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1.
The interaction of La 3+ with mitochondria in relation to respiration-coupled Ca 2+ transport 总被引:9,自引:0,他引:9
La3+ inhibits the respiration-dependent accumulation of Ca2+ by rat liver mitochondria when added in very small amounts (0.1–l.0 nmole per mg protein). However, La3+ itself does not activate respiration. With the use of 140La3+ it was found that La3+ is very rapidly bound to rat liver mitochondria in a respiration-independent process accompanied by loss of H+ to the medium. When both La3+ and Ca2+ are added to mitochondria simultaneously, most of the La3+ but little Ca2+ are bound. La3+ added to mitochondria previously loaded with Ca2+ is tightly bound without discharge of Ca2+. Conversely, when Ca2+ is added to La3+-loaded mitochondria it is not bound nor is the La3+ discharged. La3+ inhibits both high-affinity and low-affinity respiration-independent Ca2+ binding. Isotopic experiments showed that La3+ is, in fact, bound to the same high-affinity sites as Ca2+, in both intact mitochondria and in mitochondrial extracts. It is concluded (1) that La3+ binds to and inhibits the Ca2+ carrier; (2) that La3+ is not transported by the Ca2+ carrier; and (3) that La3+ is, in addition, bound to a large number of external sites on mitochondria for which Ca2+ is not a strong competitor. 相似文献
2.
《Journal of receptor and signal transduction research》2013,33(1-2):177-190
Abstract3H-Nimodipine (3H -NIM) is a high affinity radioligand suitable to study Ca2+ -channels in a variety of tissues. The binding is saturable, reversible, and stereospecific in purified bovine heart and partially purified guinea-pig brain membranes. In the latter a Bmax of 600fmol/mg protein, dissociation constants (KD) of 0.4-0.8nM and a Hill slope of 1.0 are found. At 37C the optimal pH in 50mM TRIS-HCl buffer is 7.1-7.4. The calcium channel is a metalloprotein, and the divalent cation which is essential for the binding of 3 H -NIM can be removed by EDTA (EC50 20μM); the nimodipine binding site of the channel may then be reconstituted by divalent cations with Mn2+ > Ca2+>Mg2+>Sr2+. Ca2+-antagonist drugs can be divided into three main classes based on their interaction with the 3H -NIM binding site: Class I has one site law of mass action-displacement isotherms with 3H -NIM, Class II exhibits complex biphasic inhibition profiles and Class III drugs increase the affinity of 1,4 dihydropyridines for the Ca2+ -channel. Diltiazem is a Class III Ca2+ -antagonist. Our in vitro studies lead us to conclude that the Ca2+ -channel contains multiple regulatory sites at which drugs can act. 相似文献
3.
Acid sensing ion channels (ASICs) are cation-selective membrane channels activated by H+ binding upon decrease in extracellular pH. It is known that Ca2+ plays an important modulatory role in ASIC gating, competing with the ligand (H+) for its binding site(s). However, the H+ or Ca2+ binding sites involved in gating and the gating mechanism are not fully known. We carried out a computational study to investigate potential cation and H+ binding sites for ASIC1 via all-atom molecular dynamics simulations on five systems. The systems were designed to test the candidacy of some acid sensing residues proposed from experiment and to determine yet unknown ligand binding sites. The ion binding patterns reveal sites of cation (Na+ and Ca2+) localization where they may compete with protons and influence channel gating. The highest incidence of Ca2+ and Na+ binding is observed at a highly acidic pocket on the protein surface. Also, Na+ ions fill in an inner chamber that contains a ring of acidic residues and that is near the channel entrance; this site could possibly be a temporary reservoir involved in ion permeation. Some acidic residues were observed to orient and move significantly close together to bind Ca2+, indicating the structural consequences of Ca2+ release from these sites. Local structural changes in the protein due to cation binding or ligand binding (protonation) are examined at the binding sites and discussed. This study provides structural and dynamic details to test hypotheses for the role of Ca2+ and Na+ ions in the channel gating mechanism. 相似文献
4.
