Modulation of the kinetic characteristics of the sarcoplasmic reticulum ATPase by membrane fluidity

(Ca²⁺ + Mg²⁺)-ATPase from sarcoplasmic reticulum has been reconstituted with dipalmitoylphosphatidylcholine, and the activating effect of ATP and Ca²⁺ on this enzyme has been studied at different temperatures. It has been found that two kinetic forms of the enzyme are interconverted at about 31°C, and this is possibly related to a phase change in the phospholipid which is more directly associated with the protein. Above 31°C the enzyme is less dependent on ATP activation at high ATP concentrations but shows positive cooperativity for Ca²⁺ activation. On the other hand, below 31°C, the reconstituted enzyme is more dependent on ATP for activation at high ATP concentrations than the purified ATPase and does not show cooperativity for Ca²⁺ activation.     

Ca2+ Modulation of sarcoplasmic reticulum Ca2+ release in rat skeletal muscle fibers

Ca²⁺ transients and the rate of Ca²⁺ release (dCa_REL/dt) from the sarcoplasmic reticulum (SR) in voltage-clamped, fast-twitch skeletal muscle fibers from the rat were studied with the double Vaseline gap technique and using mag-fura-2 and fura-2 as Ca²⁺ indicators. Single pulse experiments with different returning potentials showed that Ca²⁺ removal from the myoplasm is voltage independent. Thus, the myoplasmic Ca²⁺ removal (dCa_REM/dt) was studied by fitting the decaying phase of the Ca²⁺ transient (Melzer, Ríos & Schneider, 1986) and dCa_REL/dt was calculated as the difference between dCa/dt and dCa_REM/dt. The fast Ca²⁺ release decayed as a consequence of Ca²⁺ inactivation of Ca²⁺ release. Double pulse experiments showed inactivation of the fast Ca²⁺ release depending on the prepulse duration. At constant interpulse interval, long prepulses (200 msec) induced greater inactivation of the fast Ca²⁺ release than shorter depolarizations (20 msec). The correlation (r) between the myoplasmic [Ca²⁺]_i and the inhibited amount of Ca²⁺ release was 0.98. The [Ca²⁺]_i for 50% inactivation of dCa_REL/dt was 0.25 m, and the minimum number of sites occupied by Ca²⁺ to inactivate the Ca²⁺ release channel was 3.0. These data support Ca²⁺ binding and inactivation of SR Ca²⁺ release.This work was supported by Grant-in-Aid from the American Heart Association (National) and Muscular Dystrophy Association (USA). Part of this work was developed in Dr. Stefani's laboratory at Baylor College of Medicine.     

Modulation of the sarcoplasmic reticulum (Ca2+ + Mg2+)-ATPase by pentobarbital

The dependence of the (Ca2+ + Mg2+)-ATPase activity of sarcoplasmic reticulum vesicles upon the concentration of pentobarbital shows a biphasic pattern. Concentrations of pentobarbital ranging from 2 to 8 mM produce a slight stimulation, approximately 20-30%, of the ATPase activity of sarcoplasmic reticulum vesicles made leaky to Ca2+, whereas pentobarbital concentrations above 10 mM strongly inhibit the activity. The purified ATPase shows a higher sensitivity to pentobarbital, namely 3-4-fold shift towards lower values of the K0.5 value of inhibition by this drug. These effects of pentobarbital are observed over a wide range of ATP concentrations. In addition, this drug shifts the Ca2+ dependence of the (Ca2+ + Mg2+)-ATPase activity towards higher values of free Ca2+ concentrations and increases several-fold the passive permeability to Ca2+ of the sarcoplasmic reticulum membranes. At the concentrations of pentobarbital that inhibit this enzyme in the sarcoplasmic reticulum membrane, pentobarbital does not significantly alter the order parameter of these membranes as monitored with diphenylhexatriene, whereas the temperature of denaturation of the (Ca2+ + Mg2+)-ATPase is decreased by 4-5 C degrees, thus, indicating that the conformation of the ATPase is altered. The effects of pentobarbital on the intensity of the fluorescence of fluorescein-labeled (Ca2+ + Mg2+)-ATPase in sarcoplasmic reticulum also support the hypothesis of a conformational change in the enzyme induced by millimolar concentrations of this drug. It is concluded that the inhibition of the sarcoplasmic reticulum ATPase by pentobarbital is a consequence of its binding to hydrophobic binding sites in this enzyme.     

