Proton gradient formation during transport of Ca2+ by sarcoplasmic reticulum.

(i) Hydrolysis of ATP by leaky sarcoplasmic reticulum vesicles generates less protons than does hydrolysis of intact membranes. Even under conditions where the hydrolysis of ATP does not produce protons (pH 6.0) intact membranes still release protons during Ca²⁺ uptake. (ii) The fluorescence of 9-amino-6-chloro-2-methoxyacridine increases suddenly after triggering the transport of Ca²⁺ and declines when Ca²⁺ has been accumulated by the vesicles. The behavior of atebrin is opposite to that of 9-aminoacridine. The rate of the fluorescence change is much higher than the rate of Ca²⁺ translocation, and the maximum fluorescence does not depend on the amount of Ca²⁺ accumulated. (iii) The fluorescence of 3,3′-dipentyloxadicarbocyanine increases at a rate similar to that of Ca²⁺ uptake and declines when 70–80% of Ca²⁺ has been accumulated. (iv) The results suggest quite clearly that a proton gradient, which forms rapidly, is the motive force for sustaining the Ca²⁺ transport. A transient transmembrane electrical potential develops as a secondary effect during Ca²⁺ translocation.     

Ligand-gated channel of the sarcoplasmic reticulum Ca2+ transport ATPase

In resting muscle, cytoplasmic Ca²⁺ concentration is maintained at a low level by active Ca²⁺ transport mediated by the Ca²⁺ ATPase from sarcoplasmic reticulum. The region of the protein that contains the catalytic site faces the cytoplasmic side of the membrane, while the transmembrane helices form a channel-like structure that allows Ca²⁺ translocation across the membrane. When the coupling between the catalytic and transport domains is lost, the ATPase mediates Ca²⁺ efflux as a Ca²⁺ channel. The Ca²⁺ efflux through the ATPase channel is activated by different hydrophobic drugs and is arrested by ligands and substrates of the ATPase at physiological pH. At acid pH, the inhibitory effect of cations is no longer observed. It is concluded that the Ca²⁺ efflux through the ATPase may be sufficiently fast to support physiological Ca²⁺ oscillations in skeletal muscle, that occur mainly in conditions of intracellular acidosis.     

Differentiation between Ca2+ transport and ATP-induced Ca2+ binding by sarcoplasmic reticulum

The Ca²⁺ actively accumulated by sarcoplasmic reticulum isolated from skeletal muscle is composed of two fractions; one represented by intravesicular free Ca²⁺ and another represented by Ca²⁺ selectively bound to the membranes. Both of these Ca²⁺ fractions depend on ATP, although it is not clear whether ATP hydrolysis is essential for accumulation of the second Ca²⁺ fraction. The existence of the membrane-bound Ca²⁺ induced by ATP is clearly shown in experiments in which the Ca²⁺ retention by sarcoplasmic reticulum is measured in the presence and in the absence of X-537A, a Ca²⁺ ionophore, which makes the membrane permeable to Ca²⁺. Thus, in the presence of X-537A all Ca²⁺ accumulated due to ATP is bound to the membranes. This membrane-bound Ca²⁺ represents about 30 nmol/mg protein in the range of external $\text{p}\text{Ca}$ values of 7 to 3.5. The magnitude of this Ca²⁺ fraction is slightly higher whether or not the experiments are performed in the presence of oxalate, which greatly increased the intravesicular Ca²⁺ accumulation. Furthermore, taking advantage of the impermeability of sarcoplasmic reticulum to EGTA, it is possible to show the existence of the membrane-bound Ca²⁺ as a distinct fraction from that which exists intravesicularly.     

