Terminal Cisternae Of Sarcoplasmic Reticulum Research Articles

The identification of sarcoplasmic reticulum terminal cisternae proteins in platelets

The identification of sarcoplasmic reticulum terminal cisternae proteins in platelets

Denervation-induced proliferative changes of triads in rabbit skeletal muscle.

Protein compositional and functional differences exist between longitudinal and junctional sarcoplasmic reticulum (SR) in relation to Ca transport and to Ca release. In light of this knowledge, we have reinvestigated the effects of denervation on SR of rabbit gastrocnemius, a predominantly fast muscle. Electron microscopy of 2-weeks denervated muscle showed proliferation of transverse tubules (TT), forming junctional contacts with SR terminal cisternae (TC). At coincident periods, the yield of muscle microsomes was increased, and their fractionation by sucrose-density centrifugation demonstrated a relative increase of heavy vesicles. Thin-section electron microscopy of heavy SR from denervated muscle showed an increased number of vesicles containing calsequestrin (CS) as compared with control muscle. Electrophoretic analysis confirmed the relative decrease of Ca-ATPase protein and the striking increase of CS both in total microsomes and in heavy SR vesicles. Calcium loading and Ca-ATPase activity as well as the density of Ca-ATPase protein were decreased to a similar extent (20-30%) in denervated muscle microsomes. Stimulation of Ca-ATPase activity by Ca-ionophore A23187 showed that the vesicles were tightly sealed. When probed by competitive ELISA with antibody to SR Ca-ATPase from pure fast muscle, the Ca-ATPase of denervated microsomes was found to be highly cross reactive. Cleveland's peptide maps of the Ca-ATPase protein after partial digestion with S. aureus V8 protease also showed no significant change after denervation. Changes in cholesterol content and in the ratio of Mg-ATPase to Ca-ATPase activity of denervated muscle microsomes indicated a 4-fold increase of TT protein, i.e., from about 3% to not more than 12% of total protein, at 2 weeks after denervation. All these changes were totally reversed upon reinnervation of muscle fibers, and the consequent muscle recovery, as obtained by nerve crushing instead of nerve sectioning. From these results, we conclude that denervated adult fast muscle, similarly to immature fast muscle, contains more junctional SR. However, the molecular and catalytic properties of the Ca-ATPase are unaffected by denervation.

Ultrastructure of the calcium release channel of sarcoplasmic reticulum.

This study is concerned with the characterization of the morphology of the calcium release channel of sarcoplasmic reticulum (SR) from fast-twitch skeletal muscle, which is involved in excitation-contraction coupling. We have previously purified the ryanodine receptor and found it to be equivalent to the feet structures, which are involved, in situ, in the junctional association of transverse tubules with terminal cisternae of SR. The receptor is an oligomer of a single high molecular weight polypeptide and when incorporated into phospholipid bilayers, has channel conductance which is characteristic of calcium release in terminal cisternae of SR. The purified channel can be observed by electron microscopy using different methods of sample preparation, with complementary views being observed by negative staining, double staining, thin section and rotary shadowing electron microscopy. Three views can be observed and interpreted: (a) a square face which, in situ, is junctionally associated with the transverse tubule or junctional face membrane; (b) a rectangle equivalent to the side view; and (c) a diamond shape equivalent to the side view, of which the base portion appears to be equivalent to the transmembrane segment. Negative staining reveals detailed substructure of the channel. A computer averaged view of the receptor displays fourfold symmetry and ultrastructural detail. The dense central mass is divided into four domains with a 2-nm hole in the center, and is enclosed within an outer frame which has a pinwheel appearance. Double staining shows substructure of the square face in the form of parallel linear arrays (six/face). The features of the isolated receptor can be correlated with the structure observed in terminal cisternae vesicles. Sections tangential to the junctional face membrane reveal that the feet structures (23-nm squares) overlap so as to enclose smaller square spaces of approximately 14 nm/side. We suggest that this is equivalent to the transverse tubule face and that the terminal cisternae face is smaller (approximately 17 nm/face) and has larger alternating spaces as a consequence of the tapered sides of the foot structures. Image reconstruction analysis appears to be feasible and should provide the three-dimensional structure of the channel.

