Conformational change of the foot protein of sarcoplasmic reticulum as an initial event of calcium release

Heavy sarcoplasmic reticulum vesicles were labeled with the thiol-reacting fluorescent probe N-(7-dimethylamino-4-methyl-4-coumarinyl)maleimide (DACM), and the DACM-labeled foot protein moiety was purified. The fluorescence intensity of the DACM attached to the foot protein decreased by the addition of low (activating) concentrations of ryanodine, while it increased at higher (inhibitory) concentrations, suggesting that the lower fluorescence represents the active state of the foot protein, while the higher fluorescence, its inactive state. Under conditions that induce Ca2+ release from SR (Ca2+ jump, addition of Ca2+ release inducing reagents such as caffeine and polylysine), the fluorescence intensity of the protein-attached DACM decreased rapidly (e.g. k congruent to 70 s-1 under optimum conditions). The initial rate of Ca2+ release from the DACM-labeled SR showed a close correlation with the amplitude of the fluorescence change of the foot protein-attached DACM under variety of conditions; e.g. in the presence of Ca2+, polylysine, ATP, and ruthenium red, etc. The fluorescence change of the foot protein was much faster than Ca2+ release from SR under a variety of conditions of Ca2+ release. We propose that the binding of release triggering reagents to the foot protein induces a rapid conformational change, which in turn regulates Ca2+ release.     

Intravesicular calcium transient during calcium release from sarcoplasmic reticulum

The time course of changes in the intravesicular Ca2+ concentration ([Ca2+]i) in terminal cisternal sarcoplasmic reticulum vesicles upon the induction of Ca2+ release was investigated by using tetramethylmurexide (TMX) as an intravesicular Ca2+ probe. Upon the addition of polylysine at the concentration that led to the maximum rate of Ca2+ release, [Ca2+]i decreased monotonically in parallel with Ca2+ release. Upon induction of Ca2+ release by lower concentrations of polylysine, [Ca2+]i first increased above the resting level, followed by a decrease well below it. The release triggers polylysine, and caffeine brought about dissociation of calcium that bound to a nonvesicular membrane segment consisting of the junctional face membrane and calsequestrin bound to it, as monitored with TMX. No Ca2+ dissociation from calsequestrin-free junctional face membranes or from the dissociated calsequestrin was produced by release triggers, but upon reassociation of the dissociated calsequestrin and the junctional face membrane, Ca2+ dissociation by triggers was restored. On the basis of these results, we propose that the release triggers elicit a signal in the junctional face membrane, presumably in the foot protein moiety, which is then transmitted to calsequestrin, leading to the dissociation of the bound calcium; and in SR vesicles, to the transient increase of [Ca2+]i, and subsequently release across the membrane.     

Identification of a region of calsequestrin that binds to the junctional face membrane of sarcoplasmic reticulum

The interaction of calsequestrin (CSQ) with the channel-containing region of the sarcoplasmic reticulum (junctional face membrane, jfm) is involved in Ca2+ release, and it seemed of interest to identify the jfm-binding region of the CSQ molecule. For this purpose, CSQ was digested with trypsin, and peptides were screened for jfm binding. Partial amino acid sequencing of selected peptides allowed us to localize a critical site for jfm binding in the stretch encompassing residues 86-191.     

Intraluminal Ca2+ dependence of Ca2+ and ryanodine-mediated regulation of skeletal muscle sarcoplasmic reticulum Ca2+ release.

The action of ryanodine upon sarcoplasmic reticulum (SR) Ca2+ handling is controversial with evidence for both activation and inhibition of SR Ca2+ release. In this study, the role of the intraluminal SR Ca2+ load was probed as a potential regulator of ryanodine-mediated effects upon SR Ca2+ release. Through dual-wavelength spectroscopy of Ca2+:antipyrylazo III difference absorbance, the intraluminal Ca2+ dependence of ryanodine and Ca(2+)-induced Ca2+ release (CICR) from skeletal SR vesicles was examined. Ryanodine addition after initiation of Ca2+ uptake (a) increased the intraluminal Ca2+ sensitivity of CICR and (b) stimulated spontaneous Ca2+ release with a delayed onset. These ryanodine effects were inversely proportional to the intraluminal Ca2+ load. Ryanodine also inhibited subsequent CICR after reaccumulation of Ca2+ released from the initial CICR. These results provide evidence that ryanodine inhibits transitions between low and high affinity Ca2+ binding states of an intraluminal Ca2+ compartment, possibly calsequestrin. Conformational transitions of calsequestrin may be reciprocally coupled to transitions between open and closed states of the Ca2+ release channel.     

