Calcium fluxes across the membrane of sarcoplasmic reticulum vesicles

The relationship between calcium exchange across the membrane of sarcoplasmic reticulum vesicles and phosphoenzyme (EP) was examined in calcium transport reactions using a limited amount of ATP as substrate. Rapid calcium influx and efflux (approximately 385 nmol.(mg.min)-1), measured in reactions in which ATP concentration fell from 20 microM, was accompanied by a shift in the equilibrium between an ADP-sensitive EP and an ADP-insensitive EP toward the former. Rapid exchange between ATP and ADP (approximately 1500 nmol.(mg.min)-1) was also observed under conditions where no significant incorporation of Pi into ATP took place, suggesting that ATP in equilibrium ADP exchange can occur without Cao in equilibrium Cai exchange. Ca2+ permeability during the calcium transport reaction was estimated in reactions carried out with acetylphosphate, which produces a hydrolytic product that does not participate in the backward reaction of the calcium pump. Under conditions where the calcium content exceeded 43 nmol.mg-1, a level that may reflect the binding of calcium ions to sites inside the sarcoplasmic reticulum, the rate constant for Ca2+ efflux was 0.33 min-1. These data allow the rate of passive Ca2+ efflux to be estimated as approximately 17 nmol.(mg.min)-1 at the time when calcium content was maximal and a rapid Cao in equilibrium Cai was observed. It is concluded that the majority of the rapid Ca2+ efflux is mediated by partial backward reactions of the calcium pump ATPase.     

Fusion of isolated cardiac sarcolemmal and sarcoplasmic reticulum vesicles. An approach to the mechanism

A procedure for the fusion of isolated cardiac sarcolemmal and sarcoplasmic reticulum vesicles is described. When the mixture of vesicles was incubated in a medium containing CaCl₂ and ATP, membrane fusion rather than vesicle aggregation or molecular exchange was demonstrated. This was achieved either by studying changes in vesicle density using sucrose gradients, fluorescence quenching using fluorescamine labeled sarcoplasmic reticulum, or by separation of the different vesicle sizes using gel-filtration. Although extensive fusion was observed when inside-out sarcolemmal vesicles were used, right-side-out vesicles showed no capacity to fuse with sarcoplasmic reticulum vesicles. The relationship between fusion and other aspects of cardiac sarcolemmal function was discussed.     

Ca2+ uptake and membrane potential in sarcoplasmic reticulum vesicles

The rate of calcium uptake by sarcoplasmic reticulum vesicles isolated from rabbit skeletal muscle was stimulated by inside-negative membrane potential generated by K+ gradients in the presence of valinomycin. The increase in the calcium transport rate was accompanied by a proportional increase in the rate of calcium-dependent ATP hydrolysis, without significant change in the steady state level of the phosphorylated enzyme intermediate. Changes in the sarcoplasmic reticulum membrane potential during calcium transport were monitored with the optical probe, 3,3'-diethylthiadicarbocyanine. The decrease in the absorbance of 3,3'-diethylthiadicarbocyanine at 660 nm following generation of inside-negative membrane potential was reversed during ATP-induced calcium uptake. These observations support an electrogenic mechanism for the transport of calcium by the sarcoplasmic reticulum.     

Energy-dependent redistribution of a lipophilic anion in sarcoplasmic reticulum vesicles and Ca2-ATPase molecules

The distribution of lipophilic anion of phenyldicarbaundecarborane (PCB-) between water phase and fragments of sarcoplasmic reticulum (SR) from skeletal muscle was studied, using a bilayer lipid membrane (BLM) as a selective electrode. Addition of ATP leads to an increase in PCB- binding to SR vesicles. The ATP effect is totally reversible only in the presence of both EGTA and A23187. Chlorides, in contrast with oxalate and phosphate, do not reduce the ATP-dependent PCB- binding. Oxalate decreases also the energy-dependent extrusion of protons from SR into the medium. Preliminary incubation of SR fragments with calcium gluconate leads to a decrease in PCB- binding. Addition of ATP to purified Ca2+-ATPase is coupled with a release of PCB- and calcium from the enzyme. It is suggested that ATP-dependent binding of PCB- to SR membranes reflects calcium incorporation into the hydrophobic region of Ca2+-ATPase molecules.     

Crystals of sarcoplasmic reticulum Ca(2+)-ATPase

High-resolution structures of the Ca(2+)-ATPase have over the last 5 years added a structural dimension to our understanding of the function of this integral membrane protein. The Ca(2+)-ATPase is now by far the membrane protein where the most functionally different conformations have been described in precise structural detail. Here, we review our experience from solving Ca(2+)-ATPase structures: a purification scheme involving minimum handling of the protein to preserve natural and essential lipids, a rational approach to screening for crystals based on a limited number of polyethyleneglycols and many different salts, improving crystal quality using additives, collecting the data and finally solving the structures. We argue that certain of the lessons learned in the present study are very likely to be useful for crystallisation of eukaryotic membrane proteins in general.     