The ryanodine receptors (RyR) are essential to calcium signaling in striated muscles. A deep understanding of the complex Ca2+-activation/inhibition mechanism of RyRs requires detailed structural and dynamic information for RyRs in different functional states (eg, with Ca2+ bound to activating or inhibitory sites). Recently, high-resolution structures of the RyR isoform 1 (RyR1) were solved by cryo-electron microscopy, revealing the location of a Ca2+ binding site for activation. Toward elucidating the Ca2+-modulation mechanism of RyR1, we performed extensive molecular dynamics simulation of the core RyR1 structure in the presence and absence of activating and solvent Ca2+ (total simulation time is >5 μs). In the presence of solvent Ca2+, Ca2+ binding to the activating site enhanced dynamics of RyR1 with higher inter-subunit flexibility, asymmetric inter-subunit motions, outward domain motions and partial pore dilation, which may prime RyR1 for subsequent channel opening. In contrast, the solvent Ca2+ alone reduced dynamics of RyR1 and led to inward domain motions and pore contraction, which may cause inhibition. Combining our simulation with the map of disease mutation sites in RyR1, we constructed a wiring diagram of key domains coupled via specific hydrogen bonds involving the mutation sites, some of which were modulated by Ca2+ binding. The structural and dynamic information gained from this study will inform future mutational and functional studies of RyR1 activation and inhibition by Ca2+. 相似文献
5.
Inesi G 《Journal of cell communication and signaling》2011,5(3):227-237
The calcium transport ATPase and the copper transport ATPase are members of the P-ATPase family and retain an analogous catalytic
mechanism for ATP utilization, including intermediate phosphoryl transfer to a conserved aspartyl residue, vectorial displacement
of bound cation, and final hydrolytic cleavage of Pi. Both ATPases undergo protein conformational changes concomitant with
catalytic events. Yet, the two ATPases are prototypes of different features with regard to transduction and signaling mechanisms.
The calcium ATPase resides stably on membranes delimiting cellular compartments, acquires free Ca2+ with high affinity on one side of the membrane, and releases the bound Ca2+ on the other side of the membrane to yield a high free Ca2+ gradient. These features are a basic requirement for cellular Ca2+ signaling mechanisms. On the other hand, the copper ATPase acquires copper through exchange with donor proteins, and undergoes
intracellular trafficking to deliver copper to acceptor proteins. In addition to the cation transport site and the conserved
aspartate undergoing catalytic phosphorylation, the copper ATPase has copper binding regulatory sites on a unique N-terminal
protein extension, and has also serine residues undergoing kinase assisted phosphorylation. These additional features are
involved in the mechanism of copper ATPase intracellular trafficking which is required to deliver copper to plasma membranes
for extrusion, and to the trans-Golgi network for incorporation into metalloproteins. Isoform specific glyocosylation contributes
to stabilization of ATP7A copper ATPase in plasma membranes. 相似文献
6.
The sarcoplasmic reticulum Ca2+-ATPase (SERCA1a) pumps Ca2+ and countertransport protons. Proton pathways in the Ca2+ bound and Ca2+-free states are suggested based on an analysis of crystal structures to which water molecules were added. The pathways are indicated by chains of water molecules that interact favorably with the protein. In the Ca2+ bound state Ca2E1, one of the proposed Ca2+ entry paths is suggested to operate additionally or alternatively as proton pathway. In analogs of the ADP-insensitive phosphoenzyme E2P and in the Ca2+-free state E2, the proton path leads between transmembrane helices M5 to M8 from the lumenal side of the protein to the Ca2+ binding residues Glu-771, Asp-800 and Glu-908. The proton path is different from suggested Ca2+ dissociation pathways. We suggest that separate proton and Ca2+ pathways enable rapid (partial) neutralization of the empty cation binding sites. For this reason, transient protonation of empty cation binding sites and separate pathways for different ions are advantageous for P-type ATPases in general. 相似文献
7.
