Electron probe X-ray microanalysis of post-tetanic Ca2+ and Mg2+ movements across the sarcoplasmic reticulum in situ

Ca2+ and Mg2+ movements across the sarcoplasmic reticulum (SR) of frog skeletal muscle fibers were measured in situ by electron probe microanalysis of muscles rapidly frozen following a tetanus. At 400 ms following a 1.2-s tetanus at room temperature, the force had relaxed to base-line, and 0.3 mmol of Ca2+/liter of cytoplasmic H2O had been pumped by the SR, indicating that the in situ pumping of the SR Ca-ATPase is sufficiently high to account for the removal of Ca2+ from the Ca2+-specific sites of troponin (0.18 mmol of Ca2+-specific sites/liter of cytoplasmic H2O) and for the rate of relaxation from a tetanus at room temperature. The half-time of the return of the total 1.0 mmol of Ca2+/liter of cytoplasmic H2O released during a tetanus was 1.1 s, comparable to the slow Koff rate of Ca2+ from (carp) parvalbumin (1.0 s-1) and consistent with the hypothesis that the return of this Ca2+ to the terminal cisternae is rate-limited by the Ca2+ off-rate from parvalbumin. The return of the Mg2+ taken up by the terminal cisternae during a tetanus to resting levels was significantly slower than the time course of the Ca2+ movements, suggesting that the Mg2+ permeability of the SR in situ is low and may be transiently increased during tetanic stimulation.     

Calcium and magnesium contents and volume of the terminal cisternae in caffeine-treated skeletal muscle

(a) The effects of caffeine on the composition and volume of the terminal cisternae (TC) of the sarcoplasmic reticulum (SR) in frog skeletal muscle were determined with rapid freezing, electron microscopy, and electron probe analysis. (b) Caffeine (5 mM) released approximately 65% of the Ca content of the TC in 1 min and 84% after 3 min. The release of Ca from the TC was associated with a highly significant increase in its Mg content. This increase in Mg was not reduced by valinomycin. There was also a small increase in the K content of the TC at 1 min, although not after 3 min of caffeine contracture. (c) On the basis of the increase in Mg content during caffeine contracture and during tetanus (Somlyo, A. V., H. Gonzalez- Serratos, H. Shuman, G. McClellan, and A. P. Somlyo, 1981, J. Cell Biol., 90:577-594), we suggest that both mechanisms of Ca release are associated with an increase in the Ca and Mg permeability of the SR membranes, the two ions possibly moving through a common channel. (d) There was a significant increase in the P content of the TC during caffeine contracture, while in tetanized muscle (see reference above) there was no increase in the P content of the TC. (e) Mitochondrial Ca content was significantly increased (at 1 and at 3 min) during caffeine contracture. Valinomycin (5 microM) blocked this mitochondrial Ca uptake. (f) The sustained Ca release caused by caffeine in situ contrasts with the transient Ca release observed in studies of fragmented SR preparations, and could be explained by mediation of the caffeine-induced Ca release by a second messenger produced more readily in intact muscle than in isolated SR. (g) The TC were not swollen in rapidly frozen, caffeine-treated muscles, in contrast to the swelling of the TC observed in conventionally fixed, caffeine-treated preparation, the latter finding being in agreement with previous studies. (h) The fractional volume of the TC in rapidly frozen control (resting) frog semitendinosus muscles (approximately 2.1%) was less than the volume (approximately 2.5%) after glutaraldehyde-osmium fixation.     

Characterization of junctional and longitudinal sarcoplasmic reticulum from heart muscle