Effect of tris(hydroxymethyl)aminomethane on isolated sarcoplasmic reticulum vesicles.

The preincubation of isolated sarcoplasmic reticulum vesicles in Tris-Cl (pH 7.3) increases their (Ca²⁺ + Mg²⁺)-dependent adenosine triphosphatase activity and decreases their ATP-dependent Ca uptake capacity. These effects of Tris are dependent on the preincubation time and the Tris concentration; they are maximal below 10 μm Ca and decrease upon the increase of Ca concentration in the preincubation media, and they increase upon the increase of the preincubation pH. Differences in ATPase activity between preincubated and control vesicles are abolished by A23187 but not by carbonyl cyanide p-trifluoromethoxy phenyl hydrazone. The results suggest that: (i) Preincubation of the vesicles in Tris causes an increase of their permeability for Ca, or a membrane damage. (ii) Tris must diffuse within the vesicles to promote these effects. (iii) Ca prevents these effects by decreasing the membrane permeability for Tris. The basic findings were reproduced replacing Tris by imidazole.     

Functional reconstitution of the cardiac sarcoplasmic reticulum Ca2(+)-ATPase with phospholamban in phospholipid vesicles

The Ca2(+)-ATPase in cardiac sarcoplasmic reticulum (SR) is under regulation by phospholamban, an oligomeric proteolipid. To determine the molecular mechanism by which phospholamban regulates the Ca2(+)-ATPase, a reconstitution system was developed, using a freeze-thaw sonication procedure. The best rates of Ca2+ uptake (700 nmol/min/mg reconstituted vesicles compared with 800 nmol/min/mg SR vesicles) were observed when cholate and phosphatidylcholine were used at a ratio of cholate/phosphatidylcholine/Ca2(+)-ATPase of 2:80:1. The EC50 values for Ca2+ were 0.05 microM for both Ca2+ uptake and Ca2(+)-ATPase activity in the reconstituted vesicles compared with 0.63 microM Ca2+ in native SR vesicles. Inclusion of phospholamban in the reconstituted vesicles was associated with a significant inhibition of the initial rates of Ca2+ uptake at pCa 6.0. However, phosphorylation of phospholamban by the catalytic subunit of the cAMP-dependent protein kinase reversed the inhibitory effect on the Ca2+ pump. Similar findings were observed when a peptide, corresponding to amino acids 1-25 of phospholamban, was used. These findings indicate that phospholamban is an inhibitor of the Ca2(+)-ATPase in cardiac SR and phosphorylation of phospholamban relieves this inhibition. The mechanism by which phospholamban inhibits the Ca2+ pump is unknown, but our findings with the synthetic peptide suggest that a direct interaction between the Ca2(+)-ATPase and the hydrophilic portion of phospholamban may be one of the mechanisms for regulation.     

Proton paths in the sarcoplasmic reticulum Ca(2+) -ATPase

The sarcoplasmic reticulum Ca(2+)-ATPase (SERCA1a) pumps Ca(2+) and countertransport protons. Proton pathways in the Ca(2+) bound and Ca(2+)-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 Ca(2+) bound state Ca(2)E1, one of the proposed Ca(2+) entry paths is suggested to operate additionally or alternatively as proton pathway. In analogs of the ADP-insensitive phosphoenzyme E2P and in the Ca(2+)-free state E2, the proton path leads between transmembrane helices M5 to M8 from the lumenal side of the protein to the Ca(2+) binding residues Glu-771, Asp-800 and Glu-908. The proton path is different from suggested Ca(2+) dissociation pathways. We suggest that separate proton and Ca(2+) 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.     

Possible effects of membrane polarization on calcium uptake by and release from sarcoplasmic reticulum vesicles

Rates of calcium uptake by and calcium release from sarcoplasmic reticulum vesicles isolated from skeletal muscle of the crab seem to depend on membrane potential generated by potassium (K) and chloride (Cl) gradients. This does not appear to be due to an effect of the ions themselves since media of different ionic compositions leading to the same membrane potential, also lead to the same ATP hydrolysis and the same Ca uptake by SR vesicles. From a large positive intravesicular potential (conditions termed "normal" in this paper), membrane depolarization of passively Ca loaded vesicles, produced by changes in K and Cl concentrations in the media, resulted in: i) decrease in rate of calcium uptake; ii) decrease in calcium loading; iii) increase in rate of calcium release despite a decrease in the driving force for calcium ions. Moreover, the addition of caffeine (5 mmol/l) to the different polarization media resulted in a increase in calcium release.     