Ca 2+ outflow from sarcoplasmic reticulum vesicles during changes in membrane potential, Ca2+ concentration and pH

The concentration gradient Ca2+ outflux from the vesicles of the fragmented sarcoplasmic reticulum of rabbit skeletal muscles has been studied under conditions of the induced membrane potential, the concentrations of Ca2+ and H+ in the medium washing over the vesicles being different. The Ca2+ outflux from vesicles is shown to be the same with a decrease of the membrane potential from--80 down to -10 mV and gets higher with the zero and subsequent positive values of the latter. A significant intensification of the Ca2+ outflux from vesicles under the effect of external-vesicular Ca2+ has been observed at its concentration of 10(-5) M. Against this background of external-vesicular Ca2+ and zero value of the membrane potential either exogenous AMP or the pH increase from 6.5 up to 7.8 favour a release of more than 70% of passively accumulated Ca2+. The pH effect grows with a decrease in the external-vesicular concentration of Ca2+. A conclusion is drawn on the significance of protons in the regulation of the Ca2+ release from the sarcoplasmic reticulum.     

Inactivation of a Ca2+-induced Ca2+ release channel from skeletal muscle sarcoplasmic reticulum during active Ca2+ transport

ATP-dependent Ca2+ uptake by subfractions of skeletal muscle sarcoplasmic reticulum (SR) was studied with the Ca2+ indicator dye, antipyrylazo III. Ca2+ uptake by heavy SR showed two phases, a slow uptake phase and a fast uptake phase. By contrast, Ca2+ uptake by light SR exhibited a monophasic time course. In both fractions a steady state of Ca2+ uptake was observed when the concentration of free Ca2+ outside the vesicles was reduced to less than 0.1 microM. In the steady state, the addition of 5 microM Ca2+ to the external medium triggered rapid Ca2+ release from heavy SR but not from light SR, indicating that the heavy fraction contains a Ca2+-induced Ca2+ release channel. During Ca2+ uptake, heavy SR showed a constant Ca2+-dependent ATPase activity (1 mumol/mg protein X min) which was about 150 times higher than the rate of Ca2+ uptake in the slow uptake phase. Ruthenium red, an inhibitor of Ca2+-induced Ca2+ release, enhanced the rate of Ca2+ uptake during the slow phase without affecting Ca2+-dependent ATPase activity. Adenine nucleotides, activators of Ca2+ release, reduced the Ca2+ uptake rate. These results suggest that the rate of Ca2+ accumulation by heavy SR is not proportional to ATPase activity during the slow uptake phase due to the activation of the channel for Ca2+-induced Ca2+ release. In addition, they suggest that the release channel is inactivated during the fast Ca2+ uptake phase.     

Regulation of Ca2+ transport by sarcoplasmic reticulum Ca2+-ATPase at limiting [Ca2+