Longitudinal tubules and junctional sarcoplasmic reticulum (SR) were prepared from heart muscle microsomes by Ca2+-phosphate loading followed by sucrose density gradient centrifugation. The longitudinal SR had a high Ca2+ loading rate (0.93 +/- 0.08 mumol.mg-1.min) which was unchanged by addition of ruthenium red. Junctional SR had a low Ca2+ loading rate (0.16 +/- 0.02 mumol.mg-1.min) which was enhanced about 5-fold by ruthenium red. Junctional SR had feet structures observed by electron microscopy and a high molecular weight protein with Mr of 340,000, whereas longitudinal SR was essentially devoid of both. Thus, these subfractions have similar characteristics to longitudinal and junctional terminal cisternae of SR from fast twitch skeletal muscle. Ryanodine binding was localized to junctional cardiac SR as determined by [3H]ryanodine binding. Scatchard analysis of the binding data showed two types of binding (high affinity, Kd approximately 7.9 nM; low affinity, Kd approximately 1 microM), contrasting with skeletal junctional terminal cisternae where only one site with Kd of approximately 50 nM was observed. The ruthenium red enhancement of Ca2+ loading rate in junctional cardiac SR was blocked by pretreatment with low concentrations of ryanodine as reported for junctional terminal cisternae of skeletal muscle SR. The Ca2+ loading rate of junctional cardiac SR was enhanced by preincubation with high concentrations of ryanodine. The apparent inhibition constant (Ki approximately 7 nM) and stimulation constant (Km approximately 1.1 microM) for ryanodine on junctional SR corresponded to the Kd for high affinity binding (Kd approximately 7.9 nM) and low affinity binding (Kd approximately 1.1 microM), respectively. These results suggest that high affinity ryanodine binding locks the Ca2+ release channels in the open state and that low affinity binding closes the Ca2+ release channels of the junctional cardiac SR. The characteristics of the Ca2+ release channels of junctional cardiac SR appear to be similar to that of skeletal muscle SR, but the Ca2+ release channels of cardiac SR are more sensitive to ryanodine.     

Purification of the ryanodine receptor and identity with feet structures of junctional terminal cisternae of sarcoplasmic reticulum from fast skeletal muscle

The ryanodine receptor has been purified from junctional terminal cisternae of fast skeletal muscle sarcoplasmic reticulum (SR). The ryanodine receptor was solubilized with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) and stabilized by addition of phospholipids. The solubilized receptor showed the same [3H]ryanodine binding properties as the original SR vesicles in terms of affinity, Ca2+ dependence, and salt dependence. Purification of the ryanodine receptor was performed by sequential column chromatography on heparin-agarose and hydroxylapatite in the presence of CHAPS. The purified receptor bound 393 +/- 65 pmol of ryanodine/mg of protein (mean +/- S.E., n = 5). The purified receptor showed three bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with Mr of 360,000, 330,000, and 175,000. Densitometry indicates that these are present in the ratio of 2/1/1, suggesting a monomer Mr of 1.225 X 10(6) and supported by gel exclusion chromatography in CHAPS. Electron microscopy of the purified preparation showed the square shape of 210 A characteristic of and comparable in size and shape to the feet structures of junctional terminal cisternae of SR, indicating that ryanodine binds directly to the feet structures. From the ryanodine binding data, the stoichiometry between ryanodine binding sites to the number of feet structures is estimated to be about 2. Since the ryanodine receptor is coupled to Ca2+ gating, the present finding suggests that the ryanodine receptor and Ca2+ release channel represent a functional unit, the structural unit being the foot structure which, in situ, is junctionally associated with the transverse tubules. It is across this triad junction that the signal for Ca2+ release is expressed. Thus, the foot structure appears to directly respond to the signal from transverse tubules, causing the release of Ca2+ from the junctional face membrane of the terminal cisternae of SR.     

Scanning electron-microscopic studies on the three-dimensional structure of sarcoplasmic reticulum in the mammalian red,white and intermediate muscle fibers

Summary The three-dimensional structure of the sarcoplasmic reticulum (SR) in the red, white and intermediate striated muscle fibers of the extensor digitorum longus muscle of the rat was examined under a field-emission type scanning electron microscope after removal of cytoplasmic matrices by the osmium-DMSO-osmium procedure.In all three types of fibers, the terminal cisternae and transverse tubules form triads at the level of the A-I junction. Numerous slender sarcotubules, originating from the A-band side terminal cisternae, extend obliquely or longitudinally and form oval or irregular shaped networks of various sizes in front of the A-band, then become continuous with the tiny mesh (fenestrated collar) in front of the H-band. The A-and H-band SR appears as a single sheet of anastomotic tubules. Numerous sarcotubules, originating from the I-band side terminal cisternae, extend in threedimensional directions and form a multilayered network over the I-band and Z-line regions. At the I-band level, paired transversely oriented mitochondria partly embrace the myofibril. The I-band SR network is poorly developed in the narrow space between the paired mitochondria, but is well developed in places devoid of these mitochondria.The three-dimensional structure of the SR is basically the same in all three muscle fiber-types. However, the SR is sparse on the surface of mitochondria, so the mitochondria-rich red fiber has a much smaller total volume of SR than the mitochondria-poor white fiber. Moreover, the volume of SR of the intermediate fiber is intermediate between the two.     