Trypsin digestion of junctional sarcoplasmic reticulum vesicles.

A putative constituent of the junctional processes, connecting the terminal cisternae of sarcoplasmic reticulum and the transverse tubules of skeletal muscle fibers, is a greater than or equal to 350,000-dalton (Da) protein that displays ryanodine binding and Ca2+ channel properties. Ryanodine modulation of Ca2+ fluxes suggests that the ryanodine receptor and calcium channel are integral parts of one functional unit corresponding to the greater than or equal to 350,000-Da protein [Inui, M., Saito, E., & Fleischer, S. (1987) J. Biol. Chem. 262, 1740-1747; Campbell, K. P., Knudson, C. M., Imagawa, T., Leung, A. L., Sutko, J. L., Kahl, S. D., Raab, C. R., & Madson, L. (1987) J. Biol. Chem. 262, 6460-6463]. We subjected vesicular fragments of junctional-cisternal membrane to stepwise trypsin digestion. The greater than or equal to 350,000-Da protein is selectively cleaved in the early stage of digestion, with consequent disappearance of the corresponding band in electrophoretic gels. The Ca2+-ATPase is cleaved at a later stage, while calsequestrin is not digested under the same experimental conditions. While the Ca2+-ATPase yields two complementary fragments that are relatively resistant to further digestion, the greater than or equal to 350,000-Da protein yields fragments that are rapidly broken down to small peptides. Under conditions producing extensive digestion of the greater than or equal to 350,000-Da protein, the junctional processes are still visualized by electron microscopy, with no discernible alterations of their ultrastructure. The functional properties of the Ca2+ release channel are also maintained following trypsin digestion, including blockage by Mg2+ and ruthenium red and activation by Ca2+ and nucleotides.(ABSTRACT TRUNCATED AT 250 WORDS)

Ryanodine sensitivity of the calcium release channel of sarcoplasmic reticulum

Ryanodine modulates Ca 2+ permeability in isolated terminal cisternae of sarcoplasmic reticulum, suggesting that it is a specific ligand for the calcium release channel. Our laboratory has purified the ryanodine receptor and demonstrated it to be equivalent to the feet structures, which are involved in the junctional association of the transverse tubule with the terminal cisternae. Recently, Smith, Coronado and Meissner have incorporated sarcoplasmic reticulum into bilayers and found a high conductivity channel (∼ 100 pS) which has a number of characteristics expected of the Ca 2+ release channels in SR. We now find that the high conductivity channel in the bilayer is sensitive to ryanodine. Low concentrations of ryanodine (sub μM): (1) lock the channels in an open state; (2) prevent the action of ruthenium red (μM) to completely close the channel; and (3) much higher concentrations of ryanodine (300μM) close the channel. In these three respects ryanodine acts similarly on the channel in the bilayer as in vesicles. Further, the bilayer studies provide new insight into the action of ryanodine on the channel in that: (1) ryanodine locks the channel in the open state, but the conductivity is reduced to about 40%; (2) ryanodine prevents ruthenium red from closing the channel, although there is a further decrease in the open current. These studies provide support that the high conductivity calcium channel in sarcoplasmic reticulum is involved in excitation-contraction coupling. By the same token the pharmacological action of ryanodine is pinpointed to the calcium release channel.

5] Isolation of the functional face membrane of sarcoplasmic reticulum

This chapter focuses on the isolation of the junctional face membrane of sarcoplasmic reticulum. The sarcoplasmic reticulum (SR) of skeletal muscle controls the myoplasmic free calcium concentration and—thereby, mediates muscle contraction and relaxation. The molecular machinery involved in the release of calcium from the SR remains the key unknown link in the chain of events underlying excitation-contraction coupling in skeletal muscle. It has long been inferred that the immediate signal for calcium release is transmitted across the triad junction between the transverse tubule and the terminal cisternae of SR, in response to the depolarization of the transverse tubule with release of Ca 2+ from the terminal cisternae of SR. Transverse tubules are narrow invaginations of the plasma membrane of the muscle fiber, which are situated in register with the underlying sarcomeres. The transverse tubules are junctionally associated with the terminal cisternae by way of the junctional face membrane (JFM) containing the junctional feet structures. It is of obvious interest to be able to isolate and study the JFM. A method is presented for the isolation of the JFM of the junctional terminal cisternae (JTC) of sarcoplasmic reticulum.