Calcium binding proteins of junctional sarcoplasmic reticulum: detection by 45Ca ligand overlay

In skeletal muscle, the junctional sarcoplasmic reticulum (JFM) plays a crucial role in excitation-contraction coupling and Ca2+ release. In the present report, the sarcoplasmic reticulum (SR) was fractionated into longitudinal SR (LSR), terminal cisternae (TC), and JFM. Each fraction had a unique protein profile as detected by SDS-polyacrylamide gel electrophoresis as well as specific Ca2+ binding proteins as judged by 45Ca ligand overlay of nitrocellulose blots. Ca2+ binding proteins of LSR were the Ca2+ ATPase (Mr of 115K), an 80K polypeptide, and the intrinsic glycoprotein (Mr of 160K); Ca2+ binding proteins of JFM were polypeptides with the following Mr values: 350K and 325K (feet components), 200K, 170K, a doublet of 140K, 118K, 65K (calsequestrin), and 52K. Measurements of Ca2+ binding to SR fractions by equilibrium dialysis indicated that 8-17 nmol Ca2+/mg of protein was specifically bound. After EDTA extraction of calsequestrin, JFM still bound Ca2+ (5-6 nmol/mg of protein), suggesting the existence of specific Ca2+ binding sites. The Ca2+ binding sites of Ca2+-gated Ca2+ release channels might be on two JFM polypeptides (Mr's of 350K and 170K) which are putative channel constituents (F. Zorzato, A. Margreth, and P. Volpe (1986) J. Biol. Chem. 261, 13252-13257).     

Ca2+ binding effects on protein conformation and protein interactions of canine cardiac calsequestrin

Calsequestrin is a Ca2+-binding protein located intraluminally in the junctional sarcoplasmic reticulum (SR) of striated muscle. In this study, Ca2+ binding to cardiac calsequestrin was assessed directly by equilibrium dialysis and correlated with effects on protein conformation and calsequestrin's ability to interact with other SR proteins. Cardiac calsequestrin bound 800-900 nmol of Ca2+/mg of protein (35-40 mol of Ca2+/mol of calsequestrin). Associated with Ca2+ binding to cardiac calsequestrin was a loss in protein hydrophobicity, as revealed with use of absorbance difference spectroscopy, fluorescence emission spectroscopy, and photoaffinity labeling with the hydrophobic probe 3-(trifluoromethyl)-3-(m-[125]iodophenyl)diazirine. Ca2+ binding to cardiac calsequestrin also caused a large change in its hydrodynamic character, almost doubling the sedimentation coefficient. We observed that cardiac calsequestrin was very resistant to several proteases after binding Ca2+, consistent with a global effect of Ca2+ on protein conformation. Moreover, Ca2+ binding to cardiac calsequestrin completely prevented its interaction with several calsequestrin-binding proteins, which we identified in cardiac junctional SR vesicles for the first time. The principal calsequestrin-binding protein identified in junctional SR vesicles exhibited an apparent Mr of 26,000 in sodium dodecyl sulfate-polyacrylamide gels. This 26-kDa calsequestrin-binding protein was greatly reduced in free SR vesicles and absent from sarcolemmal vesicles and was different from phospholamban, an SR regulatory protein exhibiting a similar molecular weight. Our results suggest that the specific interaction of calsequestrin with this 26-kDa protein may be regulated by Ca2+ concentration in intact cardiac muscle, when the Ca2+ concentration inside the junctional SR falls to submillimolar levels during coupling of excitation to contraction.     