Preparation and characterization of longitudinal tubules of sarcoplasmic reticulum from fast skeletal muscle

Sarcoplasmic reticulum (SR) serves a central role in calcium uptake and release, thereby regulating muscle relaxation and contraction, respectively. Recently, we have isolated fractions referable to longitudinal tubules (R2) and terminal cisternae (R4), the two major types of sarcoplasmic reticulum (A. Saito et al. (1984) J. Cell Biol. 99, 875-885). The terminal cisternae contain two types of membranes, the calcium pump membrane and the junctional face membrane. The terminal cisternae are filled with electron-opaque contents which serve as a Ca2+ reservoir. The longitudinal tubules consist mainly of the calcium pump membrane. In this study, we describe a new longitudinal tubule fraction (F2) and characterize it together with the R2 and R4 SR fractions. The calcium pump membrane of the longitudinal tubules is a highly specialized membrane consisting of about 90% calcium pump protein as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Extensive changes in morphology can be observed in the SR fractions referable to osmotic differences during the fixation conditions using either glutaraldehyde-tannic acid or osmium tetroxide fixatives. The changes include swelling or shrinkage and aggregation of the compartmental contents when the fixative contains calcium ions. The two types of SR have different osmotic permeability to the same medium, as indicated by differential swelling or shrinkage. Both longitudinal tubule and terminal cisternae vesicles of SR appear larger and are spherical vesicles when the glutaraldehyde-tannic acid fixative is isotonic as compared with the "standard" fixation method. We have previously reported that the ruthenium red-sensitive calcium release channels are localized to the terminal cisternae. The terminal cisternae as isolated are leaky to Ca2+ since these channels are in the "open state" (S. Fleischer et al. (1985) Proc. Natl. Acad. Sci USA 82, 7256-7259). Thus, the Ca2+, Mg2+-dependent ATPase (Ca2+ ATPase) rate is only slightly enhanced in the presence of a Ca2+ ionophore, which dissipates the Ca2+ gradient across the SR membrane. We now find that preincubation with ruthenium red restores the tight coupling of the Ca2+ ATPase activity to Ca2+ transport. That is to say, ATPase activity is reduced and the addition of ionophore stimulates the Ca2+ ATPase activity 4- to 7-fold. The Ca2+ ATPase activity in longitudinal tubules is already tightly coupled. It is minimal after a Ca2+ gradient has been generated, but can be stimulated 9- to 20-fold when the Ca2+ gradient is dissipated with ionophore. This finding suggests that the Ca2+ ATPase activity in SR is tightly coupled to Ca2+ transport in situ.     

Regulation of the ryanodine receptor calcium release channel of the sarcoplasmic reticulum in skeletal muscle

In striated muscle contraction is under the tight control of myoplasmic calcium concentration ([Ca2+]i): the elevation in [Ca2+]i and the consequent binding of calcium to troponin C enables, while the decrease in [Ca2+]i prevents the actin-myosin interaction. Calcium ions at rest are stored in the sarcoplasmic reticulum (SR) from which they are rapidly released upon the depolarisation of the sarcolemmal and transverse (T-) tubular membranes of the muscle cell. The protein responsible for this controlled and fast release of calcium is the calcium release channel found in the membrane of the terminal cisternae of the SR. This review focuses on the physiological and pharmacological modulators of the calcium release channel and tries to draw an up-to-date picture of the events that occur between T-tubular depolarisation and the release of calcium from the SR.     

