Ca-ATPase self-fluorescence in the sarcoplasmic reticulum of the rabbit skeletal muscle

The quenching of the intrinsic protein fluorescence of sarcoplasmic reticulum Ca-ATPase from the rabbit skeletal muscles by hydrophylic (NaI, CsCl) or hydrophobic (pyrene, fluorescamine) substances has been studied. CsCl (up to 1 M) has been shown not to affect the intrinsic protein fluorescence while NaI (250 mM) quenches it at 15%, pyrene (8 mkM) decreases the intrinsic fluorescence of Ca-ATPase at 35% and fluorescamine (up to 40 mkM)--at 80%. Possible mechanisms of the interaction of the quenchers with the intrinsic fluorescence of sarcoplasmic reticulum Ca-ATPase are being discussed.     

Effect of phenothiazines on Ca-ATPase in the sarcoplasmic reticulum of rabbit skeletal muscles

The phenothiazines trifluoroperazine , chlorpromazine and etmozine inhibit Ca-ATPase of the sarcoplasmic reticulum of rabbit skeletal muscles. The inhibitory action decreases in the order of trifluoroperazine greater than chlorpromazine greater than etmozine . The data are provided, indicating that the inhibitory effects of the phenothiazines on Ca-ATPase of the reticulum of the skeletal muscles are not mediated via calmodulin.     

Distribution of sarcoplasmic reticulum Ca-ATPase and of calsequestrin in rabbit and rat skeletal muscle fibers

Muscle fibers in rabbit extensor digitorum longus (EDL), tibialis anterior (TA) and soleus, and rat soleus, were examined immunohistochemically for two proteins of the sarcoplasmic reticulum. Ca-ATPase and calsequestrin (CaS). Fibers were typed with the histochemical reaction for actomyosin ATPase. In the rabbit EDL and TA, type I fibers clearly reacted less for Ca-ATPase and CaS than type II fibers, but the difference was less with CaS than with Ca-ATPase. Although the differences were relatively small, IIB fibers consistently presented greater amounts of Ca-ATPase than IIA fibers. No type II subgroups could be recognized after incubation with anti-CaS. These findings confirm results from previous immunochemical measurements on whole muscles containing different proportions of IIA and IIB fibers (Leberer and Pette 1986). Type IIA and IIC in the rabbit and rat soleus reacted stronger for Ca-ATPase and for CaS than type I fibers. Small differences in Ca-ATPase, but not in CaS, were recognized within the type I fiber population. Therefore, type I fibers in the rabbit and rat soleus are not a homogeneous population.     

Distribution of sarcoplasmic reticulum Ca-ATPase and of calsequestrin in rabbit and rat skeletal muscle fibers

Summary Muscle fibers in rabbit extensor digitorum longus (EDL), tibialis anterior (TA) and soleus, and rat soleus, were examined immunohistochemically for two proteins of the sarcoplasmic reticulum, Ca-ATPase and calsequestrin (CaS). Fibers were typed with the histochemical reaction for actomyosin ATPase. In the rabbit EDL and TA, type I fibers clearly reacted less for Ca-ATPase and CaS than type II fibers, but the difference was less with CaS than with Ca-ATPase. Although the differences were relatively small, HB fibers consistently presented greater amounts of Ca-ATPase than IIA fibers. No types II subgroups could be recognized after incubation with anti-CaS. These findings confirm results from previous immunochemical measurements on whole muscles containing different proportions of IIA and IIB fibers (Leberer and Pette 1986). Type IIA and IIC in the rabbit and rat soleus reacted stronger for Ca-ATPase and for CaS than type I fibers. Small differences in Ca-ATPase, but not in CaS, were recognized within the type I fiber population. Therefore, type I fibers in the rabbit and rat soleus are not a homogeneous population.     

Decreased conformational stability of the sarcoplasmic reticulum Ca-ATPase in aged skeletal muscle