Calsequestrin and the calcium release channel of skeletal and cardiac muscle

Calsequestrin is by far the most abundant Ca(2+)-binding protein in the sarcoplasmic reticulum (SR) of skeletal and cardiac muscle. It allows the Ca2+ required for contraction to be stored at total concentrations of up to 20mM, while the free Ca2+ concentration remains at approximately 1mM. This storage capacity confers upon muscle the ability to contract frequently with minimal run-down in tension. Calsequestrin is highly acidic, containing up to 50 Ca(2+)-binding sites, which are formed simply by clustering of two or more acidic residues. The Kd for Ca2+ binding is between 1 and 100 microM, depending on the isoform, species and the presence of other cations. Calsequestrin monomers have a molecular mass of approximately 40 kDa and contain approximately 400 residues. The monomer contains three domains each with a compact alpha-helical/beta-sheet thioredoxin fold which is stable in the presence of Ca2+. The protein polymerises when Ca2+ concentrations approach 1mM. The polymer is anchored at one end to ryanodine receptor (RyR) Ca2+ release channels either via the intrinsic membrane proteins triadin and junctin or by binding directly to the RyR. It is becoming clear that calsequestrin has several functions in the lumen of the SR in addition to its well-recognised role as a Ca2+ buffer. Firstly, it is a luminal regulator of RyR activity. When triadin and junctin are present, calsequestrin maximally inhibits the Ca2+ release channel when the free Ca2+ concentration in the SR lumen is 1mM. The inhibition is relieved when the Ca2+ concentration alters, either because of small changes in the conformation of calsequestrin or its dissociation from the junctional face membrane. These changes in calsequestrin's association with the RyR amplify the direct effects of luminal Ca2+ concentration on RyR activity. In addition, calsequestrin activates purified RyRs lacking triadin and junctin. Further roles for calsequestrin are indicated by the kinase activity of the protein, its thioredoxin-like structure and its influence over store operated Ca2+ entry. Clearly, calsequestrin plays a major role in calcium homeostasis that extends well beyond its ability to buffer Ca2+ ions.     

Fluorescence conformational probe study of calcium release from sarcoplasmic reticulum

Sarcoplasmic reticulum isolated from rabbit skeletal muscle was labeled with a limited (0.625 nmol/mg sarcoplasmic reticulum protein) amount of the fluorescent thiol reagent N-(7-dimethylamino-4-methyl-3-coumarinyl)maleimide (DACM). The fluorescence intensity of the membrane-attached DACM decreased concurrently with (Ca2+ and caffeine)-induced Ca2+ release, depolarization-induced Ca2+ release and Ca2+-dependent dependent passive efflux of Ca2+. The decreased DACM fluorescence level initiated by a Ca2+ jump was subsequently reversed under passive efflux conditions when there was no ATP-dependent Ca2+ uptake, suggesting spontaneous closing of the channels. Therefore, the higher fluorescence level corresponds to a larger population of closed channels, whereas the lower level represents a larger population of opened channels. Under conditions when the Ca2+ release-coupled fluorescence change was maximal, a stoichiometric incorporation of DACM took place only into a 32-kDa protein. Furthermore, reconstituted vesicles, in which purified DACM-labeled 32-kDa protein was incorporated into unlabeled sarcoplasmic reticulum vesicles, were capable of both (Ca2+ and caffeine)-induced Ca2+ release and the release-coupled DACM fluorescence change. These results suggest that the 32-kDa protein is a constituent of the Ca2+ release channel or a protein which is in close contact with the channel.     