Ultrastructural organisation and the sites of calcium localisation in the lamprey striated muscle

The present paper examines the ultrastructure of the sarcoplasmic recitulum (SR) and the T system in the striated muscle of the lamprey. The pyroantimonate method was used to visualise the sites of intracellular calcium localisation. Characteristic for the muscle studied are the presence of numerous intricately shaped invaginations on the surface membrane of muscle fibres and peripheral contacts between SR cisternae and the sarcolemma. In addition to calcium localised in the terminal cisternae of SR and N-bands of the I-disk, as typical of vertebrate muscles, a great amount of calcium is present in the subsarcolemmal region, corresponding to the area of invaginations, and in longitudinal elements of SR.     

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.     

Isolation of the ryanodine receptor from cardiac sarcoplasmic reticulum and identity with the feet structures

Ryanodine, a highly toxic alkaloid, reacts specifically with the Ca2+ release channels which are localized in the terminal cisternae of sarcoplasmic reticulum (SR). In this study, the ryanodine receptor from cardiac SR has been purified, characterized, and compared with that of skeletal muscle SR. The ryanodine receptor was solubilized with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) in the presence of phospholipids. Purification was performed by sequential affinity chromatography followed by gel permeation chromatography in the presence of CHAPS and phospholipids. The enrichment of the receptor from cardiac microsomes was about 110-fold. The purified receptor contained a major polypeptide band of Mr 340,000 with a minor band of Mr 300,000 (absorbance ratio 100/8) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Electron microscopy of the purified receptor from heart showed square structures of 222 +/- 21 A/side, which is the unique characteristic of feet structures of junctional face membrane of terminal cisternae of SR. Recently, we isolated the ryanodine receptor from skeletal muscle (Inui, M., Saito, A., and Fleischer, S. (1987) J. Biol. Chem. 262, 1740-1747). The ryanodine receptors from heart and skeletal muscle have similar characteristics in terms of protein composition, morphology, chromatographic behavior, and Ca2+, salt, and phospholipid dependence of ryanodine binding. However, there are distinct differences: 1) the Mr of the receptor is slightly larger for skeletal muscle (Mr approximately 360,000); 2) the purified receptor from heart contains two different affinities for ryanodine binding with Kd values in the nanomolar and micromolar ranges, contrasting with that of skeletal muscle SR which shows only the high affinity binding; 3) the affinity of the purified cardiac receptor for ryanodine was 4-5-fold higher than that of skeletal muscle, measured under identical conditions. The greater sensitivity in ryanodine in intact heart can be directly explained by the tighter binding of the ryanodine receptor from heart. The present study suggests that basically similar machinery (the ryanodine receptor and foot structure) is involved in triggering Ca2+ release from cardiac and skeletal muscle SR, albeit there are distinct differences in the sensitivity to ryanodine and other ligands in heart versus skeletal muscle.     

Divergent effects of the malignant hyperthermia-susceptible Arg(615)-->Cys mutation on the Ca(2+) and Mg(2+) dependence of the RyR1

The sarcoplasmic reticulum (SR) Ca(2+) release channel (RyR1) from malignant hyperthermia-susceptible (MHS) porcine skeletal muscle has a decreased sensitivity to inhibition by Mg(2+). This diminished Mg(2+) inhibition has been attributed to a lower Mg(2+) affinity of the inhibition (I) site. To determine whether alterations in the Ca(2+) and Mg(2+) affinity of the activation (A) site contribute to the altered Mg(2+) inhibition, we estimated the Ca(2+) and Mg(2+) affinities of the A- and I-sites of normal and MHS RyR1. Compared with normal SR, MHS SR required less Ca(2+) to half-maximally activate [(3)H]ryanodine binding (K(A,Ca): MHS = 0.17 +/- 0.01 microM; normal = 0.29 +/- 0.02 microM) and more Ca(2+) to half-maximally inhibit ryanodine binding (K(I,Ca): MHS = 519.3 +/- 48.7 microM; normal = 293.3 +/- 24.2 microM). The apparent Mg(2+) affinity constants of the MHS RyR1 A- and I-sites were approximately twice those of the A- and I-sites of the normal RyR1 (K(A,Mg): MHS = 44.36 +/- 4.54 microM; normal = 21.59 +/- 1.66 microM; K(I,Mg): MHS = 660.8 +/- 53.0 microM; normal = 299.2 +/- 24.5 microM). Thus, the reduced Mg(2+) inhibition of the MHS RyR1 compared with the normal RyR1 is due to both an enhanced selectivity of the MHS RyR1 A-site for Ca(2+) over Mg(2+) and a reduced Mg(2+) affinity of the I-site.     