Ca2+-ATPase membrane crystals in sarcoplasmic reticulum. The effect of trypsin digestion

Vanadate induces the formation of two-dimensional crystalline arrays of Ca2+-ATPase molecules in sarcoplasmic reticulum. The Ca2+-ATPase membrane crystals are evenly distributed among the terminal cisternae and longitudinal tubules of sarcoplasmic reticulum, but very few crystals were observed in the T tubules. Tryptic cleavage of the Ca2+ transport ATPase into two major fragments (A and B) did not interfere with the vanadate-induced formation of membrane crystals. The ability of Ca2+-ATPase to crystallize was lost after further cleavage of the A fragment into the A1 and A2 subfragments that is known to be accompanied by loss of Ca2+ uptake. Vanadate (0.1-5 mM) inhibited the secondary cleavage of Ca2+-ATPase by trypsin suggesting that the susceptibility of the tryptic cleavage sites is influenced either by the conformation of the enzyme or by the formation of ATPase crystals.     

Characterizing phospholamban to sarco(endo)plasmic reticulum Ca2+-ATPase 2a (SERCA2a) protein binding interactions in human cardiac sarcoplasmic reticulum vesicles using chemical cross-linking

Chemical cross-linking was used to study protein binding interactions between native phospholamban (PLB) and SERCA2a in sarcoplasmic reticulum (SR) vesicles prepared from normal and failed human hearts. Lys²⁷ of PLB was cross-linked to the Ca²⁺ pump at the cytoplasmic extension of M4 (at or near Lys³²⁸) with the homobifunctional cross-linker, disuccinimidyl glutarate (7.7 Å). Cross-linking was augmented by ATP but abolished by Ca²⁺ or thapsigargin, confirming in native SR vesicles that PLB binds preferentially to E2 (low Ca²⁺ affinity conformation of the Ca²⁺-ATPase) stabilized by ATP. To assess the functional effects of PLB binding on SERCA2a activity, the anti-PLB antibody, 2D12, was used to disrupt the physical interactions between PLB and SERCA2a in SR vesicles. We observed a tight correlation between 2D12-induced inhibition of PLB cross-linking to SERCA2a and 2D12 stimulation of Ca²⁺-ATPase activity and Ca²⁺ transport. The results suggest that the inhibitory effect of PLB on Ca²⁺-ATPase activity in SR vesicles results from mutually exclusive binding of PLB and Ca²⁺ to the Ca²⁺ pump, requiring PLB dissociation for catalytic activation. Importantly, the same result was obtained with SR vesicles prepared from normal and failed human hearts; therefore, we conclude that PLB binding interactions with the Ca²⁺ pump are largely unchanged in failing myocardium.     

Crystal structure of sarcoplasmic reticulum Ca2+-ATPase (SERCA) from bovine muscle

The SERCA pump, a membrane protein of about 110kDa, transports two Ca(2+) ions per ATP hydrolyzed from the cytoplasm to the lumen of the sarcoplasmic reticulum. In muscle cells, its ability to remove Ca(2+) from the cytosol induces relaxation. The transport mechanism employed by the enzyme from rabbit muscle has been extensively studied, and several crystal structures representing different conformational states are available. However, no structure of the pump from other sources is known. In this paper we describe the crystal structure of the bovine enzyme, crystallized in the E1 conformation and determined at 2.9? resolution. The overall molecular model is very similar to that of the rabbit enzyme, as expected by the high amino acid sequence identity. Nevertheless, the bovine enzyme has reduced catalytic activity with respect to the rabbit enzyme. Subtle structural modifications, in particular in the region of the long loop that protrudes into the SR lumen connecting transmembrane α-helices M7 and M8, may explain the difference.     

The effects of n-alkanols on the lipid/protein interface of Ca(2+)-ATPase of sarcoplasmic reticulum vesicles.

The effects of ethanol, n-butanol, n-hexanol and n-octanol on lipid-protein interactions in sarcoplasmic reticulum vesicles (SRV) are investigated using the C-14 nitroxide spin-labeled phosphatidylcholine. n-Alkanols, which activate the Ca(2+)-dependent ATPase of sarcoplasmic reticulum but decrease net Ca2+ uptake by the vesicles, are shown to affect the lipids interacting with the protein surface. Spectral analysis revealed that increasing concentrations of the alcohols progressively displace and mobilize lipids from the lipid/protein interface. For butanol, hexanol and octanol maximally activated SRV, 23 to 30% of the protein-interacting lipids are displaced. Thus, the displacement of more than 30% of the annular lipids by these alkanols cause inhibition of the enzyme. The motional properties of the labels that remain restricted by the protein surface are unaffected by the alcohols. The degree of mobilization attained by the labels displaced from the interface is much greater than that observed in alcohol-treated dispersions of extracted lipids. We propose that the alcohol molecules interfere with the protein-lipid interactions creating fluid clusters around the proteins. These fluidized regions would affect the enzyme conformation, perturbing its function. Fluidized annular lipids apparently increase the number of ion-conducting defects around the enzyme, increasing Ca2+ efflux, and thereby reducing net uptake.     