Isolation of terminal cisternae of frog skeletal muscle. Calcium storage and release properties

Sarcoplasmic reticulum (SR) terminal cisternae (TC) of frog (Rana esculenta) fast-twitch skeletal muscle have been purified by isopycnic sucrose density gradient centrifugation. Biochemical characteristics and Ca2+ release properties have been investigated and compared to those of the homologous fraction of rabbit skeletal muscle TC. The frog SR fraction obtained at the 38/45% sucrose interface appears to be derived from the terminal cisternae region as judged by: (a) thin section electron microscopy showing vesicles containing electron opaque material and squarelike (feet) projections at the outer surface; (b) protein composition (Ca2+-ATPase, calsequestrin, and high Mr proteins); (c) Ca2+ fluxes properties. The content of calsequestrin was higher in frog TC by 50% and the Ca2+ binding capacity (624 or 45 nmol of Ca2+/mg of TC protein, depending upon experimental conditions) was 3-4 times that of rabbit TC. Species-specific antigenic differences were found between junctional SR proteins of frog and rabbit TC. After active Ca2+ preloading in the presence of pyrophosphate (Palade, P. (1987) J. Biol. Chem. 262, 6135-6141), caffeine and doxorubicin elicited Ca2+ release from either TC fraction but with much faster rates in frog TC than in rabbit TC (14 versus 3 mumol of Ca2+/min/mg of protein). The present results provide new evidence for the existence of marked differences in Ca2+ release properties between TC of amphibian and mammalian fast-twitch muscle. Higher Ca2+ binding capacity and faster release rates in frog TC might compensate for the comparably greater diffusion distance being covered by the released Ca2+ from the Z-line to the actomyosin cross-bridges in the A-I overlap region.     

Characterization of the sarcoplasmic reticulum proteins in the thermogenic muscles of fish

Marlins, sailfish, spearfishes, and swordfish have extraocular muscles that are modified into thermogenic organs beneath the brain. The modified muscle cells, called heater cells, lack organized myofibrils and are densely packed with sarcoplasmic reticulum (SR), transverse (T) tubules, and mitochondria. Thermogenesis in the modified extraocular muscle fibers is hypothesized to be associated with increased energy turnover due to Ca2+ cycling at the SR. In this study, the proteins associated with sequestering and releasing Ca2+ from the SR (ryanodine receptor, Ca2+ ATPase, calsequestrin) of striated muscle cells were characterized in the heater SR using immunoblot and immunofluorescent techniques. Immunoblot analysis with a monoclonal antibody that recognizes both isoforms of nonmammalian RYRs indicates that the fish heater cells express only the alpha RYR isoform. The calcium dependency of [3H]ryanodine binding to the RYR isoform expressed in heater indicates functional identity with the non-mammalian alpha RYR isoform. Fluorescent labeling demonstrates that the RYR is localized in an anastomosing network throughout the heater cell cytoplasm. Measurements of oxalate supported 45Ca2+ uptake, Ca2+ ATPase activity, and [32P]phosphoenzyme formation demonstrate that the SR contains a high capacity for Ca2+ uptake via an ATP dependent enzyme. Immunoblot analysis of calsequestrin revealed a significant amount of the Ca2+ binding protein in the heater cell SR. The present study provides the first direct evidence that the heater SR system contains the proteins necessary for Ca2+ release, re-uptake and sequestration, thus supporting the hypothesis that thermogenesis in the modified muscle cells is achieved via an ATP-dependent cycling of Ca2+ between the SR and cytosolic compartments.     

Increased Ca2+ storage capacity of the skeletal muscle sarcoplasmic reticulum of transgenic mice over-expressing membrane bound calcium binding protein junctate

Junctate is an integral sarco(endo)plasmic reticulum protein expressed in many tissues including heart and skeletal muscle. Because of its localization and biochemical characteristics, junctate is deemed to participate in the regulation of the intracellular Ca2+ concentration. However, its physiological function in muscle cells has not been investigated yet. In this study we examined the effects of junctate over-expression by generating a transgenic mouse model which over-expresses junctate in skeletal muscle. Our results demonstrate that junctate over-expression induced a significant increase in SR Ca2+ storage capacity which was paralleled by an increased 4-chloro-m-cresol and caffeine-induced Ca2+ release, whereas it did not affect SR Ca2+-dependent ATPase activity and SR Ca2+ loading rates. In addition, junctate over-expression did not affect the expression levels of SR Ca2+ binding proteins such as calsequestrin, calreticulin and sarcalumenin. These findings suggest that junctate over-expression is associated with an increase in the SR Ca2+ storage capacity and releasable Ca2+ content and support a physiological role for junctate in intracellular Ca2+ homeostasis.     