Phosphatidylinositol 4,5-bisphosphate-induced Ca2+ release from skeletal muscle sarcoplasmic reticulum terminal cisternal membranes. Ca2+ flux and single channel studies.

We report here that the inositol 1,4,5-trisphosphate (IP3) precursor, L-alpha-phosphatidylinositol 4,5-bisphosphate (PIP2) is a potent molecule (1 microM) which activates the ryanodine-sensitive Ca2+ release channel from rabbit skeletal muscle terminal cisternae incorporated into a phospholipid bilayer. It also stimulates Ca2+ release from these membrane vesicles. Therefore, it may play a modulating role in excitation-contraction coupling. In the bilayer, PIP2 added on the cytoplasmic side increased the mean channel opening probability 2-12-fold in the presence and absence of physiological Mg2+ and ATP. From flux studies, PIP2-induced Ca2+ release, occurring through the ryanodine-sensitive Ca2+ release channel, displayed saturation kinetics. The rate of Ca2+ release induced by PIP2 was approximately greater than 50% slower than the rates induced by other agents (e.g. caffeine, Ca2+, ATP). PIP2, and not IP3, effectively elicited Ca2+ release from terminal cisternae. On the contrary, IP3, and not PIP2, specifically mediated Ca2+ release from dog brain cerebellum microsomes, where IP3 receptors are known to be found. The PIP2-induced Ca2+ release from muscle membranes was not dependent on medium [Ca2+] (from less than 10(-9) to approximately 10(-4) M). However, IP3 could activate the terminal cisternae Ca2+ channel in the bilayer when there was low Ca2+ (less than 10(-7) M). The data suggest that the ionic microenvironment around the Ca2+ channel may be different for observing the two phosphoinositide actions.     

Electron probe analysis of vascular smooth muscle. Composition of mitochondria, nuclei, and cytoplasm

Electron probe analysis of dry cryosections was used to determine the composition of the cytoplasm and organelles of rabbit portal-anterior mesenteric vein (PAMV) smooth muscle. All analytical values given are in mmol/kg wt +/- SEM. Cytoplasmic concentrations in normal, resting muscles were: K, 611 +/- 1.7; Na, 167 +/- 2.7; Cl, 278 +/- 1.0; Mg, 36 +/- 1.1; Ca, 1.9 +/- 0.5; and P, 247 +/- 1.1. Hence, the sum of intracellular Na + K exceeded cytoplasmic Cl by 500 mmol/kg dry wt, while the calculated total, nondiffusible solute was approximately 50 mmol/kg. Cytoplasmic K and Cl were increased in smooth muscles incubated in solutions containing an excess (80 mM) of KCl. Nuclear and cytoplasmic Na and Ca concentrations were not significantly different. The mitochondrial Ca content in normal fibers was low, 0.8 +/- 0.5, and there was no evidence of mitochondrial Ca sequestration in muscles frozen after a K contracture lasint 30 min. Transmitochondrial gradients of K, Na, and Cl were small (0.9--1.2). In damaged fibers, massive mitochondrial Ca accumulation of up to 2 mol/kg dry wt in granule form and associated with P could be demonstrated. Our findings suggest (a) that the nonDonnan distribution of Cl in smooth muscle is not caused by sequestration in organelles, and that considerations of osmotic equilibrium and electroneutrality suggest the existence of unidentified nondiffusible anions in smooth muscle, (b) that nuclei do not contain concentrations of Na or Ca in excess of cytoplasmic levels, (c) that mitochondria in PAMV smooth muscle do not play a major role in regulating cytoplasmic Ca during physiological levels of contraction but can be massively Ca loaded in damaged cells, and (d) that the in situ transmitochondrial gradients of K, Na, and Cl do not show these ions to be distributed according to a large electromotive Donnan force.     

