein or protein-ion interactions with multisite proteins are essential to the regulation of intracellular and extracellular events. There is, however, limited understanding of how ligand-multisite protein interactions selectively regulate the activities of multiple protein targets. In this paper, we focus on the important calcium (Ca(2+)) binding protein calmodulin (CaM), which has four Ca(2+) ion binding sites and regulates the activity of over 30 other proteins. Recent progress in structural studies has led to significant improvements in the understanding of Ca(2+)-CaM-dependent regulation mechanisms. However, no quantitative model is currently available that can fully explain how the structural diversity of protein interaction surfaces leads to selective activation of protein targets. In this paper, we analyze the multisite protein-ligand binding mechanism using mathematical modelling and experimental data for Ca(2+)-CaM-dependent protein targets. Our study suggests a potential mechanism for selective and differential activation of Ca(2+)-CaM targets by the same CaM molecules, which are involved in a variety of intracellular functions. The close agreement between model predictions and experimental dose-response curves for CaM targets available in the literature suggests that such activation is due to the selective activity of CaM conformations in complexes with variable numbers of Ca(2+) ions. Although the paper focuses on the Ca(2+)-CaM pair as a particularly data rich example, the proposed model predictions are quite general and can easily be extended to other multisite proteins. The results of the study may therefore be proposed as a general explanation for multifunctional target regulation by multisite proteins.     

Calcium signal transduction from caveolae

Caveolae are specialized membrane microdomains that are found on the plasma membrane of most cells. Recent studies indicate that a variety of signaling molecules are highly organized in caveolae, where their interactions initiate specific signaling cascades. Molecules enriched in this membrane include G protein-coupled receptors, heterotrimeric GTP binding proteins, IP3 receptor-like protein, Ca2+ ATPase, eNOS, and several PKC isoforms. Direct measurements of calcium changes in endothelial cells suggest that caveolae may be sites that regulate intracellular Ca2+ concentration and Ca2+ dependent signal transduction. This review will focus on the role of caveolae in controlling the spatial and temporal pattern of intracellular Ca2+ signaling.     

Lanthanum influx into cultured human keratinocytes: effect on calcium flux and terminal differentiation.

Trivalent cation lanthanum (La) binds to calcium binding sites of cells and either mimics the properties of calcium or inhibits the effects of calcium by displacing calcium from its binding sites. Extracellular calcium induces differentiation of human epidermal keratinocytes in culture, in part by increasing the intracellular calcium levels (Cai). Therefore, in this study we determined the effect of La on differentiation and intracellular calcium levels of keratinocytes. We observed that La inhibited the production of cornified envelopes, a marker for terminal differentiation of keratinocytes. La inhibited the calcium requiring envelope cross-linking enzyme, transglutaminase, in a direct manner, presumably, by displacing calcium from its binding site on the enzyme. La inhibited the influx and the efflux of 45Ca from keratinocytes. Paradoxically, extracellular La appeared to increase the Cai levels of keratinocytes as measured by the fluorescent probe indo-1. However, subsequent experiments revealed that indo-1 bound La with a higher affinity than Ca and emitted fluorescence in the same wavelength as the Ca bound form. Using this probe, we observed that La enters keratinocytes in a dose-dependent fashion and achieves concentrations exceeding 80 nM when the external La concentration is raised to 300 microM. This fully accounted for the apparent increase in Cai when La was added to the cells. Treatment of cells with ionomycin increased indo-1 fluorescence maximally in the presence of La indicating influx of La via this Ca specific ionophore. Our results indicate that La enters cells and inhibits calcium mediated keratinocyte differentiation both by blocking Ca influx and by blocking calcium regulated intracellular processes such as transglutaminase directed cornified envelope formation.     

Depletion of intracellular calcium stores by calcium ionophore A23187 induces the genes for glucose-regulated proteins in hamster fibroblasts

Animal cells respond to calcium ionophore (A23187) treatment with the coordinate induction of a set of genes encoding proteins identical to the glucose-regulated proteins (GRPs). By monitoring the intracellular free calcium with the fluorescent indicator fura-2 while employing both intracellular and extracellular calcium buffers, we demonstrated that A23187 can induce the GRP94 and GRP78 genes without an increase in cytoplasmic calcium ([Ca2+]i). Induction of GRP mRNA during glucose starvation was also independent of [Ca2+]i. Instead, gene induction by A23187 was closely correlated with the depletion of intracellular calcium stores. We conclude that perturbations of sequestered calcium ions by A23187 can serve as a stimulus for gene expression.     