Adenosinetriphosphatase site stoichiometry in sarcoplasmic reticulum vesicles and purified enzyme

The stoichiometry of phosphorylation (catalytic) sites in sarcoplasmic reticulum vesicles ( SRV ) and SR ATPase purified by differential solubilization with deoxycholate was found to be 4.77 +/- 0.4 and 6.05 +/- 0.18 nmol/mg of protein, respectively, when phosphorylation was carried out under conditions permitting 32P labeling of nearly all sites. Assuming that each site corresponds to a single 115K ATPase chain, the observed site stoichiometry accounts only for 55% and 70% of the total protein. Failure to obtain higher phosphorylation levels was due to the presence of nonspecific protein contaminants in SRV or to the presence of inactive aggregates in the ATPase purified with deoxycholate. This was demonstrated by dissolving SRV and purified ATPase with lithium dodecyl sulfate, subjecting them to molecular sieve HPLC, and collecting the elution fractions for determination of protein, measurement of 32P-labeled sites, and electrophoretic analysis. In fact, in the specific elution peak containing the 115K ATPase chains, phosphorylation levels were 6.62 +/- 0.33 and 7.03 +/- 0.18 in SRV and purified ATPase, corresponding to 68% and 86% of the protein in the specific elution peak. An alternate purification method was then developed, based on solubilization of SRV with dodecyl octaethylene glycol monoether ( C12E8 ), separation of delipidated ATPase by anion-exchange chromatography, and enzyme reactivation with phosphatidylcholine. This preparation yields 7.3 +/- 0.44 nmol of phosphorylation site/mg of protein of the SRV fraction before HPLC.(ABSTRACT TRUNCATED AT 250 WORDS)     

Testing the versatility of the sarcoplasmic reticulum Ca(2+)-ATPase reaction cycle when p-nitrophenyl phosphate is the substrate

A detailed characterization of p-nitrophenyl phosphate as energy-donor substrate for the sarcoplasmic reticulum Ca(2+)-ATPase was undertaken in this study. The fact that p-nitrophenyl phosphate can be hydrolyzed in the presence or absence of Ca(2+) by the purified enzyme is consistent with the observed phenomenon of intramolecular uncoupling. Under the most favorable conditions, which include neutral pH, intact microsomal vesicles, and low free Ca(2+) in the lumen, the Ca(2+)/P(i) coupling ratio was 0.6. A rise or decrease in pH, high free Ca(2+) in the lumenal space, or the addition of dimethyl sulfoxide increase the intramolecular uncoupling. Alkaline pH and/or high free Ca(2+) in the lumen potentiate the accumulation of enzyme conformations with high Ca(2+) affinity. Acidic pH and/or dimethyl sulfoxide favor the accumulation of enzyme conformations with low Ca(2+) affinity. Under standard assay conditions, two uncoupled routes, together with a coupled route, are operative during the hydrolysis of p-nitrophenyl phosphate in the presence of Ca(2+). The prevalence of any one of the uncoupled catalytic cycles is dependent on the working conditions. The proposed reaction scheme constitutes a general model for understanding the mechanism of intramolecular energy uncoupling.     

Characterization of the palytoxin effect on Ca2+-ATPase from sarcoplasmic reticulum (SERCA)

The effect of palytoxin was studied in a microsomal fraction enriched in longitudinal tubules of the sarcoplasmic reticulum membrane. Half-maximal effect of palytoxin on Ca²⁺-ATPase activity yielded an apparent inhibition constant of approx. 0.4 μM. The inhibition process exhibited the following characteristics: (i) the degree of inhibition was dependent on membrane protein concentration; (ii) no protection was observed when the ATP concentration was raised; (iii) dependence on Ca²⁺ concentration with a decreased maximum catalytic rate; (iv) it occurred in the absence of Ca²⁺ ionophoric activity. Likewise, the inhibition mechanism was linked to: (i) rapid enzyme phosphorylation from ATP in the presence of Ca²⁺ but lower steady-state levels of phosphoenzyme; (ii) more drastic effect on phosphoenzyme levels when the toxin was added to the enzyme in the absence of Ca²⁺; (iii) decreased phosphoenzyme levels at saturating Ca²⁺ concentrations; (iv) no effect on kinetics of phosphoenzyme decomposition. The palytoxin effect is related with lock of the enzyme in the Ca²⁺-free conformation so that progression of the catalytic cycle is impeded.