ATP-driven Calcium Pump Research Articles

Multiyear Simulation Study Provides Breakthrough in Membrane Protein Research

A research team based at the University of Chicago used supercomputing resources at the US Department of Energy's (DOE's) Argonne National Laboratory to gain a deep mechanistic view of a critical ATP-driven calcium pump. The team's recent discovery fills in essential information inaccessible to experimentalists, and potentially crucial to new therapeutic drug design.

Exploring the Conformational Transitions of Biomolecular Systems Using a Simple Two-State Anisotropic Network Model

Biomolecular conformational transitions are essential to biological functions. Most experimental methods report on the long-lived functional states of biomolecules, but information about the transition pathways between these stable states is generally scarce. Such transitions involve short-lived conformational states that are difficult to detect experimentally. For this reason, computational methods are needed to produce plausible hypothetical transition pathways that can then be probed experimentally. Here we propose a simple and computationally efficient method, called ANMPathway, for constructing a physically reasonable pathway between two endpoints of a conformational transition. We adopt a coarse-grained representation of the protein and construct a two-state potential by combining two elastic network models (ENMs) representative of the experimental structures resolved for the endpoints. The two-state potential has a cusp hypersurface in the configuration space where the energies from both the ENMs are equal. We first search for the minimum energy structure on the cusp hypersurface and then treat it as the transition state. The continuous pathway is subsequently constructed by following the steepest descent energy minimization trajectories starting from the transition state on each side of the cusp hypersurface. Application to several systems of broad biological interest such as adenylate kinase, ATP-driven calcium pump SERCA, leucine transporter and glutamate transporter shows that ANMPathway yields results in good agreement with those from other similar methods and with data obtained from all-atom molecular dynamics simulations, in support of the utility of this simple and efficient approach. Notably the method provides experimentally testable predictions, including the formation of non-native contacts during the transition which we were able to detect in two of the systems we studied. An open-access web server has been created to deliver ANMPathway results.

Controlling the Inhibition of the Sarcoplasmic Ca2+-ATPase by Tuning Phospholamban Structural Dynamics

Cardiac contraction and relaxation are regulated by conformational transitions of protein complexes that are responsible for calcium trafficking through cell membranes. Central to the muscle relaxation phase is a dynamic membrane protein complex formed by Ca2+-ATPase (SERCA) and phospholamban (PLN), which in humans is responsible for approximately 70% of the calcium re-uptake in the sarcoplasmic reticulum. Dysfunction in this regulatory mechanism causes severe pathophysiologies. In this report, we used a combination of nuclear magnetic resonance, electron paramagnetic resonance, and coupled enzyme assays to investigate how single mutations at position 21 of PLN affects its structural dynamics and, in turn, its interaction with SERCA. We found that it is possible to control the activity of SERCA by tuning PLN structural dynamics. Both increased rigidity and mobility of the PLN backbone cause a reduction of SERCA inhibition, affecting calcium transport. Although the more rigid, loss-of-function (LOF) mutants have lower binding affinities for SERCA, the more dynamic LOF mutants have binding affinities similar to that of PLN. Here, we demonstrate that it is possible to harness this knowledge to design new LOF mutants with activity similar to S16E (a mutant already used in gene therapy) for possible application in recombinant gene therapy. As proof of concept, we show a new mutant of PLN, P21G, with improved LOF characteristics in vitro.

The Ca(2+)-ATPase of the sarcoplasmic reticulum in skeletal and cardiac muscle. An overview from the very beginning to more recent prospects.

The discovery of the ATP-driven calcium pump in the sarcoplasmic reticulum membranes reaches back to the postwar (World War II) years and would not be possible without the generous support by the American scientific community. It was this community that in pre- and postwar years gave shelter to many European scientists, which in return stimulated scientific development in the United States. These pre- and postwar relations helped to establish the calcium pump as a physiologically relevant mechanism in all kinds of cells. The pump and its counterpart, the calcium release channel, proved to be controlled by various intrinsic mechanisms. Rising hydrogen concentrations as occurring in ischemic muscles switch off pump activity and counteract allosterically caffeine-induced calcium release (CICR). Rising phosphate or the presence of other calcium-precipitating anions, on the other hand, prevents pump inhibition by intraluminal calcium precipitation, which, simultaneously, can increase the quantity of releasable calcium. The inactivation of CICR by removing medium chloride must be considered as a hint of additional mechanisms by which calcium-dependent activity regulation can be modified.