Morphology and molecular composition of sarcoplasmic reticulum surface junctions in the absence of DHPR and RyR in mouse skeletal muscle

Calcium release during excitation-contraction coupling of skeletal muscle cells is initiated by the functional interaction of the exterior membrane and the sarcoplasmic reticulum (SR), mediated by the "mechanical" coupling of ryanodine receptors (RyR) and dihydropyridine receptors (DHPR). RyR is the sarcoplasmic reticulum Ca(2+) release channel and DHPR is an L-type calcium channel of exterior membranes (surface membrane and T tubules), which acts as the voltage sensor of excitation-contraction coupling. The two proteins communicate with each other at junctions between SR and exterior membranes called calcium release units and are associated with several proteins of which triadin and calsequestrin are the best characterized. Calcium release units are present in diaphragm muscles and hind limb derived primary cultures of double knock out mice lacking both DHPR and RyR. The junctions show coupling between exterior membranes and SR, and an apparently normal content and disposition of triadin and calsequestrin. Therefore SR-surface docking, targeting of triadin and calsequestrin to the junctional SR domains and the structural organization of the two latter proteins are not affected by lack of DHPR and RyR. Interestingly, simultaneous lack of the two major excitation-contraction coupling proteins results in decrease of calcium release units frequency in the diaphragm, compared with either single knockout mutation.     

Polylysine induces a rapid Ca2+ release from sarcoplasmic reticulum vesicles by mediation of its binding to the foot protein

The addition of polylysine to a heavy fraction of sarcoplasmic reticulum (SR) vesicles produces a rapid Ca2+ release with no appreciable lag period. The polylysine concentration for half-maximal activation (C1/2) is approximately 0.99 micrograms/ml, or 0.3 microM, the lowest C 1/2 for Ca2+ release-inducing reagents reported in the literature. The time course and the [Ca2+] dependence of polylysine-induced release are similar to those of caffeine-induced Ca2+ release. At higher concentrations of polylysine (e.g., 10 micrograms/ml), however, little or no Ca2+ release occurs. Upon photolysis of SR vesicles with the photocrosslinkable radiolabeled polylysine derivative, [3H]succinimidyl azido benzoate polylysine, 0.28 and 0.52-1.2 mol polylysine were bound to 1 mol of the 400-kDa foot protein at activating (3 micrograms/ml) and inhibitory (10 micrograms/ml) concentrations of polylysine, respectively. On the other hand, the amounts of polylysine bound to the other SR proteins (mol/mol) were negligible (e.g., less than or equal to 0.0127 mol polylysine/mol calsequestrin). This suggests that the binding of polylysine to the foot protein is responsible not only for the induction of release but also for inactivation. These results provide direct evidence that the receptor for the chemical trigger of Ca2+ release is localized within the foot protein. Ruthenium red, which inhibits polylysine-induced Ca2+ release, does not inhibit polylysine binding to the foot protein, suggesting that the polylysine binding domain of the foot protein is different from the channel domain.     

Rapid purification of calsequestrin from cardiac and skeletal muscle sarcoplasmic reticulum vesicles by Ca2+-dependent elution from phenyl-sepharose

Treatment of cardiac or skeletal muscle sarcoplasmic reticulum vesicles with 0.1 M sodium carbonate selectively extracts both the Ca2+-binding protein calsequestrin and the two "intrinsic glycoproteins," while leaving the Ca2+-dependent ATPase membrane bound. Phenyl-Sepharose chromatography in the presence of ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) and high salt (0.5 M NaCl) readily fractionates these solubilized proteins into a Ca2+-elutable fraction, which contains purified calsequestrin, and a low ionic strength elutable fraction, which contains one of the two intrinsic glycoproteins. Elution of calsequestrin from phenyl-Sepharose occurs near 1 mM Ca2+. Copurifying with calsequestrin are an homologous set of high molecular weight proteins, which like calsequestrin stain blue with Stains-All. These proteins are present in trace amounts and do not correspond to any sarcoplasmic reticulum proteins previously identified. Elution of calsequestrin from phenyl-Sepharose is consistent with the Ca2+-binding protein losing its hydrophobic character in the presence of millimolar Ca2+. This behavior is converse to that observed for several calmodulin-like proteins, which are eluted from hydrophobic gels in the presence of EGTA. The high yield and purity of calsequestrin prepared by this method makes possible a unique system for studying what may be a distinct class of Ca2+-binding proteins.     