Immunocytochemistry of calciosomes in liver and pancreas

Calciosomes are small cytoplasmic vacuoles identified in various nonmuscle cell types by their content of protein(s) similar to calsequestrin (CS), the Ca2+ storage protein of the muscle sarcoplasmic reticulum (SR). These entities have been interpreted as the "primitive" counterpart of the SR, and suggested to be the organelle target of inositol-1,4,5-triphosphate action (Volpe, P., K. H. Krause, S. Hashimoto, F. Zorzato, T. Pozzan, J. Meldolesi, and D. P. Lew. Proc. Natl. Acad. Sci. USA. 85:1091-1095). Immunoperoxidase and immunogold experiments carried out in both thick and ultrathin cryosections of rat hepatocytes and pancreatic acinar cells by using antimuscle CS antibodies revealed a specific labeling widely distributed in the entire cytoplasm, while nuclei were negative. Individual calciosomes appeared as small (105 nm) membrane-bound vacuoles intermingled with, and often apposed to ER cisternae and mitochondria. Other calciosomes were scattered in the Golgi area, in between zymogen granules and beneath the plasma membrane. The cumulative volume of the CS-positive organelles was measured to account for the 0.8 and 0.45% of the cytoplasm in liver and pancreas cells, respectively. The real total volume of the calciosome compartment is expected to be approximately twice as large. In hepatocytes, structures similar to CS-positive calciosomes were decorated by antibodies against the Ca2+ ATPase of muscle SR, while ER cisternae were not. By dual labeling, colocalization was revealed in 53.6% of the organelles, with 37.6% positive for the ATPase only. CS appeared preferentially confined to the content, and the Ca2+ ATPase to the contour of the organelle. The results suggested a partial segregation of the two antigens, reminiscent of their well-known segregation in muscle SR. Additional dual-label experiments demonstrated that hepatic calciosomes express neither two ER markers (cytochrome-P450 and NADH-cytochrome b5 reductase) nor the endolysosome marker, luminal acidity (revealed by 3- [2,4-dinitroanilino]-3'-amino-N-methyl dipropylamine). Calciosomes appear as unique cytological entities, ideally equipped to play a role in the rapid-scale control of the cytosolic-free Ca2+ in nonmuscle cells.     

Effects of disuse on the function of fragmented sarcoplasmic reticulum of rabbit M. gastrocnemius

A preparation method has been described to obtain a relatively pure and functionally intact fragmented sarcoplasmic reticulum (SR) vesicles fraction from normal and atrophied muscles. Purified SR preparations from rabbit gastrocnemius muscle atrophied by disuse showed similar protein composition (gel electrophoresis; Laemmli 1970) and similar vanadate induced crystallization (Dux and Martonosi 1983) properties of Ca2+-ATPase as those of control preparations. In the early period of atrophy (1-2 weeks) both the Ca2+-ATPase activity and Ca2+ uptake showed a 2-3-fold increase (from 3.42 +/- 0.24 to 7.34 +/- 0.25 mumol Pi X mg-1 prot X min-1 and from 1.26 +/- 0.10 to 3.36 +/- 0.22 mumol/l Ca2+ X min-1 X mg-1 prot. respectively).     

Antibodies to junctional sarcoplasmic reticulum proteins: probes for the Ca2+-release channel.