6] Isolation of triads from skeletal muscle

This chapter describes methodology for the isolation of morphologically intact triad structures. Excitation-contraction coupling in skeletal muscle is initiated at the neuromuscular junction. The action potential then proceeds longitudinally along the length of the fiber and transversely to within the fiber by way of invaginations from the plasmalemma, referred to as transverse tubules. The latter are junctionally associated with the terminal cisternae of sarcoplasmic reticulum via "feet" structures. By an unknown mechanism, Ca 2+ is then released from the terminal cisternae, elevating the myoplasmic calcium concentration, and—thereby, triggering muscle contraction. The structure formed by the junctional association of the transverse tubule with the terminal cisternae is referred to as the "triad," and the junction is the triad junction. The methodology is presented in two sections: (1) Procedures for triad isolation, and (2) characterization of the isolated triads. A method of sample preparation for electron microscopy was developed to estimate the purity and morphological integrity of our subcellular fractions. Dextran is added as a nonosmophilic spacer to the prefixed sample and filtration is used in the preparation for thin sectioning.

36] Purification of Ca2+ release channel (ryanodine receptor) from heart and skeletal muscle sarcoplasmic reticulum

Publisher Summary This chapter describes the isolation of the Ca 2+ release channel from heart and skeletal muscle sarcoplasmic reticulum (SR). Significant progress has been made in the past several years with regard to characterizing the Ca 2+ release machinery of SR from both skeletal muscle and heart. (1) Junctional terminal cisternae of sarcoplasmic reticulum have been isolated and characterized. (2) Ca 2+ permeability in terminal cisternae was found to be modulated by ryanodine in pharmacologically significant concentrations. (3) Ryanodine binding in the same concentration range has been localized to the terminal cisternae of SR. (4) The ryanodine receptor has been isolated and found to be equivalent to the feet structures of the junctional terminal cisternae. (5) SR vesicles have been fused into black lipid films and found to contain Ca 2+ channels. These channels have characteristics of the Ca 2+ release observed in vesicles. (6) Ryanodine has been found to modulate the channel in the bilayer—thereby, reinforcing its role in the Ca 2+ release process. (7) The ryanodine receptor incorporated into a bilayer has the Ca 2+ channel characteristics, consistent with calcium release observed in isolated terminal cisternae. The purification procedure of the ryanodine receptor from JTC of skeletal muscle SR is summarized in the chapter. Ryanodine binding is localized to the JTC of SR both in heart and skeletal muscle.

10] Functional and longitudinal sarcoplasmic reticulum of heart muscle

This chapter focuses on the junctional and longitudinal sarcoplasmic reticulum of heart muscle. In heart, muscle contraction is triggered by calcium, which derives from two sources: (1) Extracellular Ca 2+ enters via the plasmalemma during excitation; which then (2) induces Ca 2+ release from sarcoplasmic reticulum (SR). This type of release is referred to as Ca 2+ -induced Ca 2+ release. In fast twitch skeletal muscle, essentially all of the Ca 2+ , which triggers muscle contraction derives from the sarcoplasmic reticulum subsequent to depolarization at the plasmalemma/transverse tubule. This type of release is referred to as "depolarization-induced calcium release. '' In order to study the calcium release mechanism in skeletal muscle, a fraction of junctional terminal cisternae containing well-defined feet structures has been isolated. The feet structures in situ are involved in junctional association with the terminal cisternae to form the triad junction. The calcium release mechanism was found to be modulated by ryanodine at pharmacologically significant concentrations, and has been localized to the terminal cisternae of sarcoplasmic reticulum.

Purified ryanodine receptor of skeletal muscle sarcoplasmic reticulum forms Ca2+-activated oligomeric Ca2+ channels in planar bilayers.