Structure of the Ca 2+ Pump of Sarcoplasmic Reticulum: A View along the Lipid Bilayer at 9-Å Resolution

We have used multilamellar crystals of the ATP-driven calcium pump from sarcoplasmic reticulum to address the structural effects of calcium binding to the enzyme. They are stacks of disk-shaped two-dimensional crystals. A density map projected along the lipid bilayer was obtained at 9-Å resolution by frozen-hydrated electron microscopy. Although only in projection, much more details of the structure were revealed than previously available, especially in the transmembrane region. Quantitative comparison was made with the model obtained from the tubular crystals of this enzyme formed in the absence of calcium. Unexpectedly large differences in conformation were found, particularly in the cytoplasmic domain.

Three-dimensional cryo-electron microscopy of the calcium ion pump in the sarcoplasmic reticulum membrane

The ATP-driven calcium pump (Ca(2+)-ATPase) is an integral membrane protein (M(r) 110K) which relaxes striated muscle by pumping calcium out of the cytoplasm into the sarcoplasmic reticulum against a large concentration gradient. Recent efforts have attempted to relate the sequence of Ca(2+)-ATPase to its structure and function. In particular, site-directed mutagenesis has identified critical amino-acid residues, and its predicted secondary structure, which includes ten transmembrane helices, has gained experimental support. But direct visualization of the molecule has so far been limited to the cytoplasmic domains at low resolution. We present here the three-dimensional structure of Ca(2+)-ATPase in the native sarcoplasmic reticulum membrane at 14 A resolution, determined by cryo-electron microscopy and helical image analysis. The structure shows an unexpected transmembrane organization, consisting of three distinct segments, one of which is highly inclined. These features can be related to earlier predictions of secondary structure.

Effects of dietary salt on sodium-calcium exchange and ATP-driven calcium pump in arterial smooth muscle of Dahl rats.

To compare the effects of dietary salt on sodium-calcium exchange and the ATP-driven calcium pump in arterial smooth muscle between Dahl salt-sensitive (DS) and salt-resistant (DR) rats. Aortic rings freshly excised from 16 DS rats and 16 DR rats on a low- (0.3%) or high- (8%) NaCl diet for 4 weeks were superfused with physiological saline and isometric tension was measured. In the presence of 10 mumol/l phentolamine, 10 mumol/l verapamil and 5 mmol/l caffeine, relaxation of a low-Na+ contraction was promoted by external calcium removal. On the high-salt diet, the rate of relaxation at 1.2 mmol/l extracellular sodium (calcium extrusion by calcium ATPase) was significantly lower in aortic rings from DS rats than from DR rats. The increase in the rates of relaxation from 1.2 mmol/l to normal (139.2 mmol/l) extracellular sodium (calcium extrusion by sodium-calcium exchange) was significantly greater with the high-salt diet than with the low-salt diet in rings from DR rats, but it was not different between the high- and low-salt diets in DS rats. The rate of increase in tonic tension by reducing extracellular Na+ from normal to 1.2 mmol/l in the presence of verapamil, caffeine and phentolamine (calcium entry by sodium-calcium exchange) was significantly lower in rings from DS rats than in those from DR rats on the high-salt diet. These observations suggest that the effects of dietary salt on the sodium-calcium exchange system in arterial smooth muscle differ between DS and DR rats and that calcium extrusion by the calcium pump is decreased in DS rats compared with DR rats. The lack of an increase in sodium-calcium exchange in salt-fed DS rats might lead to an elevation in cellular calcium and contribute to the mechanism of hypertension.

ATP-driven calcium transport in membrane vesicles of Streptococcus sanguis.

Calcium transport was investigated in membrane vesicles prepared from the oral bacterium Streptococcus sanguis. Procedures were devised for the preparation of membrane vesicles capable of accumulating 45Ca2+. Uptake was ATP dependent and did not require a proton motive force. Calcium transport in these vesicles was compared with 45Ca2+ accumulation in membrane vesicles from Streptococcus faecalis and Escherichia coli. The data support the existence of an ATP-driven calcium pump in S. sanguis similar to that in S. faecalis. This pump, which catalyzes uptake into membrane vesicles, would be responsible for extrusion of calcium from intact cells.

Steady state of an ATP-driven calcium pump: limitations on kinetic and thermodynamic parameters.