Properties of the sarcoplasmic reticulum Ca2+-pump in coronary artery skinned smooth muscle

Pig coronary artery cultured smooth muscle cells were skinned using saponin. In the presence of an ATP-regenerating system and oxalate, the skinned cells showed an ATP-dependent azide insensitive Ca²⁺-uptake which increased linearly with time for >1 h. The Ca²⁺-uptake occurred with K_m values of 0.20±0.03 M for Ca²⁺ and 400±34 M for MgATP^2–. Thapsigargin and cyclopiazonic acid inhibited this uptake with IC₅₀ values of 0.13±0.02 and 0.56±0.04 M, respectively. These properties of SR Ca²⁺-pump are similar to those reported for membrane fractions isolated from fresh smooth muscle of coronary artery and other arteries. However, optimum pH of the uptake in the skinned cells (6.2) was lower than that reported previously using isolated membranes (6.4–6.8).Abbreviations SR sarcoplasmic reticulum - ER endoplasmic reticulum - PM plasma membrane - CPA cyclopiazonic acid - DTT dithiothreitol     

Reconstitution of sarcoplasmic reticulum Ca2+-ATPase vesicles lacking ion channels and demonstration of electrogenicity of Ca2+-pump

Sarcoplasmic reticulum (SR) vesicles were reconstituted by the salting out method in the presence of excess phospholipids: the lipid-to-protein ratio ranged from 10 to 100. It was found that the reconstituted vesicles could be separated by KC1 density gradient centrifugation into four types: those having both cation and anion channels (CASR), those having only cation channels (CSR), those having only anion channels (ASR), and those having no ion channels (PSR). From the yield of these vesicles, it was estimated that one native SR vesicle contains 19 cation channels and 1.4 anion channels on average; the amount of cation channels is 14 times larger than that of anion channels. Although all vesicles thus prepared are considered to contain the Ca2+-ATPase protein, the PSR vesicles alone did not take up Ca2+, but they did do so in the presence of valinomycin. This result indicates that the Ca2+-ATPase takes up Ca2+ in an electrogenic manner. The electromotive force was estimated to be about 50 mV.     

Ca2+-ATPase from sarcoplasmic reticulum: acylphosphatase activity

Fractionation of sarcoplasmic reticulum vesicles from rabbit skeletal muscle was performed by solubilization of the vesicles in the presence of deoxycholate, followed by sucrose density gradient centrifugation and gel filtration chromatography. This procedure permitted the isolation of essentially pure Ca²⁺-ATPase; this enzyme showed ATPase as well as acylphosphatase activity, both activities being clearly enhanced by deoxycholate. The acylphosphatase activity of the purified Ca²⁺-ATPase was characterized with regard to some kinetic properties, such as pH, Mg²⁺, Ca²⁺, and deoxycholate dependence, and substrate affinity, determined in the presence of acetylphosphate, succinylphosphate, carbamylphosphate, and benzoylphosphate; in addition, the stability of both activities was checked in time-course experiments. The main similarities between the two activities, such as the Mg²⁺ requirement, the deoxycholate activation, and the pH dependence, together with the competitive inhibition of the benzoylphosphatase activity by ATP, the inhibition of both activities by tris(bathophenanthroline)-Fe²⁺, and the relief of this inhibitory effect by carbonylcyanide-4-trifluoromethoxyphenyl hydrazone support the hypothesis that acylphosphatase and ATPase activities of sarcoplasmic reticulum vesicles reside in the same active site of the enzyme. With regard to possible relationships between acylphosphatase activity of the purified Ca²⁺-ATPase and “soluble” acylphosphatase present in the 100,000g supernatant fraction, comparison of some kinetic and structural parameters indicate that these two activities are supported by quite different enzymes.     

Organization of lipids in sarcoplasmic reticulum membrane and Ca2+-dependent ATPase activity.

The organization of lipids in sarcoplasmic reticulum membrane was studied with a variety of stearic spin labels and a phosphatidylcholine spin label. The ESR spectra of the spin-labeled membranes consisted of two components, one due to labels in lipid bilayer structure and the other due to more immobilized labels. The relative intensity of the immobilized component increased when the lipid content of the membrane was decreased by treatment with phospholipase A [EC 3.1.1.4] and subsequent washing with bovine serum albumin. Membrane containing 30% of the intact phospholipid, i.e.0.15 mg of phospholipid per mg of protein, showed a spectrum consisting only of the immobilized component (the overall splitting ranged from 58.5 G to 60.5 G). The immobilized component was ascribed to lipids complexed with protein. The fraction of lipids in the two different organizations was determined from the ESR spectrum. The activity of the Ca2+-Mg2+ dependent ATPase [ATP phosphohydrolase, EC 3.6.1.3] was found to increase almost linearly with the lipid bilayer content in the membrane, whereas phosphoenzyme formation was almost independent of the bilayer content. This indicated that the bilayer structure is necessary for the ATPase to attain its full transport activity.     