The ryanodine receptor of sarcoplasmic reticulum (SR) from fast-twitch skeletal muscle has been purified and found by electron microscopy to be equivalent to the feet structures that are involved in situ in the junctional association of transverse tubules with terminal cisternae of SR. We now find that when the purified receptor is incorporated into vesicle-derived planar bilayers, it forms Ca2+-specific channels, which are dependent on submicromolar Ca2+ for activity. In the presence of 1 mM ATP, the channel shows essentially no activity at 10 nM Ca2+ but becomes highly activated at 50 nM Ca2+. At suboptimal Ca2+ levels (100 nM), the channel is strongly activated by 1 mM ATP and can be blocked by ruthenium red, both effects being prevented by higher Ca2+ levels (1 microM). Mg2+, added from the cis side at millimolar concentrations, blocks Ca2+ flux through the channel from trans to cis (equivalent to flux from luminal to myoplasmic compartment). Ryanodine stabilizes the open state of the channel and blocks the action of ruthenium red to close the channel. Thus, the purified ryanodine receptor incorporated into a bilayer has the Ca2+-channel characteristics consistent with the calcium release observed in isolated terminal cisternae vesicles. Furthermore, ryanodine induced the appearance of a sublevel gating mode characterized by long open conductance states, which were integral multiples of the smallest observed conductance, 3.8 pS in 50 mM Ca2+. The purified receptor consists essentially of a single-sized high molecular weight polypeptide (Mr. approximately equal to 360,000), which on reconstitution forms the square rectangles diagnostic of the feet structures. We conclude that the identity of the Ca2+-release channel of SR is the foot structure, which consists of an oligomer of the high molecular weight polypeptide.

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.

Target size of the ryanodine receptor from junctional terminal cisternae of sarcoplasmic reticulum.

Target inactivation analysis was carried out on the ryanodine receptor. This receptor recently has been implicated as the channel involved in the calcium release process in excitation-contraction coupling and was localized to the junctional terminal cisternae of sarcoplasmic reticulum from skeletal muscle [Fleischer, S., Ogunbunmi, E. M., Dixon, M. C., & Fleer, E.A.M. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 7256-7259]. Irradiation of the junctional terminal cisternae resulted in an exponential decrease in ryanodine binding with radiation dose, thereby consistent with target theory. The target molecular weight was found to be 138,000 +/- 21,000, i.e., smaller than the polypeptide that binds ryanodine. The calcium pump protein in the same membrane preparation served as an internal control to validate the methodology.

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.

Etiopathogenetic defect of malignant hyperthermia: Hypersensitive calcium-release channel of skeletal muscle sarcoplasmic reticulum