Rescue of contractile parameters and myocyte hypertrophy in calsequestrin overexpressing myocardium by phospholamban ablation

Cardiac-specific overexpression of murine cardiac calsequestrin results in depressed cardiac contractile parameters, low Ca(2+)-induced Ca(2+) release from sarcoplasmic reticulum (SR) and cardiac hypertrophy in transgenic mice. To test the hypothesis that inhibition of phospholamban activity may rescue some of these phenotypic alterations, the calsequestrin overexpressing mice were cross-bred with phospholamban-knockout mice. Phospholamban ablation in calsequestrin overexpressing mice led to reversal of the depressed cardiac contractile parameters in Langendorff-perfused hearts or in vivo. This was associated with increases of SR Ca(2+) storage, assessed by caffeine-induced Na(+)-Ca(2+) exchanger currents. The inactivation time of the L-type Ca(2+) current (I(Ca)), which has an inverse correlation with Ca(2+)-induced SR Ca(2+) release, and the relation between the peak current density and half-inactivation time were also normalized, indicating a restoration in the ability of I(Ca) to trigger SR Ca(2+) release. The prolonged action potentials in calsequestrin overexpressing cardiomyocytes also reversed to normal upon phospholamban ablation. Furthermore, ablation of phospholamban restored the expression levels of atrial natriuretic factor and alpha-skeletal actin mRNA as well as ventricular myocyte size. These results indicate that attenuation of phospholamban function may prevent or overcome functional and remodeling defects in hypertrophied hearts.     

High molecular weight proteins in cardiac and skeletal muscle junctional sarcoplasmic reticulum vesicles bind calmodulin, are phosphorylated, and are degraded by Ca2+-activated protease

A unique set of high molecular weight proteins was identified in junctional sarcoplasmic reticulum (SR) vesicles isolated from both cardiac muscle and skeletal muscle. These high Mr proteins were not present in free SR vesicles isolated from either tissue, nor were they observed in purified sarcolemmal fractions. The junctional SR high Mr proteins migrated as doublets in sodium dodecyl sulfate-polyacrylamide gels and exhibited apparent Mr values between 290,000 and 350,000. The high Mr proteins bound calmodulin; they were the principal proteins labeled in the cardiac and skeletal muscle SR subfractions by azido-125I-calmodulin. The high Mr proteins were also substrates for an endogenous Ca2+-calmodulin-dependent protein kinase activity, as well as exogenously added catalytic subunit of cAMP-dependent protein kinase. In addition, the junctional SR high Mr proteins were the major SR proteins degraded by a Ca2+-activated protease purified from smooth muscle. Control experiments verified the separation of junctional SR vesicles and free SR vesicles from both muscle types. Junctional SR vesicles were enriched in calsequestrin, and they exhibited Ca2+ uptake which was stimulated up to 10-fold by either ryanodine or ruthenium red. Free SR vesicles were deficient in calsequestrin and were insensitive to these two agents. Localization of the cardiac and skeletal muscle high Mr proteins to the junctional SR, coupled with demonstration of their nearly identical biochemical properties, suggests that the proteins are homologous and are likely to have similar functions in both types of striated muscle.     

Effect of luminal calcium on Ca2+ release channel activity of sarcoplasmic reticulum in situ.