The factors regulating Ca2+ transport by isolated sarcoplasmic reticulum (SR) vesicles have been studied using the fluorescent indicator Fluo-3 to monitor extravesicular free [Ca2+]. ATP, in the presence of 5 mM oxalate, which clamps intravesicular [Ca2+] at approximately 10 microM, induced a rapid decline in Fluo-3 fluorescence to reach a limiting steady state level. This corresponds to a residual medium [Ca2+] of 100 to 200 nM, and has been defined as [Ca2+]lim, whilst thermodynamic considerations predict a level of less than 1 nM. This value is similar to that measured in intact muscle with Ca2+ fluophores, where it is presumed that sarcoplasmic free [Ca2+] is a balance between pump and leaks. Fluorescence of Fluo-3 at [Ca2+]lim was decreased 70% to 80% by histidine, imidazole and cysteine. The K0.5 value for histidine was 3 mM, suggesting that residual [Ca2+]lim fluorescence is due to Zn2+. The level of Zn2+ in preparations of SR vesicles, measured by atomic absorption, was 0.47+/-0.04 nmol/mg, corresponding to 0.1 mol per mol Ca-ATPase. This is in agreement with findings of Papp et al. (Arch. Biochem. Biophys., 243 (1985) 254-263). Histidine, 20 mM, included in the buffer, gave a corrected value for [Ca2+]lim of 49+/-1.8 nM, which is still higher than predicted on thermodynamic grounds. A possible 'pump/leak' mechanism was tested by the effects of varying active Ca2+ transport 1 to 2 orders with temperature and pH. [Ca2+]lim remained relatively constant under these conditions. Alternate substrates acetyl phosphate and p-NPP gave similar [Ca2+]lim levels even though the latter substrate supported transport 500-fold slower than with ATP. In fact, [Ca2+]lim was lower with 10 mM p-NPP than with 5 mM ATP. The magnitude of passive efflux from Ca-oxalate loaded SR during the steady state of [Ca2+]lim was estimated by the unidirectional flux of 45Ca2+, and directly, following depletion of ATP, by measuring release of 40Ca2+, and was 0.02% of Vmax. Constant infusion of CaCl2 at [Ca2+]lim resulted in a new steady state, in which active transport into SR vesicles balances the infusion rate. Varying infusion rates allows determination of [Ca2+]-dependence of transport in the absence of chelating agents. Parameters of non-linear regression were Vmax=853 nmol/min per mg, K0.5(Ca)=279 nM, and nH(Ca)=1.89. Since conditions employed in this study are similar to those in the sarcoplasm of relaxed muscle, it is suggested that histidine, added to media in studies of intracellular Ca2+ transients, and in the relaxed state, will minimise contribution of Zn2+ to fluophore fluorescence, since it occurs at levels predicted in this study to cause significant overestimation of cytoplasmic free [Ca2+] in the relaxed state. Similar precautions may apply to non-muscle cells as well. This study also suggests that [Ca2+]lim in the resting state is a characteristic feature of Ca2+ pump function, rather than a balance between active transport and passive leakage pathways.     

Ca2+-controlled conformational states of the Ca2+ transport enzyme of sarcoplasmic reticulum.

The fluorescent reagent, S-mercuric N-dansyl-cysteine, reacts specifically with thiols of the purified Ca2+-ATPase of the sarcoplasmic reticulum, producing an increase of fluorescence of fluorescence intensity at 500 nm (lambda ex = 335 nm). The reaction is stoichiometric, and the increase of the fluorescence intensity is proportional to the number of blocked thiols. Twelve reactive thiols per 10(5) daltons of ATPase peptide fall into roughly three classes. Blocking of the most reactive thiol entails little inhibition of enzyme activity. Blocking of the five thiols reacting next (intermediate class) results in almost complete inhibition of both phosphorylated intermediate formation and ATP hydrolysis. The second order rate constants of the reaction of thiols have been determined by stopped flow studies. The most reactive thiol and the six least reactive thiols can each be treated as a single class with respect to the rate constant; five thiols of intermediate reactivity appear to have different rate constants (k2, k3, ..k6). Of these constants, k1, corresponding to the most reactive thiol, does not change with [Ca2+]. Upon increasing [Ca2+] from 10(-9) to 10(-5) M, k2 increase and k7-12 decreases; the changes roughly parallel the activation of ATPase activity and the Ca2+ binding to the high affinity alpha sites (Ikemoto, N. (1975) J. Biol. Chem. 250, 7219-7224). Upon further increase of [Ca2+] k2 decreases and k7-12 increase, in parallel with the inhibition of ATPase activity and with the Ca2+ binding to the low affinity gamma sites.     

Effects of alkaline pH on sarcoplasmic reticulum Ca2+ release and Ca2+ uptake.

Alkalinization-induced Ca2+ release from isolated frog or rabbit sarcoplasmic reticulum vesicles appears to consist of two distinct components: 1) a direct activation of ruthenium red-sensitive Ca2+ release channels in terminal cisternae and 2) an increased ruthenium red-insensitive Ca2+ efflux through some other efflux pathway distributed throughout the sarcoplasmic reticulum. The first of these releases exhibits an alkalinization-induced inactivation process and does not depend on the ruthenium red-insensitive form of Ca2+ release as a triggering agent for secondary Ca(2+)-induced Ca2+ release. Both releases are inhibited when the extravesicular (i.e. cytoplasmic) free [Ca2+] is reduced. This may reflect an increased sensitivity of the Ca2+ release channels to Ca2+ at alkaline pH. The pH sensitivity of the ruthenium red-sensitive Ca2+ release channels could be of significance during excitation-contraction coupling. The ruthenium red-insensitive form of Ca2+ release is less likely to be physiologically relevant, but it probably has contributed greatly to reports of alkalinization-induced decreases in net sarcoplasmic reticulum Ca2+ uptake, particularly under conditions where oxalate supported Ca2+ uptake is much less affected, as here.     