Overwhelming clinical, physiological, pharmacological, and biochemical evidence indicates that MH occurs due to a genetic defect of intracellular calcium homeostasis in skeletal muscle. Specifically, the balance of data clearly and plausibly implicates the channel through which calcium is released from the terminal cisternae of sarcoplasmic reticulum as the site of the underlying molecular lesion of MH. This putative channel is commonly believed to be ligand-gated, that is, opened by the binding of specific agonists (Meisner, 1984). Apparently, the gating of the MH channel is abnormally sensitized, that is more tightly coupled than normal, to the binding of agonists such as calcium (and ATP). Consequently, the MH channel requires less trigger calcium or ATP for activation. Caffeine may act as weak agonist of the putative, purinergic (ATP) receptor of the ligand-gated calcium channel of the sarcoplasmic reticulum (Meisner, 1984). Persistent elevations of myoplasmic free calcium in the vicinity of the channel, which would be subliminal for normal muscle, may trigger massive and regenerative calcium-induced calcium-release in MHS muscle. Uncontrollable and sustained activation of the contractile apparatus and metabolic machinery follows. The source of the ‘trigger’ calcium may be the lumen of the transverse tubules via their voltage-gated calcium-channels (Ikemoto et al., 1984; Curtis & Catteral, 1984; Graf & Schatzmann, 1984), dislodged calcium from binding sites on the inner leaflet of the transverse tubular membrane (Frank, 1979; Curtis & Eisenberg, 1985), or the lumen of the terminal cisternae of sarcoplasmic reticulum via their ligand-gated calcium channels. Such persistent, but normally subliminal, elevations of ‘trigger’ calcium at the site of the calcium-release channel presumably occurs in MHS muscle with administration of potent volatile anesthetics, especially halothane, and depolarizing muscle relaxants, especially succinylcholine. The gaseous anesthetics apparently act indirectly on the calcium channel by virtue of their being potent but non-specific membrane perturbing agents (Ohnishi et al., 1986) which increase the passive permeability of the sarcoplasmic reticulum, and possibly surface membrane, to calcium. In individuals which are homozygous for the genetic defect the less potent muscle stimuli of anoxia, heat, and exertion may be sufficient to increase the local ‘trigger’ calcium concentration to a level resulting in uncontrollable calcium-release. Such homozygosity occurs rarely in people but commonly in swine due to inbreeding and the high frequency of the MH gene. Homozygosity for the MH gene may explain the occurrence of porcine (Topel, 1968), human (Wingard, 1974), and canine (O'Brien & Rand, 1985) stress syndromes: a double dosage of the genetic defect may render homozygotes hypersensitive to stimuli less potent than volatile anesthetics. Dantrolene, the MH-preventive and reversing (if administered early enough in the syndrome) drug, apparently acts by a different mechanism than drugs such as ruthenium red and the amide local anesthetics which appear to physically block the pore of the ligand-gated calcium channel (Ohnishi et al., 1986). Dantrolene's effect on the calcium channel is apparently indirect. It blocks the membrane-perturbing effects of halothane (Nelson & Denborough, 1977; Ohnishi et al., 1986) perhaps by ‘stabilizing’ muscle membranes and therefore their calcium channels (voltage-gated in transverse tubules and ligand-gated in terminal cisternae). However, this muscle relaxant is less effective at inhibiting the calcium-releasing effects of calcium or caffeine probably because these act directly on the calcium channel (Nelson & Denborough, 1977; Nelson, 1984; Britt et al., 1984; Araki et al., 1985; Ohnishi et al., 1986). Modelling the MH defect as a hypersensitive calcium-release channel also provides a plausible explanation for observations of gradations of susceptibility to triggering of MH for individuals and within populations (Gronert, 1980; Ellis & Heffron, 1985). This model predicts gradations due to gene dosage (homozygous versus heterozygous) as well as due to environmental, pharmacologic, and genetic factors which would compromise or challenge calcium homeostatic potential, such as: heat, anoxia, exertion, drugs which stimulate muscle, and other myopathies. Thus, MH is initiated due to a hypersensitive calcium-release mechanism of skeletal muscle sarcoplasmic reticulum. Malignant hyperthermia may be reversed by dantrolene and symptomatic treatment up until a critical irreversible point in the development of the syndrome. This point of irreversibility probably corresponds to the occurrence of significant degenerative structural and functional changes within the muscle fiber. Loss of calcium-sequestration capabilities by sarcoplasmic reticulum is likely the most important factor which contributes to the irreversibility of MH.

External Ca2+-Dependent Ca2+ Release in Directly Stimulated Diaphragm Muscles of Mice

Intracellular Ca2+ release in directly stimulated diaphragm muscles of mice was found to be dependent on external Ca2+, using the intracellular Ca2+aequorin luminescence transient technique. The Ca2+ mobilization into the cells may operate via a voltage- and external Ca2+-dependent mechanism, and via a caffeine- and external Ca2+-independent one, from the Ca2+ pools in plasma membranes or in the terminal cisternae of sarcoplasmic reticulum.

External Ca2+-dependent Ca2+ release in directly stimulated diaphragm muscles of mice.

Intracellular Ca2+ release in directly stimulated diaphragm muscles of mice was found to be dependent on external Ca2+, using the intracellular Ca2+ aequorin luminescence transient technique. The Ca2+ mobilization into the cells may operate via a voltage- and external Ca2+-dependent mechanism, and via a caffeine- and external Ca2+-independent one, from the Ca2+ pools in plasma membranes or in the terminal cisternae of sarcoplasmic reticulum.

