A kinetic model for Ca2+ efflux mediated by the Ca2+ + Mg2+-activated ATPase of sarcoplasmic reticulum.
Ontology highlight
ABSTRACT:
SUBMITTER: McWhirter JM
PROVIDER: S-EPMC1148190 | biostudies-other | 1987 Aug
REPOSITORIES: biostudies-other
Similar Datasets
Project description:The Ca2+ + Mg2+-activated ATPase of sarcoplasmic reticulum exhibits complex kinetics of activation with respect to ATP. ATPase activity is pH-dependent, with similar pH-activity profiles at high and low concentrations of ATP. Low concentrations of Ca2+ in the micromolar range activate the ATPase, whereas activity is inhibited by Ca2+ at millimolar concentrations. The pH-dependence of this Ca2+ inhibition and the effect of the detergent C12E8 (dodecyl octaethylene glycol monoether) on Ca2+ inhibition are similar to those observed on activation by low concentrations of Ca2+. On the basis of these and other studies we present a kinetic model for the ATPase. The ATPase is postulated to exist in one of two conformations: a conformation (E1) of high affinity for Ca2+ and MgATP and a conformation (E2) of low affinity for Ca2+ and MgATP. Ca2+ binding to E2 and to the phosphorylated form E2P are equal. Proton binding at the Ca2+-binding sites in the E1 and E2 conformations explains the pH-dependence of Ca2+ effects. Binding of MgATP to the phosphorylated intermediate E1'PCa2 and to E2 modulate the rates of the transport step E1'PCa-E2'PCa2 and the return of the empty Ca2+ sites to the outside surface of the sarcoplasmic reticulum, as well as the rate of dephosphorylation of E2P. Only a single binding site for MgATP is postulated.
| S-EPMC1146968 | biostudies-other
Project description:In a previous paper [Gould, East, Froud, McWhirter, Stefanova & Lee (1986) Biochem. J. 237, 217-227] we presented a kinetic model for the activity of the Ca2+ + Mg2+-activated ATPase of sarcoplasmic reticulum. Here we extend the model to account for the effects on ATPase activity of Mg2+, cations and anions. We find that Mg2+ concentrations in the millimolar range inhibit ATPase activity, which we attribute to competition between Mg2+ and MgATP for binding to the nucleotide-binding site on the E1 and E2 conformations of the ATPase and on the phosphorylated forms of the ATPase. Competition is also suggested between Mg2+ and MgADP for binding to the phosphorylated form of the ATPase. ATPase activity is increased by low concentrations of K+, Na+ and NH4+, but inhibited by higher concentrations. It is proposed that these effects follow from an increase in the rate of dephosphorylation but a decrease in the rate of the conformational transition E1'PCa2-E2'PCa2 with increasing cation concentration. Li+ and choline+ decrease ATPase activity. Anions also decrease ATPase activity, the effects of I- and SCN- being more marked than that of Cl-. These effects are attributed to binding at the nucleotide-binding site, with a decrease in binding affinity and an increase in 'off' rate constant for the nucleotide.
| S-EPMC1148191 | biostudies-other
Project description:Stop-flow fluorescence and rapid-filtration methods have been used to establish the kinetics of Ca2+ binding to, and dissociation from, the (Ca(2+)-Mg2+)-ATPase of skeletal-muscle sarcoplasmic reticulum and to define the effects of H+ and Mg2+ on Ca2+ binding and dissociation rates. The kinetics have been interpreted in terms of the scheme: E2 E2<==>E1<==>E1Ca<==>E1'Ca<==>E1'Ca2. The kinetics of the E2<==>E1 E1 transition have been determined by measuring the rate of change of the fluorescence of the ATPase labelled with 4-nitrobenzo-2-oxa-1,3-diazole after a pH jump or the addition of Ca2+ to the labelled ATPase in the presence of thapsigargin or thapsivillosin A. It has been shown that Mg2+ has a marked effect on Ca2+ dissociation at pH 7.2 and that changes in the tryptophan fluorescence of the ATPase follow the same time course as the dissociation of 45Ca2+. It is proposed that the effect of Mg2+ follows from binding to a 'gating' site, as detected by changes in the fluorescence of the ATPase labelled with 4-(bromomethyl)-6,7-dimethoxycoumarin. The rate of dissociation of Ca2+ from the ATPase increases with increasing pH. The rate of dissociation of Ca2+ decreases with increasing Ca2+ concentration in the medium, with an apparent affinity for Ca2+ greater than that seen for the change in fluorescence amplitude. It is shown that this follows if the first, inner, Ca(2+)-binding site on the ATPase has a lower affinity for Ca2+ than the second, outer, site. Effects of H+ and Mg2+ on Ca2+ dissociation can be treated by the quasiequilibrium approach. Mg2+ and H+ also affect the rate of Ca2+ binding to the ATPase, and effects of H+ and Mg2+ on the E2<==>E1 equilibrium explain the results of experiments in which the concentrations of H+ and Mg2+ are jumped.