The junctional face membrane plays a key role in excitation-contraction coupling in skeletal muscle. A protein of 350 kDa, tentatively identified as a component of the junctional feet, connects transverse tubules to terminal cisternae of sarcoplasmic reticulum [Kawamoto, Brunschwig, Kim & Caswell (1986) J. Cell Biol. 103, 1405-1414]. The membrane topology and protein composition of sarcoplasmic reticulum Ca2+-release channels of rabbit skeletal muscle were investigated using an immunological approach, with anti-(junctional face membrane) and anti-(350 kDa protein) polyclonal antibodies. Upon preincubation of the terminal cisternae with anti-(junctional face membrane) antibodies, Ca2+-ATPase and Ca2+-loading activities were not affected, whereas anti-(350 kDa protein) antibodies stimulated Ca2+-ATPase activity by 25% and inhibited Ca2+-loading activity by 50% (at an antibody/terminal cisternae protein ratio of 1:1). Specific photolabelling of terminal cisternae proteins with [14C]doxorubicin was prevented by both anti-(junctional face membrane) and anti-(350 kDa protein) antibodies. Stimulation of Ca2+ release by doxorubicin was prevented by both anti-(junctional face membrane) and anti-(350 kDa protein) antibodies. Half-maximal inhibition was obtained at an antibody/terminal cisternae protein ratio of 1:1. Kinetic measurements of Ca2+ release indicated that anti-(350 kDa protein) antibodies prevented Ca2+-induced Ca2+ release, whereas the ATP-stimulation and the inhibition by Mg2+ were not affected. These results suggest that: (i) Ca2+- and doxorubicin-induced Ca2+ release is mediated by Ca2+ channels which are selectively localized in the junctional face membrane; (ii) the 350 kDa protein is a component of the Ca2+-release channel in native terminal cisternae vesicles; and (iii) the Ca2+-activating site of the channel is separate from other allosteric sites.     

Preparation and morphology of sarcoplasmic reticulum terminal cisternae from rabbit skeletal muscle

We have developed a procedure to isolate, from skeletal muscle, enriched terminal cisternae of sarcoplasmic reticulum (SR), which retain morphologically intact junctional "feet" structures similar to those observed in situ. The fraction is largely devoid of transverse tubule, plasma membrane, mitochondria, triads (transverse tubules junctionally associated with terminal cisternae), and longitudinal cisternae, as shown by thin-section electron microscopy of representative samples. The terminal cisternae vesicles have distinctive morphological characteristics that differ from the isolated longitudinal cisternae (light SR) obtained from the same gradient. The terminal cisternae consist of two distinct types of membranes, i.e., the junctional face membrane and the Ca2+ pump protein-containing membrane, whereas the longitudinal cisternae contain only the Ca2+ pump protein-containing membrane. The junctional face membrane of the terminal cisternae contains feet structures that extend approximately 12 nm from the membrane surface and can be clearly visualized in thin section through using tannic acid enhancement, by negative staining and by freeze-fracture electron microscopy. Sections of the terminal cisternae, cut tangential to and intersecting the plane of the junctional face, reveal a checkerboardlike lattice of alternating, square-shaped feet structures and spaces each 20 nm square. Structures characteristic of the Ca2+ pump protein are not observed between the feet at the junctional face membrane, either in thin section or by negative staining, even though the Ca2+ pump protein is observed in the nonjunctional membrane on the remainder of the same vesicle. Likewise, freeze-fracture replicas reveal regions of the P face containing ropelike strands instead of the high density of the 7-8-nm particles referable to the Ca2+ pump protein. The intravesicular content of the terminal cisternae, mostly Ca2+-binding protein (calsequestrin), is organized in the form of strands, sometimes appearing paracrystalline, and attached to the inner face of the membrane in the vicinity of the junctional feet. The terminal cisternae preparation is distinct from previously described heavy SR fractions in that it contains the highest percentage of junctional face membrane with morphologically well-preserved junctional feet structures.     

Head-to-tail oligomerization of calsequestrin: a novel mechanism for heterogeneous distribution of endoplasmic reticulum luminal proteins

Many proteins retained within the endo/sarcoplasmic reticulum (ER/SR) lumen express the COOH-terminal tetrapeptide KDEL, by which they continuously recycle from the Golgi complex; however, others do not express the KDEL retrieval signal. Among the latter is calsequestrin (CSQ), the major Ca2+-binding protein condensed within both the terminal cisternae of striated muscle SR and the ER vacuolar domains of some neurons and smooth muscles. To reveal the mechanisms of condensation and establish whether it also accounts for ER/SR retention of CSQ, we generated a variety of constructs: chimeras with another similar protein, calreticulin (CRT); mutants truncated of COOH- or NH2-terminal domains; and other mutants deleted or point mutated at strategic sites. By transfection in L6 myoblasts and HeLa cells we show here that CSQ condensation in ER-derived vacuoles requires two amino acid sequences, one at the NH2 terminus, the other near the COOH terminus. Experiments with a green fluorescent protein GFP/CSQ chimera demonstrate that the CSQ-rich vacuoles are long-lived organelles, unaffected by Ca2+ depletion, whose almost complete lack of movement may depend on a direct interaction with the ER. CSQ retention within the ER can be dissociated from condensation, the first identified process by which ER luminal proteins assume a heterogeneous distribution. A model is proposed to explain this new process, that might also be valid for other luminal proteins.     