Significance of the increase in glucose 6‐phosphatase activity in skeletal muscle cells of the mouse by starvation

The effects of starvation on glucose 6-phosphatase (G6Pase; EC 3.1.3.9., D-glucose 6-phosphate phosphohydrolase) and glycogen phosphorylase (EC 2.4.1.1.) activities, and on glycogen content, were studied in skeletal muscles (m. rectus femoris) of mice. In the muscle cells from fed animals, the cytochemical reaction product for G6Pase activity was observed in moderate amounts in the terminal cisternae of sarcoplasmic reticulum and in small amounts in the nuclear envelope, and was rare or absent in the intermyofibrillar sarcoplasmic reticulum. After 4 days of starvation, however, the reaction product became abundant in all of the terminal cisternae, intermyofibrillar sarcoplasmic reticulum, and nuclear envelope. Biochemical G6Pase and glycogen phosphorylase a (active form) activities were higher in the muscles of starved mice than in those of fed animals. The glycogen content decreased markedly in the muscles of starved mice. The results suggest that the role of the increased G6Pase in skeletal muscle cells of starved mice is to release glucose into the blood by hydrolyzing glucose 6-phosphate produced through the increased phosphorylase activity.

Characterization of the junctional face membrane from terminal cisternae of sarcoplasmic reticulum.

We have recently described a preparation of junctional terminal cisternae (JTC) from fast skeletal muscle of rabbit hind leg. The fraction differs from other heavy sarcoplasmic reticulum (SR) fractions in that it contains a substantial amount of junctional face membrane (JFM) (15-20% of the membrane) with morphologically well-defined junctional feet structures. In common with other heavy SR preparations, it contains predominantly the calcium pump membrane (80-85% of the membrane) and compartmental contents (CC), consisting mainly of calcium-binding protein (calsequestrin). In this study, a modified procedure for the preparation of JTC from frozen rabbit back muscle is described. The yield is substantially greater (threefold per weight of muscle), yet retaining characteristics similar to JTC from fresh hind leg muscles. Methodology has been developed for the disassembly of the JTC. This is achieved by selectively extracting the calcium pump membrane with 0.5% Triton X-100 in the presence of 1 mM CaCl2 to yield a complex of JFM with CC. The CC are then solubilized in the presence of EDTA to yield JFM. This fraction contains unidirectionally aligned junctional feet structures protruding from the cytoplasmic face of the membrane with repeat spacings comparable to that observed in JTC. The JFM contains 0.16 mumol phosphorus (lipid) per milligram protein. Characteristic proteins include 340 and 79-kD bands, a doublet at 28 kD, and a component that migrates somewhat slower than or equivalent to the calcium pump protein. Approximately 10% of the calcium-binding protein remains bound to the JFM after EDTA extraction, indicating the presence of a specific binding component in the JFM. The JFM, which is involved in junctional association with transverse tubule and likely in the Ca2+ release process in excitation-contraction coupling, is now available in the test tube.

Localization of Ca2+ release channels with ryanodine in junctional terminal cisternae of sarcoplasmic reticulum of fast skeletal muscle.

The mechanism of Ca2+ release from sarcoplasmic reticulum, which triggers contraction in skeletal muscle, remains the key unresolved problem in excitation-contraction coupling. Recently, we have described the isolation of purified fractions referable to terminal and longitudinal cisternae of sarcoplasmic reticulum. Junctional terminal cisternae are distinct in that they have a low net energized Ca2+ loading, which can be enhanced 5-fold or more by addition of ruthenium red. The loading rate, normalized for calcium pump protein content, then approaches that of longitudinal cisternae of sarcoplasmic reticulum. We now find that the ruthenium red-enhanced Ca2+ loading rate can be blocked by the previous addition of ryanodine. The inhibition constant is in the nanomolar range (20-180 nM). Ryanodine and ruthenium red have no effect on the Ca2+ loading rate of longitudinal cisternae. Direct binding studies with [3H]ryanodine localized the receptors to the terminal cisternae and not to longitudinal cisternae. Scatchard analysis of the binding data gives a dissociation constant for ryanodine in the range of the drug action on the terminal cisternae (approximately 100 nM range) with approximately 4 to 20 pmol bound per mg of protein. Ryanodine is known to be toxic in animals, leading to irreversible muscle contractures. These studies provide evidence on the mode of action of ryanodine and its localization to the terminal cisternae. The low concentration at which the drug is effective appears to account for its toxicity. Ryanodine locks the Ca2+ release channels in the "open state," so that Ca2+ is not reaccumulated and the muscle fiber cannot relax.

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.