| S-EPMC1137879 | biostudies-other
Project description:The sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA) is a transmembrane ion transporter belonging to the P(II)-type ATPase family. It performs the vital task of re-sequestering cytoplasmic Ca(2+) to the sarco/endoplasmic reticulum store, thereby also terminating Ca(2+)-induced signaling such as in muscle contraction. This minireview focuses on the transport pathways of Ca(2+) and H(+) ions across the lipid bilayer through SERCA. The ion-binding sites of SERCA are accessible from either the cytoplasm or the sarco/endoplasmic reticulum lumen, and the Ca(2+) entry and exit channels are both formed mainly by rearrangements of four N-terminal transmembrane ?-helices. Recent improvements in the resolution of the crystal structures of rabbit SERCA1a have revealed a hydrated pathway in the C-terminal transmembrane region leading from the ion-binding sites to the cytosol. A comparison of different SERCA conformations reveals that this C-terminal pathway is exclusive to Ca(2+)-free E2 states, suggesting that it may play a functional role in proton release from the ion-binding sites. This is in agreement with molecular dynamics simulations and mutational studies and is in striking analogy to a similar pathway recently described for the related sodium pump. We therefore suggest a model for the ion exchange mechanism in P(II)-ATPases including not one, but two cytoplasmic pathways working in concert.
| S-EPMC3624456 | biostudies-literature
Project description:A kinetic model for the Ca2(+) + Mg2(+)-activated ATPase of sarcoplasmic reticulum was presented in a previous paper [Stefanova, Napier, East & Lee (1987) Biochem. J. 245, 723-730]. Here, that model is modified to account for the pH-dependence of ATPase activity and for the effects of Mg2+ on activity at high pH. It is shown that effects of Mg2+ on measurements of ATPase activity as a function of ATP concentration at pH 8.0 and pH 8.5 are consistent with binding of Mg2+ to the Ca2(+)-binding sites on the phosphorylated ATPase, such binding inhibiting dephosphorylation of the ATPase. It is also shown that slow dissociation of Ca2+ from the phosphorylated ATPase is consistent with the previously published model.
| S-EPMC1131306 | biostudies-other
Project description:Ca2+ + Mg2+-dependent ATPase from sarcoplasmic reticulum was inhibited by preincubation with vanadate. When the inhibited enzyme was preincubated in the presence of vanadate and assayed in its absence, a slow reactivation process was observed. This slow, hysteretic, process was exploited to study the influence of Ca2+ and ATP on the dissociation of vanadate. Ca2+ alone slowly displaced vanadate from the inhibited enzyme, and a rate constant of 0.1 min-1, at 25 degrees C, was calculated for this re-activation process. However, ATP re-activated with an apparent constant that hyperbolically depended on ATP concentration, and from it a rate constant for vanadate dissociation induced by ATP of 0.5 min-1 was calculated. It is deduced from the kinetic studies that ATP binds to the enzyme-vanadate complex, forming a ternary complex, with a dissociation constant of 4 microM, and that this binding accelerates vanadate dissociation. Binding experiments with [14C]ATP showed that ATP binds to the enzyme-vanadate complex with a dissociation constant of 12 microM, i.e. the affinities calculated with the isotope technique and the kinetic procedure are of the same order of magnitude.
| S-EPMC1144022 | biostudies-other
Project description:Sarcoplasmic reticulum Ca2+-ATPase (SERCA) is critical for cardiac Ca2+ transport. Reversal of phospholamban (PLB)-mediated SERCA inhibition by saturating Ca2+ conditions operates as a physiological rheostat to reactivate SERCA function in the absence of PLB phosphorylation. Here, we performed extensive atomistic molecular dynamics simulations to probe the structural mechanism of this process. Simulation of the inhibitory complex at superphysiological Ca2+ concentrations ([Ca2+] = 10 mm) revealed that Ca2+ ions interact primarily with SERCA and the lipid headgroups, but not with PLB's cytosolic domain or the cytosolic side of the SERCA-PLB interface. At this [Ca2+], a single Ca2+ ion was translocated from the cytosol to the transmembrane transport sites. We used this Ca2+-bound complex as an initial structure to simulate the effects of saturating Ca2+ at physiological conditions ([Ca2+]total ? 400 ?m). At these conditions, ?30% of the Ca2+-bound complexes exhibited structural features consistent with an inhibited state. However, in ?70% of the Ca2+-bound complexes, Ca2+ moved to transport site I, recruited Glu771 and Asp800, and disrupted key inhibitory contacts involving the conserved PLB residue Asn34 Structural analysis showed that Ca2+ induces only local changes in interresidue inhibitory interactions, but does not induce repositioning or changes in PLB structural dynamics. Upon relief of SERCA inhibition, Ca2+ binding produced a site I configuration sufficient for subsequent SERCA activation. We propose that at saturating [Ca2+] and in the absence of PLB phosphorylation, binding of a single Ca2+ ion in the transport sites rapidly shifts the equilibrium toward a noninhibited SERCA-PLB complex.