A numerical analysis was carried out to explore limitations on kinetic and thermodynamic parameters for an ATP-driven Ca pump. A conventional pump reaction cycle was employed, with a transport stoichiometry of two Ca ions per cycle. Rigid requirements were imposed to represent the needs of physiological function, defined as the ability to maintain the cytoplasmic Ca concentration below 10(-7) M against a 3 mM concentration on the opposite side of the membrane. Realistic physical limits were placed on the magnitudes of rate constants for individual reaction steps. Reversibility under laboratory conditions was assumed. The results show that these requirements can be satisfied simultaneously only if the equilibrium constant for binding Ca from the cytoplasmic (uptake) side of the membrane is much larger than the binding constant on the discharge side. More generally, the results demonstrate that limitations on rate constants make it possible for the pump to maintain an adequate rate only if steady-state levels of kinetically important (slowly reacting) reaction intermediates do not become too disparate. Experimental data for the sarcoplasmic reticulum calcium pump support these theoretical conclusions.

Glycoproteins in the Local Mechanism of Calcification

Glycoproteins in calcifiable matrices appear to play two different roles. As structural components, glycoproteins are associated with proteoglycans of which they share many properties and functions. In particular they are removed before the onset of calcification, therefore unmasking sites of calcium and phosphate deposition. As calcium and/or phosphate binding sites, glycoproteins appear to be associated with the mechanism of calcification. Glycoproteins may aid in transporting calcium and phosphate from points of initial deposition into the relatively inaccessible sites of mineralization. It is possible to assume also a nucleating effect and it cannot be excluded that Ca-binding glycoproteins are components of the "crystal ghosts." Glycoproteins also modulate calcium dependent phosphatase and ATPase activities. The recent discovery of an ATP-driven calcium pump associated with the plasma membrane of chondrocytes and the previous finding of an ATPase activity associated with the cartilage calcium-binding glycoprotein suggest that reduction in ATP content in matrix vesicles may be responsible for the initial deposits of mineral.

Physiological role of ATP-driven calcium pump in squid axon

IT has been proposed that the regulation of the ionised calcium concentration inside many excitable and non-excitable cells is mediated by a Na–Ca countertransport mechanism where the Na electrochemical gradient provides the energy to maintain low levels of intracellular Ca2+ (refs 1, 2). Two recent observations, however, provide evidence consistent with the idea that Ca extrusion in squid axons is mediated, at least in part, by an ATP-driven Ca pump of the type observed in red blood cells3,4. First, in the absence of a Na gradient across the membrane there is an ATP-dependent ‘uphill’ extrusion of Ca which persists in the absence of external Na, Ca and Mg (ref. 3). And second, in Ca-injected unpoisoned axons, 50–90% of the Ca efflux can occur as an ‘uncoupled’ flux—that is, Ca efflux not accompanied by the uptake of Na, Ca or Mg (ref. 5). The existence of two apparently separate mechanisms for Ca transport, Na–Ca exchange and an ATP driven Ca-pump, raises the question of whether, in normal conditions, both mechanisms participate in the maintenance of the physiological pCa. To be of any use in controlling the pCa of the nerve, the mechanism responsible must be able to operate at normal Ca2+ concentrations, <10−7 M (ref. 6). We now present evidence showing (1) that in squid axons with physiological levels of Ca2+i most of the Ca efflux can be accounted for by an ATP-dependent system with high affinity for Ca2+i and ATP and which can operate in the complete absence of external Na and Ca; and (2) that a Ca transport mechanism with a low affinity for Ca2+i and ATP and extremely sensitive to Nai, Nao and Cao can contribute substantially to the total Ca efflux only at Ca2+i well above the physiological Ca2+ concentrations.

Fine structural localization of adenine nucleoside phosphatase activity in the sarcoplasmic reticulum and the T system of rat myocardium

Adenosine mono-, di-, and triphosphatase activities were studied in rat cardiac muscle fibers fixed in situ by perfusion of the coronary arteries with glutaraldehyde. Enzymes splitting ATP and ADP were present in the lateral elements of triads and in flattened cisterns found in relation to the intercalated disk and the free surface of the fiber. They are referred to as subsarcolemmal cisterns. With AMP as substrate, reaction product was found in the entire T system, but none was evident in the subsarcolemmal cisterns or in the lateral elements of triads. It is suggested for reasons discussed that the ATPase-containing structures play a role in the excitation-contraction coupling in cardiac muscle cells by energizing an ATP-driven calcium pump.