Giuseppe Inesi Li Chen Carlota Sumbilla David Lewis Mary E. Kirtley 《Bioscience reports》1995,15(5):327-339
Three experimental systems are described including sarcoplasmic reticulum (SR) vesicles, reconstituted proteoliposomes, and recombinant protein obtained by gene transfer and expression in foreign cells. It is shown that the Ca2+ ATPase of sarcoplasmic reticulum (SR) includes an extramembranous globular head which is connected through a stalk to a membrane bound region. Cooperative binding of two calcium ions occurs sequentially, within a channel formed by four clustered helices within the membrane bound region. Destabilization of the helical cluster is produced following enzyme phosphorylation by ATP at the catalytic site in the extramembranous region. The affinity and orientation of the Ca2+ binding site are thereby changed, permitting vectorial dissociation of bound Ca2+ against a concentration gradient. A long range linkage between phosphorylation and Ca2+ binding sites is provided by an intervening peptide segment that retains high homology in cation transport ATPases, and whose function is highly sensitive to mutational perturbations. 相似文献
8.
《Cell calcium》2018
TRPM3 proteins assemble to Ca2+-permeable cation channels in the plasma membrane, which act as nociceptors of noxious heat and mediators of insulin and cytokine release. Here we show that TRPM3 channel activity is strongly dependent on intracellular Ca2+. Conceivably, this effect is attributed to the Ca2+ binding protein calmodulin, which binds to TRPM3 in a Ca2+-dependent manner. We identified five calmodulin binding sites within the amino terminus of TRPM3, which displayed different binding affinities in dependence of Ca2+. Mutations of lysine residues in calmodulin binding site 2 strongly reduced calmodulin binding and TRPM3 activity indicating the importance of this domain for TRPM3-mediated Ca2+ signaling. Our data show that TRPM3 channels are regulated by intracellular Ca2+ and provide the basis for a mechanistic understanding of the regulation of TRPM3 by calmodulin. 相似文献
9.
Robert J. Deschenes Dana C. Hilt Judith K. Marquis Henry G. Mautner 《生物化学与生物物理学报:生物膜》1981,641(1):166-172
Tb3+, a fluorescent trivalent cation with physicochemical properties similar to Ca2+, binds to peripheral nerve membrane vesicles prepared from the walking leg nerve bundle of the lobster (Homarus americanus). Saturable binding is measured for at least two classes of binding site. Bound Tb3+ can be displaced by other cations in the order: Ca2+ > Mg2+ = Zn2+ > NH4+. The binding of Tb3+ to the lower affinity site (KD(app) = 6.0 μM) is inhibitable by Na+, Mg2+ and Ca2+, whereas the higher affinity site (KD(app) = 2.2 μM) is only sensitive to Ca2+. Using this spectral probe the role of Ca2+ in peripheral nerve membrane function can be investigated. 相似文献
10.
Using murexide (Mx), a metallochromic indicator, and a dual wavelength spectrophotometer with a high signal-to-noise ratio, the Ca++ binding in a system containing two classes of binding sites was studied. Solutions with solute containing one or two classes of Ca++ binding sites and without such solute were titrated with Ca++ using Mx as an indicator of free Ca++ concentration. Since curvilinear Scatchard plots are obtained from titration curves of solutes containing two classes of binding sites, a computer program was developed to resolve such plots into two linear partial plots, each corresponding to a single class of binding site. The validity of the procedure was examined with solutions of ethylene glycol bis(β-aminoethyl)-N-N′-tetraacetic acid, adenosine triphosphate (EGTA, ATP), or a mixture thereof. The method was also applied to biological material and it was found that a protein fraction isolated from rat skeletal muscle sarcotubular membranes, termed Fraction-2 (Fr-2), has two classes of binding sites for Ca++; the association constants of the high affinity site and low affinity site are 4.3 × 105 M-1 and 9 × 103 M-1, respectively. The advantages and limitations of this methodology are discussed. 相似文献
11.