Sarcoplasmic reticulum (SR) membranes purified from young adult (4–6 months) and aged (26–28 months) Fischer 344 male rat skeletal muscle were compared with respect to the functional and structural properties of the Ca-ATPase and its associated lipids. While we find no age-related alterations in (1) expression levels of Ca-ATPase protein, and (2) calcium transport and ATPase activities, the Ca-ATPase isolated from aged muscle exhibits more rapid inactivation during mild (37°C) heat treatment relative to that from young muscle. Saturation-transfer EPR measurements of maleimide spin-labeled Ca-ATPase and parallel measurements of fatty acyl chain dynamics demonstrate that, accompanying heat inactivation, the Ca-ATPase from aged skeletal muscle more readily undergoes self-association to form inactive oligomeric species without initial age-related differences in association state of the protein. Neither age nor heat inactivation results in differences in acyl chain dynamics of the bilayer including those lipids at the lipid-protein interface. Initial rates of tryptic digestion associated with the Ca-ATPase in SR isolated from aged muscle are 16( ± 2)% higher relative to that from young muscle, indicating more solvent exposure of a portion of the cytoplasmic domain. During heat inactivation these structural differences are amplified as a result of immediate and rapid further unfolding of the Ca-ATPase isolated from aged muscle relative to the delayed unfolding of the Ca-ATPase isolated from young muscle. Thus age-related alterations in the solvent exposure of cytoplasmic peptides of the Ca-ATPase are likely to be critical to the loss of conformational and functional stability.     

Effect of thyroxine on the function of rabbit skeletal muscle sarcoplasmic reticulum

Thyrotoxicosis in rabbits was induced by prolonged intraperitoneal injection of L-thyroxin. The development of thyroxicosis was assoiated with a decreased Ca2+ accumulation rate by sarcoplasmic reticulum (SR) fragments and a lowered Ca2+ dependent ATPase activity. As compared to the analogous parameters in normal animals. Ca2+ accumulation rate and ATPase activity of thyrotoxicosis animals decreased by 60 and 25%, respectively. The changes in the specific parameters of SR were also observed during incubation of normal SR samples in the medium containing thyroxin (10-5 M). The changes seen in SR functioning in thyrotoxicosis animals are likely to be related to structural rearrangements of lipoprotein surroundings of Ca-ATPase.     

Calcium-permeable channel in sarcoplasmic reticulum of rabbit skeletal muscle

Membrane vesicles from sarcoplasmic reticulum of rabbit skeletal muscle were incorporated into a bilayer lipid membrane. With this system, single current fluctuation was observed in the presence of 50 mM Ba-gluconate. This channel activity was observed only in vesicles from terminal cisternae. The single channel conductance was 14.1 pS, and the channel state was almost wholly open. The open-close transition of the channel obeyed simple two-state kinetics and was voltage-independent. The ionic selectivity was also studied, and the channel showed no selectivity among Ba, Ca, Mn, and Mg. On the other hand, it was less permeable to Cs than to Ba. Based on these results, the relation of the Ca channel to excitation-contraction coupling is discussed.     

Self-aggregation of triadin in the sarcoplasmic reticulum of rabbit skeletal muscle

The 95 kDa transmembrane glycoprotein triadin is believed to be an essential component of excitation-contraction coupling in the junctional sarcoplasmic reticulum of skeletal muscle fibers. It is debatable whether triadin mediates intraluminal interactions between calsequestrin and the ryanodine receptor exclusively or whether this junctional protein provides also a cytoplasmic linkage between the Ca2+-release channel and the dihydropyridine receptor. Here, we could show that native triadin exists as disulfide-linked homo-polymers of above 3000 kDa. Under non-reducing conditions, protein bands representing the alpha1-dihydropyridine receptor and calsequestrin did not show an immunodecorative overlap with the extremely high-molecular-mass triadin clusters. Following chemical crosslinking, the ryanodine receptor and triadin exhibited a similarly decreased electrophoretic mobility. However, immunoblotting of diagonal non-reducing/reducing two-dimensional gels clearly demonstrated a lack of overlap between the immunodecorated bands representing triadin, the alpha1-dihydropyridine receptor, the ryanodine receptor and calsequestrin. Thus, in native membranes triadin appears to form large self-aggregates primarily. Although triadin exists in a close neighborhood relationship to the Ca2+-release channel tetramers, it does not seem to be directly linked to the other main triad components implicated in the regulation of the excitation-contraction-relaxation cycle and Ca2+-homeostasis. This agrees with a proposed role of triadin in the maintenance of overall triad architecture.     

Spin labels in the analysis of Ca-ATPase in the sarcoplasmic reticulum of rabbit skeletal muscles in hypercholesteremia

The method of electron paramagnetic resonance with spin-labeled maleimide was used to study variation of the structure of Ca-ATPase of the sarcoplasmic reticulum (SR) in rabbit skeletal muscles under long-term hypercholesterolemia (HC). The rate of the maleimide spin label binding with Ca-ATPase of the SR was decreased in HC, which correlated with a lesser access of spin-labeled thiol groups for potassium ferricyanide and sodium ascorbate. HC led to a considerable reduction in the lability and to enhancement of hydrophobia of the spin-labeled fragment of the enzyme. It is concluded that the disordered function of the SR Ca-pump is a consequence of structural changes in the Ca-ATPase molecule in HC.     