Characterization of the terminal cisternae and longitudinal tubules of sarcoplasmic reticulum from malignant hyperpyrexia susceptible porcine skeletal muscle

1. The sarcoplasmic reticulum (SR) from malignant hyperpyrexia susceptible (MHS) and control porcine skeletal muscle was separated into vesicular fractions enriched in the membrane elements of the terminal cisternae and longitudinal tubules. 2. The two membrane preparations were highly purified and had distinctive features which were associated with their origins in the SR membraneous network. 3. Calsequestrin and calcium were enriched in the terminal cisternae fraction (HSR), in comparison to longitudinal tubule preparations (LSR). 4. The HSR membrane also had a greater total capacity to store Ca2+ and Ca2+ release was more rapid than from LSR preparations. 5. No distinction could be made between the membrane morphology, Ca2+ -fluxes or Ca2+ -dependent ATPase activities, associated with these functionally distinct regions of MHS and control preparations.     

Inositol trisphosphate, calcium and muscle contraction

The identity of organelles storing intracellular calcium and the role of Ins(1,4,5)P3 in muscle have been explored with, respectively, electron probe X-ray microanalysis (EPMA) and laser photolysis of 'caged' compounds. The participation of G-protein(s) in the release of intracellular Ca2+ was determined in saponin-permeabilized smooth muscle. The sarcoplasmic reticulum (SR) is identified as the major source of activator Ca2+ in both smooth and striated muscle; similar (EPMA) studies suggest that the endoplasmic reticulum is the major Ca2+ storage site in non-muscle cells. In none of the cell types did mitochondria play a significant, physiological role in the regulation of cytoplasmic Ca2+. The latency of guinea pig portal vein smooth muscle contraction following photolytic release of phenylephrine, an alpha 1-agonist, is 1.5 +/- 0.26 s at 20 degrees C and 0.6 +/- 0.18 s at 30 degrees C; the latency of contraction after photolytic release of Ins(1,4,5)P3 from caged Ins(1,4,5)P3 is 0.5 +/- 0.12 s at 20 degrees C. The long latency of alpha 1-adrenergic Ca2+ release and its temperature dependence are consistent with a process mediated by G-protein-coupled activation of phosphatidylinositol 4,5 bisphosphate (PtdIns(4,5)P2) hydrolysis. GTP gamma S, a non-hydrolysable analogue of GTP, causes Ca2+ release and contraction in permeabilized smooth muscle. Ins(1,4,5)P3 has an additive effect during the late, but not the early, phase of GTP gamma S action, and GTP gamma S can cause Ca2+ release and contraction of permeabilized smooth muscles refractory to Ins(1,4,5)P3. These results suggest that activation of G protein(s) can release Ca2+ by, at least, two G-protein-regulated mechanisms: one mediated by Ins(1,4,5)P3 and the other Ins(1,4,5)P3-independent. The low Ins(1,4,5)P3 5-phosphatase activity and the slow time-course (seconds) of the contractile response to Ins(1,4,5)P3 released with laser flash photolysis from caged Ins(1,4,5)P3 in frog skeletal muscle suggest that Ins(1,4,5)P3 is unlikely to be the physiological messenger of excitation-contraction coupling of striated muscle. In contrast, in smooth muscle the high Ins(1,4,5)P3-5-phosphatase activity and the rate of force development after photolytic release of Ins(1,4,5)P3 are compatible with a physiological role of Ins(1,4,5)P3 as a messenger of pharmacomechanical coupling.