Ca 2+ transport activity in mitochondria from some plant tissues 总被引:8,自引:0,他引:8
Mitochondria isolated from some 14 different higher plants and fungi were examined for their capacity to carry out respiration-dependent accumulation of Ca2+. Additions of Ca2+ give little or no stimulation of state 4 respiration of plant mitochondria, although the added Ca2+ was largely accumulated. Accumulation of Ca2+ required phosphate and, in most cases, was stimulated by Mg2+ and ADP or ATP. Ca2+ uptake was abolished by respiratory inhibitors and uncoupling agents. The ratio of Ca2+ ions taken up per pair of electrons per energy-conserving site was normal at about 2.0 for mitochondria from sweet potato and white potato; mitochondria from other plants showed somewhat lower ratios. Accumulated Ca2+ was only very slowly released from previously loaded plant mitochondria. Respiration-inhibited sweet potato mitochondria show both high-affinity and low-affinity Ca2+ binding sites sensitive to uncouplers, La3+, and ruthenium red and thus resemble animal mitochondria. Most other plant mitochondria lack high affinity sites. In general, mitochondria from sweet potato and white potato tubers resemble those from animal tissues, but mitochondria from carrots, beets, turnips, onions, cabbage, artichokes, cauliflower, avocados, mung bean and corn seedlings, and mushrooms show rather low affinity and activity in accumulation of Ca2+, probably due to lack of a specific Ca2+ carrier. 相似文献
12.
《Journal of receptor and signal transduction research》2013,33(6):975-987
AbstractThe effects of Ca2+ ions on 3H-RO 5–4864 binding to the peripheral benzodiazepine receptor were examined. Preincubation of rat kidney membranes with Ca2+ at 37°C produced a dose-dependent inhibition of 3H-RO 5–4864 binding. No inhibition was observed in membranes preincubated at 0°C.The effect of Ca2+ was competitive in nature and was fully reversed by the addition of EGTA. At 1 mM, the maximal effect was achieved with CaCl2, whereas CoCl2 and CdCl2 had lesser effects. No other divalent cation salts examined decreased 3H-RO 5–4864 binding to rat kidney membranes. Collectively, these data demonstrate that the affinity of 3H-RO 5–4864 binding to rat kidney membranes is regulated by Ca2+ and suggest the presence of cation recognition binding sites coupled to the peripheral benzodiazepine receptor. 相似文献
13.
Chloride salts of Li+, Na+, K+, Mg2+, Ca2+, Cr3+, Mn2+, Fe2+, and Fe3+ had no effect on [3H]diazepam binding. Chloride salts of Co2+, Ni2+, Cu2+, and Zn2+ increased [3H]diazepam binding by 34 to 68% in a concentration-dependent fashion. Since these divalent cations potentiated the GABA-enhanced [3H]diazepam binding and the effect of each divalent cation was nearly additive with GABA, these cations probably act at a site different from the GABA recognition site in the benzodiazepine-receptor complex. Scatchard plots of [3H]diazepam binding without an effective divalent cation showed a single class of binding, with a Kd value of 5.3 mM. In the presence of 1 mM Co2+, Ni2+, Cu2+, or Zn2+, two distinct binding sites were evident with apparent Kd values of 1.0 nM and 5.7 nM. The higher-affinity binding was not detected in the absence of an effective divalent cation and is probably a novel, super-high-affinity binding site. 相似文献
14.