Reversible inhibition of sarcoplasmic reticulum Ca-ATPase by altered neuromuscular activity in rabbit fast-twitch muscle

A 50% decrease in both the initial rate and the total capacity of Ca2+ uptake by the sarcoplasmic reticulum (SR) occurred 2 days after the onset of chronic (10 Hz) nerve stimulation in rabbit fast-twitch muscle. Prolonged stimulation (up to 28 days) did not lead to further decreases. This reduction, which was detected in muscle homogenates using a Ca2+-sensitive electrode, was reversible after 6 days cessation of stimulation and was not accompanied by changes in the immunochemically (ELISA) determined tissue level or isozyme characteristics of the SR Ca2+-ATPase protein. However, as measured in isolated SR, it correlated with a reduced specific activity of the Ca2+-ATPase. Kinetic analyses demonstrated that affinities of the SR Ca2+-ATPase towards Ca2+ and ATP were unaltered. Positive cooperativity for Ca2+ binding (h = 1.5) was maintained. However, a 50% decrease in Ca2+-dependent phosphoprotein formation indicated the presence of inactive forms of Ca2+-ATPase in stimulated muscle. The reduced phosphorylation of the enzyme was accompanied by an approximately 50% lowered binding of fluorescein isothiocyanate, a competitor at the ATP-binding site. In view of the unaltered affinity for ATP, this finding suggests that active Ca2+-ATPase molecules coexist in stimulated muscle with inactive enzyme molecules, the latter displaying altered properties at the nucleotide-binding site.     

The cross-linking of rabbit skeletal muscle sarcoplasmic reticulum protein.

Sarcoplasmic reticulum proteins have been cross-linked in situ with two reagents, the disulphide-bridged bifunctional imido ester, dimethyl-3,3'-dithiobispropionimidate dihydrochloride and the mild oxidant cupric phenanthroline. Analysis of proteins so cross-linked by electrophoresis on agarose/acrylamide gels reveals that a series of new polypeptides, up to a molecular weight of 900 000, are formed. These have molecular weights which are multiples of 100 000. Further analysis of samples by electrophoresis in a second dimensions containing a reducing agent revealed the monomeric polypeptides from which the cross-linked polypeptides were formed. With dimethyl 3,3'-dithiobispropionimidate dihydrochloride homopolymers of the Ca2+-stimulated ATPase, calsequestrin and/or calcium binding protein were formed. With cupric phenanthroline only the Ca2+-stimulated ATPase was involved in polymer formation. It has been confirmed on another gel system that these two proteins which are involved in Ca2+ binding are not cross-linked intermolecularly with this latter reagent. We conclude that the 100 000 dalton Ca2+-stimulated ATPase polypeptides are within 2 A of each other in the membrane while calsequestrin and/or calcium binding protein are within 11 A of each other. Although there appears to be no limit to the extent of cross-linking of any of these polypeptides there is not indication of heteropolymer associations between them.     

Isolation of two proteolipids from rabbit skeletal muscle sarcoplasmic reticulum

We have isolated two proteolipids from rabbit skeletal muscle sarcoplasmic reticulum by chromatography on columns of Sepharose CL-6B and Sephadex LH-60. One, PL-II, is identical to the proteolipid previously obtained by others using organic solvent extraction. The other, PL-I, has an amino acid composition very similar to those of proteolipids we previously isolated from canine cardiac SR and lamb kidney (Na,K)-ATPase.     

The nucleotide-binding site of the sarcoplasmic reticulum Ca-ATPase is conformationally altered in aged skeletal muscle.

Cellular conditions in senescent skeletal muscle have been shown to result in the loss of conformational stability of the sarcoplasmic reticulum (SR) Ca-ATPase. To identify underlying structural features of age-modified Ca-ATPase, we have utilized the fluorescence properties of protein-bound probes to assess both local and global structure. We find conformational changes that include an age-related decrease in the apparent binding affinity to high affinity calcium sites detected by fluorescence signals in both tryptophans within nearby membrane-spanning helices and fluorescein isothiocyanate (FITC) bound distally to Lys(515) within the nucleotide-binding site. In addition, a substantial (80%) age-related increase in the accessibility to soluble quenchers of fluorescence of FITC is observed without concomitant changes in bimolecular quenching constants (k(q)) for protein-bound IAEDANS, also within the nucleotide-binding domain, and tryptophans within the membrane. Using fluorescence resonance energy transfer to measure distances between IAEDANS and FITC across the nucleotide-binding domain, we find no significant age-related change in the mean donor-acceptor distance; however, significant increases are observed in the conformational heterogeneity of this domain, as assessed by the width at half-maximum (HW) of the distance distribution, increasing with age from 29.4 +/- 0.8 A to 42.5 +/- 1. 1 A. Circular dichroism indicates that the average secondary structure is unaltered with age. Thus, these data suggest tertiary structural alterations in specific regions around the nucleotide-binding site rather than global conformational changes.     