| S-EPMC6093236 | biostudies-literature
Project description:Sarco(endo)plasmic reticulum Ca2+-ATPase catalyzes ATP-driven Ca2+ transport from the cytoplasm to the lumen and is critical for a range of cell functions, including muscle relaxation. Here, we investigated the effects of the headgroups of the 1-palmitoyl-2-oleoyl glycerophospholipids phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), and phosphatidylglycerol (PG) on sarcoplasmic reticulum (SR) Ca2+-ATPase embedded into a nanodisc, a lipid-bilayer construct harboring the specific lipid. We found that Ca2+-ATPase activity in a PC bilayer is comparable with that of SR vesicles and is suppressed in the other phospholipids, especially in PS. Ca2+ affinity at the high-affinity transport sites in PC was similar to that of SR vesicles, but 2-3-fold reduced in PE and PS. Ca2+ on- and off-rates in the non-phosphorylated ATPase were markedly reduced in PS. Rate-limiting phosphoenzyme (EP) conformational transition in 0.1 m KCl was as rapid in PC as in SR vesicles, but slowed in other phospholipids, especially in PS. Using kinetic plots of the logarithm of rate versus the square of mean activity coefficient of solutes in 0.1-1 m KCl, we noted that PC is optimal for the EP transition, but PG and especially PS had markedly unfavorable electrostatic effects, and PE exhibited a strong non-electrostatic restriction. Thus, the major SR membrane lipid PC is optimal for all steps and, unlike the other headgroups, contributes favorable electrostatics and non-electrostatic elements during the EP transition. Our analyses further revealed that the surface charge of the lipid bilayer directly modulates the transition rate.
| S-EPMC5724008 | biostudies-literature
Project description:The integral membrane protein complex between phospholamban (PLN) and sarcoplasmic reticulum Ca(2+)-ATPase (SERCA) regulates cardiac contractility. In the unphosphorylated form, PLN binds SERCA and inhibits Ca(2+) flux. Upon phosphorylation of PLN at Ser16, the inhibitory effect is reversed. Although structural details on both proteins are emerging from X-ray crystallography, cryo-electron microscopy, and NMR studies, the molecular mechanisms of their interactions and regulatory process are still lacking. It has been speculated that SERCA regulation depends on PLN structural transitions (order to disorder, i.e., folding/unfolding). Here, we investigated PLN conformational changes upon chemical unfolding by a combination of electron paramagnetic resonance and NMR spectroscopies, revealing that the conformational transitions involve mostly the cytoplasmic regions, with two concomitant phenomena: (1) membrane binding and folding of the amphipathic domain Ia and (2) folding/unfolding of the juxtamembrane domain Ib of PLN. Analysis of phosphorylated and unphosphorylated PLN with two phosphomimetic mutants of PLN (S16E and S16D) shows that the population of an unfolded state in domains Ia and Ib (T' state) is linearly correlated to the extent of SERCA inhibition measured by activity assays. Inhibition of SERCA is carried out by the folded ground state (T state) of the protein (PLN), while the relief of inhibition involves promotion of PLN to excited conformational states (Ser16 phosphorylated PLN). We propose that PLN population shifts (folding/unfolding) are a key regulatory mechanism for SERCA.
| S-EPMC3573877 | biostudies-literature
Project description:The Ca2+-transport ATPase of sarcoplasmic reticulum (SR) is an integral, transmembrane protein. It sequesters cytoplasmic calcium ions released from SR during muscle contraction, and causes muscle relaxation. Based on negative staining and transmission electron microscopy of SR vesicles isolated from rabbit skeletal muscle, we propose that the ATPase molecules might also be a calcium-sensitive membrane-endoskeleton. Under conditions when the ATPase molecules scarcely transport Ca2+, i.e., in the presence of ATP and ≤ 0.9 nM Ca2+, some of the ATPase particles on the SR vesicle surface gathered to form tetramers. The tetramers crystallized into a cylindrical helical array in some vesicles and probably resulted in the elongated protrusion that extended from some round SRs. As the Ca2+ concentration increased to 0.2 µM, i.e., under conditions when the transporter molecules fully carry out their activities, the ATPase crystal arrays disappeared, but the SR protrusions remained. In the absence of ATP, almost all of the SR vesicles were round and no crystal arrays were evident, independent of the calcium concentration. This suggests that ATP induced crystallization at low Ca2+ concentrations. From the observed morphological changes, the role of the proposed ATPase membrane-endoskeleton is discussed in the context of calcium regulation during muscle contraction.
| S-EPMC7961605 | biostudies-literature