The dimeric form of Ca2+-ATPase is involved in Ca2+ transport in the sarcoplasmic reticulum

To identify the functional unit of Ca(2+)-ATPase in the sarcoplasmic reticulum, we assessed Ca(2+)-transport activities occurring on sarcoplasmic reticulum membranes with different combinations of active and inactive Ca(2+)-ATPase molecules. We prepared heterodimers, consisting of a native Ca(2+)-ATPase molecule and a Ca(2+)-ATPase molecule inactivated by FITC labelling, by fusing vesicles loaded with each type of Ca(2+)-ATPase. The heterodimers exhibited neither Ca(2+) transport nor ATP hydrolysis, suggesting that Ca(2+) transport by the Ca(2+)-ATPase requires an interaction between functional Ca(2+)-ATPase monomers. This finding implies that the functional unit of the Ca(2+)-ATPase is a dimer.     

Inhibition of sarcoplasmic reticulum Ca2+-ATPase by Mg2+ at high pH

Steady state turnover of Ca2+-ATPase of sarcoplasmic reticulum has generally been reported to have a bell-shaped pH profile, with an optimum near pH 7.0. While a free [Mg2+] of 2 mM is optimal for activity at pH 7.0, it was found that this level was markedly inhibitory (K1/2 = 2 mM) at pH 8.0, thus accounting for the generally observed low activity at high pH. High activity was restored at pH 8.0 using an optimum free [Mg2+] of 0.2 mM. The mechanism of the Mg2+-dependent inhibition at pH 8.0 was probed. Inhibition was not due to Mg2+ competition with Ca2+ for cytoplasmic transport sites nor to inhibition of formation of steady state phosphoenzyme from ATP. Mg2+ inhibited (K1/2 = 1.8 mM) decay of steady state phosphoenzyme; thus, the locus of inhibition was one of the phosphoenzyme interconversion steps. Transient kinetic experiments showed that Mg2+ competitively inhibited (Ki = 0.7 mM) binding of Ca2+ to lumenal transport sites, blocking the ability of Ca2+ to reverse the catalytic cycle to form ADP-sensitive, from ADP-insensitive, phosphoenzyme. The data were consistent with a hypothesis in which Mg2+ binds lumenal Ca2+ transport sites with progressively higher affinity at higher pH to form a dead-end complex; its dissociation would then be rate-limiting during steady state turnover.     

The sarcoplasmic reticulum Ca2+-ATPase

Summary This review summarizes studies on the structural organization of Ca²⁺-ATPase in the sarcoplasmic reticulum membrane in relation to the function of the transport protein. Recent advances in this field have been made by a combination of protein-chemical, ultrastructural, and physicochemical techniques on membraneous and detergent solubilized ATPase. A particular feature of the ATPase (Part I) is the presence of a hydrophilic head, facing the cytoplasm, and a tail inserted in the membrane. In agreement with this view the protein is moderately hydrophobic, compared to many other integral membrane proteins, and the number of traverses of the 115 000 Dalton peptide chain through the lipid may be limited to 3–4.There is increasing evidence (Part II) that the ATPase is self-associated in the membrane in oligomeric form. This appears to be a common feature of many transport proteins. Each ATPase peptide seems to be able to perform the whole catalytic cycle of ATP hydrolysis and Ca²⁺ transport. Protein-protein interactions seem to have a modulatory effect on enzyme activity and to stabilize the enzyme against inactivation.Phospholipids (Part III) are not essential for the expression of enzyme activity which only requires the presence of flexible hydrocarbon chains that can be provided e.g. by polyoxyethylene glycol detergents. Perturbation of the lipid bilayer by the insertion of membrane protein leads to some immobilization of the lipid hydrocarbon chains, but not to the extent envisaged by the annulus hypothesis. Strong immobilization, whenever it occurs, may arise from steric hindrance due to protein-protein contacts. Recent studies suggest that breaks in Arrhenius plots of enzyme activity primarily reflect intrinsic properties of the protein rather than changes in the character of lipid motion as a function of temperature.     