Ca2+ influx into empty SR in the absence of Ca2+ pump activity was determined in skinned frog skeletal muscle fibers and compared with Ca2+ efflux from loaded SR (i.e., Ca2+ release) to deepen our understanding of the properties of the Ca2+ release channel (CRC). Calcium content in SR increased approximately in a first-order kinetics and finally reached the equilibrium level determined by cytoplasmic Ca2+ ([Ca2+]c). Because AMP caused an increase in the rate of Ca2+ influx, and procaine, Mg2+, and high concentrations of Ca2+ caused a characteristic decrease, the major Ca2+ influx pathway was concluded to be the CRC, as is true of Ca2+ release. The apparent rate constant (k(app)) of Ca2+ efflux did not significantly change when the loading level was decreased to one-third. At a given [Ca2+]c, the same equilibrium level of calcium in SR was attained with a similar k(app) by both Ca2+ influx and Ca2+ efflux. The relationship between [Ca2+]c and calcium in SR indicated the Ca2+ binding sites in SR. These results, together with the anticipated effects of these Ca2+ buffer sites on kinetics, are consistent with the idea that luminal Ca2+ inhibits the CRC.     

Potentiation of fractional sarcoplasmic reticulum calcium release by total and free intra-sarcoplasmic reticulum calcium concentration

Our aim was to measure the influence of sarcoplasmic reticulum (SR) calcium content ([Ca](SRT)) and free SR [Ca] ([Ca](SR)) on the fraction of SR calcium released during voltage clamp steps in isolated rabbit ventricular myocytes. [Ca](SRT), as measured by caffeine application, was progressively increased by conditioning pulses. Sodium was absent in both the intracellular and in the extracellular solutions to block sodium/calcium exchange. Total cytosolic calcium flux during the transient was inferred from I(Ca), [Ca](SRT), [Ca](i), and cellular buffering characteristics. Fluxes via the calcium current (I(Ca)), the SR calcium pump, and passive leak from the SR were evaluated to determine SR calcium release flux (J(rel)). Excitation-contraction (EC) coupling was characterized with respect to both gain (integral J(rel)/integral I(Ca)) and fractional SR calcium release. Both parameters were virtually zero for a small, but measurable [Ca](SRT). Gain and fractional SR calcium release increased steeply and nonlinearly with both [Ca](SRT) and [Ca](SR). We conclude that potentiation of EC coupling can be correlated with both [Ca](SRT) and [Ca](SR). While fractional SR calcium release was not linearly dependent upon [Ca](SR), intra-SR calcium may play a crucial role in regulating the SR calcium release process.     

Postnatal development of Ca2+-sequestration by the sarcoplasmic reticulum of fast and slow muscles in normal and dystrophic mice

Ca2+-uptake activities of the sarcoplasmic reticulum (SR) were determined with a Ca2+-sensitive electrode in homogenates from fast- and slow-twitch muscles from both normal and dystrophic mice (C57BL/6J strain) of different ages. Immunochemical quantification of tissue Ca2+-ATPase content allowed determination of the specific Ca2+-transport activity of the enzyme. In 3-week-old mice of the dystrophic strain specific Ca2+ transport was already significantly lower than in the normal strain. It progressively decreased with maturation and reached only 40-50% and 30-50% of the normal values in fast- and slow-twitch muscles of adult dystrophic animals, respectively. Tissue contents of calsequestrin were reduced in both types of muscle leading to an increased Ca2+-ATPase to calsequestrin protein ratio. Equal amounts of the Ca2+-ATPase protein (detected by Coomassie blue staining of polyacrylamide gels) were present in SR vesicles isolated by Ca2+-oxalate loading from adult normal and dystrophic fast-twitch muscles. However, the specific ATP-hydrolysing activity of the enzyme was approximately 50% lower in dystrophic than in normal SR. The reduced ATP-hydrolysing activity was correlated with decreased Ca2+-transport activity, phosphoprotein formation and fluorescein isothiocyanate labeling as determined in total microsomal and heavy SR fractions. Although the Ca2+ and ATP affinities of the enzyme were unaltered, its ATPase activity was reduced at all levels of ATP in the dystrophic SR. Taken together, these findings point to a markedly impaired function of the SR and an increase in the population of inactive SR Ca2+-ATPase molecules in murine muscular dystrophy.