Mechanism of inhibition of Ca2+-transport activity of sarcoplasmic reticulum Ca2+-ATPase by anisodamine

The mechanism of inhibition of Ca2+-transport activity of rabbit sarcoplasmic reticulum Ca 2+-ATPase (SERCA) by anisodamine (a drug isolated from a medicinal herb Hyoscyamuns niger L) was investigated by using ANS (1-anilino-8-naphthalenesulfonate) fluorescence probe, intrinsic fluorescence quenching and Ca 2+-transport activity assays. The number of ANS binding sites for apo Ca2+-ATPase was determined as 8, using a multiple-identical binding site model. Both anisodamine and Ca2+ at millimolar level enhanced the ANS binding fluorescence intensities. Only anisodamine increased the number of ANS molecules bound by SERCA from 8 to 14. The dissociation constants of ANS to the enzyme without any ligand, with 30 mM anisodamine and with 15 mM Ca 2 were found to be 53.0 microM, 85.0 microM and 50.1 microM, respectively. Both anisodamine and Ca2+ enhanced the ANS binding fluorescenc with apparent dissociation constants of 7.6 mM and 2.3 mM, respectively, at a constant concentration of the enzyme. Binding of anisodamine significantly decreased the binding capacity of Ca2+ with the dissociation constant of 9.5 mM, but binding of Ca2+ had no obvious effect on binding of anisodamine. Intrinsic fluorescence quenching and Ca2+-transport activity assays gave the dissociation constants of anisodamine to SERCA as 9.7 and 5.4 mM, respectively, which were consistent with those obtained from ANS-binding fluorescence changes during titration of SERCA with anisodamine and anisodamine + 15 mM Ca2+, respectively. The results suggest that anisodamine regulates Ca2+-transport activity of the enzyme, by stabilizing the trans-membrane domain in an expanded, inactive conformation, at least at its annular ring region.     

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.     

Ca2+ translocation and catalytic activity of the sarcoplasmic reticulum ATPase. Modulation by ATP, Ca2+, and Pi

The ratio between Ca2+ uptake and Ca(2+)-dependent ATP hydrolysis measured in sarcoplasmic reticulum vesicles of rabbit skeletal muscle was found to vary greatly depending on the concentrations of oxalate or Pi used. In the presence of 5 mM oxalate, 20 mM Pi, and 1 mM Pi, the ratios found were in the range of 1.4-2.3, 0.6-0.8, and 0.01-0.10, respectively. The rates of Ca2+ exchange and ATP synthesis were measured at the steady state by adding trace amounts of 45Ca and 32Pi, after the vesicles had been loaded with Ca2+. In the presence of 1 mM Pi, 10 mM MgCl2, and 0.2 mM CaCl2, the ratio between Ca2+ exchange and ATP synthesis varied from 9 to 14. This ratio approached two when Ca2+ in the medium was reduced to a very low level, or when in the presence of Ca2+, dimethyl sulfoxide was added to the assay medium, or when the Pi concentration was raised from 1 to 20 mM. A ratio of two was also measured when the steady state was attained using ITP instead of ATP. In all the conditions that led to a ratio close to two, there was an increase in the fraction of enzyme phosphorylated by Pi. It is proposed that the coupling between Ca2+ translocation and ATP hydrolysis or synthesis is modulated by the phosphorylation of the ATPase by Pi.     

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.     

Modulation of Ca2+ release channel activity from sarcoplasmic reticulum by annexin VI (67-kDa calcimedin)

The effect of annexin VI (67-kDa calcimedin) on the activity of the Ca2+ release channel was studied using heavy sarcoplasmic reticulum membranes reconstituted into planar bilayers. Annexin VI, in a range of 5-40 nM, modified the gating behavior of the Ca2+ release channel by increasing the probability of opening by 2.7-fold and the mean open time by 82-fold relative to controls. Annexin VI caused no change in the slope conductance of the channel. The modulatory effect of annexin VI on the activity of Ca2+ release channels was Ca2+ dependent, and the annexin VI-modified channel was sensitive to both ruthenium red and ryanodine. The effect of annexin VI was observed when this protein was added specifically to the trans chamber, which corresponds to the luminal side of sarcoplasmic reticulum as determined by the ATP activation of the channel. In addition, differential extraction studies demonstrated that some annexin VI is localized within the lumen of the isolated heavy sarcoplasmic reticulum vesicles prepared by several different procedures. Annexin VI did not modify, from either the cis or trans chambers, the activity of K+ or Cl- channels from sarcoplasmic reticulum or the dihydropyridine sensitive Ca2+ channel from transverse tubules. In addition, the 38-kDa core proteolytic fragments of annexin VI had no effect on the Ca2+ release channel activity. Annexin VI is therefore a candidate for a physiological modulator of the Ca2+ release channel and as such, may play an important role in the excitation-contraction coupling.     