Thermodynamics of Ca2+ transport through sarcoplasmic reticulum membranes during the transient-state of simulated reactions

The kinetics of a chemical model of Ca2+ transport and coupled ATPase activity in sarcoplasmic reticulum membranes were solved for the transient-state of simulated reactions, using a numerical integration procedure. The simulation conditions reproduced in vitro experiments using either fragmented membranes or vesicles with Ca2+ accumulating ability. The results yielded the concentrations of all the ligands and intermediates of the enzymatic cycle as a function of the reaction time. These results were applied to calculations of several thermodynamic variables: (1) the step by step profile of the standard free energy change of the cycle. (2) The step by profile of the actual free energy change of the cycle, and its evolution with the reaction time. (3) The separate contributions of ATP hydrolysis and Ca2+ transport to the overall free energy change with the reaction. (4) The dependence of the velocity of the free energy change with the reaction time. (5) The efficiency of the transport system, and its change with the reaction time. (6) The separate contributions of the Ca2+ gradient and some enzymatic intermediates as free energy stores. The main findings are: (1) the step by step diagrams of the free energy change calculated from the results of the kinetic analysis better describe the thermodynamic profile of the cycle than previously reported diagrams of the standard free energy and basic free energy changes. The relative contribution of each partial step to the driving force of the whole reactions, as well as their changes upon the advancement of the reactions, are derived from the diagrams. (2) Free energy yielded by ATP hydrolysis is stored by the system, not only as a Ca2+ gradient, but also as enzymatic intermediates of the reaction. The progressive increase of both free energy pools upon the advancement of the reaction is quantitated.     

Lipid peroxidation and disturbance in Ca2+ transport through sarcoplasmic reticulum membranes during E-avitaminosis]

The antioxidant effect of alpha-tocopherol in biolgoical membranes in vivo is described. Experimental E-avitaminosis is accompanied by accumulation of products of free-radical oxidation of phospholipids and by a loss of Ca2+-transporting ability of the muscle cells microsomal fraction. The role of alpha-tocopherol in stabilization and as a "radical trap" in biological membranes is discussed.     

A mechanism of passive transport of Ca2+ in muscle sarcoplasmic reticulum

Basing on the data available in literature and authors' investigations the mechanism of local alkalization of the myoplasm by proton efflux attended by Ca2+ influx is mic reticulum and may be the main link in the process of electrochemical coupling in the skeletal and cardiac muscle cells. Experimental evidence for participation of Ca2(+)-ATPase in the passive transport of calcium through sarcoplasmic membrane is given.     

Antioxidants--stabilizers of the Ca2+ transport enzyme system in sarcoplasmic reticulum membranes in vivo

It has been demonstrated that natural and synthetic antioxidants of different chemical structures (alpha-tocopherol, butylated hydroxytoluene, 2-ethyl-6-methyl-3-hydroxypyridine) were capable of stabilizing enzymatic Ca2+ transport in sarcoplasmic membranes of the heart and skeletal muscles in vivo. Chronic administration of water-soluble antioxidant 2-ethyl-6-methyl-3-hydroxypyridine to young and old rats resulted in the increased rate of Ca2+ transport into sarcoplasmic reticulum vesicles of the heart and skeletal muscle homogenates. Keeping rats on vitamin E-rich diets supplemented with synthetic antioxidant butylated hydroxytoluene led to stabilization of Ca2+-ATPase against thermal denaturation in sarcoplasmic reticular membranes.