Cyclic ADP-ribose induced Ca release in rabbit skeletal muscle sarcoplasmic reticulum

The Ca²⁺-mobilizing metabolite cyclic ADP-ribose (cADPR) has been shown to release Ca²⁺ from ryanodine-sensitive stores in many cells. We show that this metabolite at a concentration of 17μM, but not its precursor β-NAD⁺ nor non-cyclic ADPR at the same concentration, is active in releasing Ca²⁺ from rabbit skeletal muscle sarcoplasmic reticulum. The release was not sensitive to Ruthenium red (1μM) nor to the ryanodine receptor-specific scorpion toxin Buthotus₁-1 (10 μM). In planar bilayer single channel recordings, concentrations up to 50μM cADPR did not increase the open probability of Ruthenium red and toxin-sensitive Ca²⁺ release channels. Thus Ca²⁺ release induced by cADPR in skeletal muscle sarcoplasmic reticulum may not involve opening of ryanodine receptors.     

Shotgun proteomic analysis of sarcoplasmic reticulum preparations from rabbit skeletal muscle

To obtain a comprehensive understanding of proteins involved in excitation–contraction coupling, a catalog of proteins from sarcoplasmic reticulum (SR) membrane fractions of New Zealand white rabbit skeletal muscle was analyzed by an optimized shotgun proteomic method. Light and heavy SR membrane fractions were obtained by nonlinear sucrose gradient centrifugation and separated by 1DE followed by a highly reproducible, automated LC‐MS/MS on the hybrid linear ion trap (LTQ) Orbitrap mass spectrometer. By integrating as low as 1% false discovery rate as one of the features for quality control method, 483 proteins were identified from both of the two independent SR preparations. Proteins involved in calcium release unit complex, including ryanodine receptor 1, dihydropyridine receptor, calmodulin, triadin, junctin, and calsequestrin, were all detected, which offered validation for this protein identification method. Rigorous bioinformatics analysis was performed. Protein pI value, molecular weight range, hydrophobicity index, and transmembrane region were calculated using bioinformatics softwares. Eighty‐three proteins were classified as hydrophobic proteins and 175 proteins were recognized as membrane proteins. Based on the proteomic analysis results, we found as the first time that not only transverse tubule but also mitochondrion physically connected to SR. The complete mapping of these proteomes may help in the elucidation of the process of excitation–contraction coupling and excitation–metabolism coupling.     

Quercetin stimulation of calcium release from rabbit skeletal muscle sarcoplasmic reticulum

To elucidate the mechnism by which quercetin enhances the rate of tension development in skinned muscle fibers, effects on calcium release from longitudinal tubule-derived SR (LSR) after phosphate-supported calcium uptake were examined. In all studies, 100 μM quercetin (which inhibits initial calcium uptake velocity 85%) was added at or shortly after the time calcium content reached a maximum at various extravesicular Ca²⁺ concentrations (Ca_o). At moderate Ca_o (0.2–1.0 μM). where spontaneous calcium release rate depended on Ca_o, quercetin caused a marked stimulation of calcium release. This was accompanied by a 60% reduction in calcium influx and a 30-fold increase in calcium efflux. Thus, the previously reported quercetin-induced increase in the rate of tension development by skinned muscle fibers may result, at least in part, from sensitization of Ca²⁺-triggered calcium release to lower Ca_o.     

The effect of carnosine on Ca-channels in rabbit skeletal muscle sarcoplasmic reticulum]

Carnosine (beta-alanyl-L-histidine), which is present in millimolar concentrations in skeletal muscles, induces Ca2+ release from the heavy fraction of rabbit skeletal muscle sarcoplasmic reticulum by activation ruthenium red-sensitive Ca-release channels. The effect of carnosine is dose-dependent, which indicates the presence of saturable carnosine-binding sites in the Ca-release channel molecule. The half-maximal Ca2+ release is observed in the presence of 8.7 mM carnosine. At the same time, carnosine addition to the medium increases the affinity of sarcoplasmic reticulum Ca-channels for the Ca-release activators, caffeine and adenine nucleotides. It is concluded that carnosine is an endogenous regulator of skeletal muscle sarcoplasmic reticulum Ca-channels which modulates the affinity of these channels for different ligands.