Lactate inhibits Ca2+-activated Ca2+-channel activity from skeletal muscle sarcoplasmic reticulum

Favero, Terence G., Anthony C. Zable, David Colter, andJonathan J. Abramson. Lactate inhibits Ca²⁺-activatedCa²⁺-channel activity from skeletal muscle sarcoplasmicreticulum. J. Appl. Physiol. 82(2): 447-452, 1997.Sarcoplasmic reticulum (SR) Ca²⁺-release channelfunction is modified by ligands that are generated during about ofexercise. We have examined the effects of lactate on Ca²⁺-and caffeine-stimulated Ca²⁺ release,[³H]ryanodine binding, and singleCa²⁺-release channel activity of SR isolated from rabbitwhite skeletal muscle. Lactate, at concentrations from 10 to 30 mM,inhibited Ca²⁺- and caffeine-stimulated[³H]ryanodine binding to and inhibited Ca²⁺-and caffeine-stimulated Ca²⁺ release from SR vesicles.Lactate also inhibited caffeine activation of single-channel activityin bilayer reconstitution experiments. These findings suggest thatintense muscle activity, which generates high concentrations oflactate, will disrupt excitation-contraction coupling. This may lead todecreases in Ca²⁺ transients promoting a decline in tensiondevelopment and contribute to muscle fatigue.

     

How do Ca2+ ions pass through the sarcoplasmic reticulum membrane

We propose an overview of the mechanism of Ca²⁺ transport through the sarcoplasmic reticulum membrane via the Ca²⁺-ATPase. We describe cytoplasmic calcium binding, calcium occlusion in the membrane and lumenal calcium dissociation. A channel-like structure is discussed and related to structural data on the membranous domain of the Ca²⁺-ATPase.Abbreviations SR Sarcoplasmic Reticulum - AMPPNP adenylyl-imidodiphosphate - AMPPCP adenylyl (,-methylene)-diphosphonate - FITC fluorescein 5-isothiocyanate - NBD 4-nitrobenzo-2-oxa-1,3-diazole - DCCD dicyclohexylcarbodiimide     

Inhibition of sarcoplasmic reticulum Ca2+-ATPase by miconazole

The inhibition of sarcoplasmic reticulumCa²⁺-ATPase activity by miconazole was dependent on theconcentration of ATP and membrane protein. Half-maximal inhibition wasobserved at 12 µM miconazole when the ATP concentration was 50 µMand the membrane protein was 0.05 mg/ml. When ATP was 1 mM, a lowmicromolar concentration of miconazole activated the enzyme, whereashigher concentrations inhibited it. A qualitatively similar responsewas observed when Ca²⁺ transport was measured. Likewise,the half-maximal inhibition value was higher when the membraneconcentration was raised. Phosphorylation studies carried out aftersample preequilibration in different experimental settings shed lighton key partial reactions such as Ca²⁺ binding and ATPphosphorylation. The miconazole effect on Ca²⁺-ATPaseactivity can be attributed to stabilization of theCa²⁺-free enzyme conformation giving rise to a decrease inthe rate of the Ca²⁺ binding transition. The phosphoryltransfer reaction was not affected by miconazole.

     

Inhibition of sarcoplasmic reticulum Ca2+-ATPase by 2-mercaptoethanol

The Ca²⁺-dependent ATPase activity of sarcoplasmic reticulum was inhibited when membrane vesicles were incubated at 0°C in presence of thiols. 2-mercaptoethanol was the most effective inhibitor from the thiols tested. The effect of 2-mercaptoethanol on the ATPase activity was biphasic; enzyme inhibition originally increased and then decreased with increasing thiol concentration. The inhibitory action of this thiol was significantly higher at low membrane concentrations and the rate of inactivation at 22°C was considerably lower than that at 0°C. Ca²⁺-ATPase previously inhibited by 2-mercaptoethanol was partially reactivated by incubation with periodate.