Birhythmicity, trirhythmicity and chaos in bursting calcium oscillations

We have analyzed various types of complex calcium oscillations. The oscillations are explained with a model based on calcium-induced calcium release (CICR). In addition to the endoplasmic reticulum as the main intracellular Ca2+ store, mitochondrial and cytosolic Ca2+ binding proteins are also taken into account. This model was previously proposed for the study of the physiological role of mitochondria and the cytosolic proteins in gene rating complex Ca2+ oscillations [1]. Here, we investigated the occurrence of different types of Ca2+ oscillations obtained by the model, i.e. simple oscillations, bursting, and chaos. In a bifurcation diagram, we have shown that all these various modes of oscillatory behavior are obtained by a change of only one model parameter, which corresponds to the physiological variability of an agonist. Bursting oscillations were studied in more detail because they express birhythmicity, trirhythmicity and chaotic behavior. Two different routes to chaos are observed in the model: in addition to the usual period doubling cascade, we also show intermittency. For the characterization of the chaotic behavior, we made use of return maps and Lyapunov exponents. The potential biological role of chaos in intracellular signaling is discussed.     

High molecular weight calcium binding protein in the microsome of scallop striated muscle

In the microsome of scallop adductor striated muscle, 30K, 55K, 90K, and 360K proteins were detected as calcium binding proteins by 45Ca autoradiography on the transferred nitrocellulose membrane after sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE). The 360K protein was directly extracted with Triton X-100 from the whole homogenate of striated portion of scallop adductor muscle and purified through DEAE cellulose and hydroxyapatite column chromatography. This purified scallop high molecular weight calcium binding protein (SHCBP) showed a faster mobility in SDS PAGE in the presence of Ca2+ than in its absence. The decrease of tryptophan fluorescence had a half maximum near pCa 7 and was slightly co-operative with Mg2+. UV absorbance was slightly increased with Ca2+. The CD spectrum also changed with Mg2+ and Ca2+. These results reflect that this SHCBP binds calcium ions under near physiological conditions. SHCBP-like high molecular weight calcium binding proteins were also detected in the smooth muscle portion of adductor muscle and branchiae of scallop by 45Ca autoradiography, but not in liver. The adductor muscle of clam had a high molecular weight calcium binding protein whose molecular weight was a little smaller than that of SHCBP. The foot of turban shell had the same molecular weight calcium binding protein as SHCBP. Stains-all, a cationic carbocyanine dye, which has been reported to stain calcium binding proteins blue, stained SHCBP blue. The spectrum of SHCBP stained with Stains-all was very similar to that of calsequestrin. Although the function of SHCBP is still unknown, it might be expected to correspond to calsequestrin of vertebrate skeletal muscle, a calcium sequestering protein, in the sarcoplasmic reticulum.     

Annexins: multifunctional components of growth and adaptation

Plant annexins are ubiquitous, soluble proteins capable of Ca(2+)-dependent and Ca(2+)-independent binding to endomembranes and the plasma membrane. Some members of this multigene family are capable of binding to F-actin, hydrolysing ATP and GTP, acting as peroxidases or cation channels. These multifunctional proteins are distributed throughout the plant and throughout the life cycle. Their expression and intracellular localization are under developmental and environmental control. The in vitro properties of annexins and their known, dynamic distribution patterns suggest that they could be central regulators or effectors of plant growth and stress signalling. Potentially, they could operate in signalling pathways involving cytosolic free calcium and reactive oxygen species.     

Trk is a calmodulin-binding protein: implications for receptor processing

The tyrosine kinase receptors for the neurotrophins (Trk) are a family of transmembrane receptors that regulate the differentiation and survival of different neuronal populations. Neurotrophin binding to Trk leads to the activation of several signalling pathways including a rapid, but moderate, increase in intracellular calcium levels. We have previously described the role of calcium and its sensor protein, calmodulin, in Trk-activated intracellular pathways. Here we demonstrate that calmodulin is able to precipitate TrkA from PC12 cell lysates. Using recombinant GST-fusion proteins containing the complete intracellular domain of TrkA, or fragments of this region, we show that calmodulin binds directly to the C-terminal domain of TrkA in a Ca2+-dependent manner. We have also co-immunoprecipitated endogenous Trk and calmodulin in primary cultures of cortical neurones. Moreover, we provide evidence that calmodulin is involved in the regulation of TrkA processing in PC12 cells. Calmodulin inhibition results in the generation of a TrkA-derived p41 fragment from the cytosolic portion of the protein. This fragment is autophosphorylated in tyrosines and can recruit PLCgamma and Shc adaptor proteins. These results suggest that calmodulin binding to Trk may be important for the regulation of Trk intracellular localization and cleavage.