Calcium-activated chloride channels (CaCC) encoded by family members of transmembrane proteins of unknown function 16 (TMEM16) have recently been intensely studied for functional properties as well as their physiological roles as chloride channels in various tissues. One technical hurdle in studying these channels is the well-known channel rundown that frequently impairs the precision of electrophysiological measurements for the channels. Using experimental protocols that employ fast-solution exchange, we circumvented the problem of channel rundown by normalizing the Ca2+-induced current to the maximally-activated current obtained within a time period in which the channel rundown was negligible. We characterized the activation of the TMEM16A-encoded CaCC (also called ANO1) by Ca2+, Sr2+, and Ba2+, and discovered that Mg2+ competes with Ca2+ in binding to the divalent-cation binding site without activating the channel. We also studied the permeability of the ANO1 pore for various anions and found that the anion occupancy in the pore–as revealed by the permeability ratios of these anions–appeared to be inversely correlated with the apparent affinity of the ANO1 inhibition by niflumic acid (NFA). On the other hand, the NFA inhibition was neither affected by the degree of the channel activation nor influenced by the types of divalent cations used for the channel activation. These results suggest that the NFA inhibition of ANO1 is likely mediated by altering the pore function but not through changing the channel gating. Our study provides a precise characterization of ANO1 and documents factors that can affect divalent cation activation and NFA inhibition of ANO1. 相似文献
15.
Using 45Ca2+ and 153Gd3+ we studied the effects of binding the lanthanide ion, gadolinium, to skeletal muscle G-actin. Gd3+ can specifically displace 6–7 Ca2+ from their binding sites on actin. Furthermore, a total of 6–7 Gd3+ can be shown to bind to actin, and this result is not affected by the subsequent addition of polymerizing quantities of KCl. We conclude that Gd3+ binds only to the Ca2+-binding sites of actin. The number of these Gd3+ sites closely corresponds to the known number of high and low affinity sites for divalent cations such as Ca2+. 相似文献
16.
Zhenhui Chen Brandy L. Akin Larry R. Jones 《The Journal of biological chemistry》2010,285(5):3253-3260
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 Pi 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. 相似文献
17.
Robert F. Steiner 《Biopolymers》1984,23(6):1121-1135
The interactive binding by calmodulin of Ca2+ and 1-anilinonaphthalene-8-sulfonate (1,8-ANS) has been examined. In the presence of saturating levels of Ca2+, calmodulin develops one moderately strong binding site for 1,8-ANS, plus one or more weaker sites. The binding of 1,8-ANS by unliganded, or singly liganded, calmodulin is slight; the development of a strong binding site, as well as the characteristic fluorescence enhancement and CD spectrum, requires the binding of two Ca2+ ions. Little further change occurs on binding additional Ca2+ ions. 相似文献
18.
The protein-sensitized fluorescence of Tb3+ was used as a probe for cation binding sites on synaptic vesicles. Competition studies show that the order of affinity for the sites is Cu2+ > Mn2+ > Ca2+ > Mg2+ and Zn2+ is inactive. Fluorescence quenching studies indicate that the site is superficial and the effect of pH suggests that histidine is involved in the binding. Measurements of enzyme activities in the presence of lanthanides reveal that the metal binding site identified by Tb3+ fluorescence is not the Cu2+ site associated with dopamine-β-hydroxylase. Terbium inhibits Ca2+-stimulated ATPase but not Mg2+-stimulated ATPase activities of the synaptic vesicle fraction. A kinetic analysis indicates that the site monitored by Tb3+ fluorescence may be a component of the Ca2+-stimulated ATPase. It is also suggested that Mg2+ and especially Cu2+ may bind to the sites in vivo, serving as a bridge between vesicles and other synaptic components such as the presynaptic plasma membrane. 相似文献
19.
Gerhard Meissner 《生物化学与生物物理学报:生物膜》1973,298(4)
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+. 相似文献
20.
《Channels (Austin, Tex.)》2013,7(2):125-129
The 30+ members of the family of TRP channels are diverse in their physiological roles, yet the mechanisms that regulate their gating may be conserved. In particular, all TRP channels show an activity-dependent inhibition which is mediated by Ca2+. The mechanism by which Ca2+ inhibits TRP channels is currently a matter of intense debate, with Ca2+-regulated kinases, phosphatases, phospholipases, and calmodulin all proposed to be involved. In this review, we will discuss different mechanisms for Ca2+-dependent desensitization in TRP channels. We will conclude with a model that focuses on Ca2+-dependent activation of phospholipase C and Ca2+ binding to calmodulin and propose that the phospholipase C and calmodulin pathways are structurally and functionally coupled. 相似文献