Cisternae Of Sarcoplasmic Reticulum Research Articles

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.

Characterization of Glucose Transporter-Enriched Membranes from Rat Skeletal Muscle: Assessment of Endothelial Cell Contamination and Presence of Sarcoplasmic Reticulum and Transverse Tubules*

The subcellular origin of membranes from rat skeletal muscle that contain insulin-responsive glucose transporters was investigated. Rat skeletal muscle membranes were prepared by isopycnic centrifugation in sucrose gradients. In vivo insulin treatment increased the content of GLUT-4 glucose transporters in the 25% sucrose fraction (enriched in the plasma membrane marker 5'-nucleotidase) and decreased it in the 35% sucrose fraction (devoid of plasma membrane markers). The possibility of endothelial cell membrane contamination in these fractions was investigated using a mouse monoclonal antibody, MRC OX-43, raised against a cell surface protein specific to rat vascular endothelium. MRC OX-43 did not react with any of the muscle membrane fractions, but did recognize a protein of around 100 kDa in extracts of human endothelial cells and rat aorta. An antibody to the dihydropyridine receptor of skeletal muscle, IIC12, was used to determine the presence of transverse tubules in these fractions. IIC12 reacted positively with a 180-kDa protein in purified rat transverse tubules. In contrast, this antibody did not cross-react with the 25% or 35% sucrose fractions. The 25% sucrose fraction was devoid of calsequestrin and ryanodine receptor, cisternal sarcoplasmic reticulum markers. However, small amounts of these proteins were detected in the 35% sucrose fraction. The results suggest that the 25% sucrose fraction represents plasma membranes, while the 35% sucrose fraction is an insulin-sensitive intracellular fraction that contains, but is not enriched in, sarcoplasmic reticulum cisternae. The results further show that insulin-induced recruitment of GLUT-4 transporters in skeletal muscles can be demonstrated independently of GLUT-4 recruitment in endothelial cells.

High‐resolution scanning electron microscopic studies on the three‐dimensional structure of the transverse‐axial tubular system, sarcoplasmic reticulum and intercalated disc of the rat myocardium

The three-dimensional structure of the transverse-axial tubular system, sarcoplasmic reticulum (SR), and intercalated disc of the rat left ventricle was examined by high-resolution scanning electron microscopy after removal of the cytoplasmic matrices by the osmium-DMSO-osmium procedure. In the intermyofibrillar space, the transverse tubules (T-tubules) are accompanied by longitudinally oriented axial tubules and together form a transverse-axial system. The junctional SR is usually small but occasionally medium or large in size and couples with the T- or with the axial tubules. On the surface of the junctional SR facing the T- or the axial tubule, tiny junctional processes are seen. One or two sarcotubules, the so-called Z-tubules, frequently run parallel to the T-tubule. The sarcotubules derived from the junctional SR or from the Z-tubule run longitudinally or obliquely and form polygonal meshes around the myofibrils. On the surface of the SR at the H-band level, small fenestrations of 12-40 nm in diameter, and tiny hollows 8-20 nm in diameter are seen. Bulbous swellings of the SR, the corbular SR, are preferentially seen near the Z-band. The large and flat SR, known as the cisternal SR, intercalates among the SR meshes. In the subsarcolemmal space, the sarcotubules form a multilayered network (peripheral SR). The cisternal SR is frequently intercalated in these meshes and closely associated with the inner surface of the sarcolemma. The intercalated disc appears as a prominently undulated membrane demarcating the border between two adjacent heart muscle cells, and occasionally small projections 60-90 nm in diameter and 200-600 nm in length display on its surface.

The atrial myocardial cells of mouse heart: A structural and stereological study

Structural and stereological studies of mouse atrial myocardial cells, carried out in the same fashion as our previous investigations on mouse ventricle, demonstrate an extremely well-developed sarcoplasmic reticulum (SR) in atrial cells. The volume fraction ( V v) of the SR exceeds 12% in mouse atrial cells; perimyofibrillar network SR constitutes the major portion. We have confirmed the findings of Bossen et al. (1981, Tissue Cell 13, 71–77) of a difference between atria in terms of coupling density, the right atrium having a significantly lower incidence of interior junctional SR than the left. The SR of mouse atrium comprises a rich variety of specialized segments, including the IJSR, peripheral junctional SR, corbular SR, cisternal SR (including regions similar to fenestrated collars of striated skeletal muscle SR), as well as a peculiar form of extended junctional SR (EJSR). Although less frequent in occurrence than corbular SR, the EJSR seems closely related, since it occurs in multiple clusters at or near the Z-line regions, contains internal granular densities, and bears surface-connected structures resembling junctional processes. Seen in thin sections, mouse atrial EJSR elements are more complex than corbular SR, being larger in diameter and frequently circular in profile. Thick-section and serial-section analyses reveal that bodies of EJSR are in fact hollow spheroids. The transverse-axial tubular system of mouse atrium is rather poorly developed in comparison to its ventricular counterpart. The Golgi apparatus and associated specific atrial granules are prominent cell components. “Focal ellipsoidal deposits” (FEDs) previously described by Page and co-workers (1986, Amer. J. Physiol.) are consistently located adjacent to the Golgi region, but immunocytochemical staining for two different segments of atrial natriuretic peptide reveals no specific reaction in FEDs, whereas the SAGS are densely labeled for both antibodies.

Polymyositis in mice experimentally inoculated with Getah virus.

Mice inoculated intracerebrally with parent, large-plaque (LP) and small-plaque (SP) strains of Kanagawa strain of Getah virus showed clinically recumbency and paralysis. The LP strain caused recumbency more rapidly and killed mice more early after inoculation than the parent and SP strains. Microscopically, skeletal muscles of the whole body were involved showing degenerative or inflammatory changes. In mice inoculated with the parent or SP strains, there were degeneration and necrosis of the muscle fibers with inflammatory cell infiltration and regenerative reaction. The lesions were particularly conspicuous in muscles of the hind legs. In mice inoculated with the LP strain, most of the muscle fibers revealed degeneration and necrosis, but reactive changes were poor. In addition, the periosteum and muscular connective tissue were thickened with karyorrhexis. Electron microscopically, virus particles were recognized mainly in cisternae of sarcoplasmic reticulum in skeletal muscle fibers of mice inoculated with the LP strain, while they were rare in those of animals injected with the parent and SP strains. From these finding, it was suggested that Kanagawa strain of Getah virus has the virulence to skeletal muscles of mice.

Bimodal operation of the ryanodine-sensitive transducer calcium channel

Ryanodine binds to the transducer calcium channel complex that links depolarization of the transverse tubule to calcium release from the terminal cisternae during excitation-contraction coupling of skeletal muscle. Ryanodine exerts a bimodal action on the transducer calcium channel complex depending upon membrane potential and concentration. When the transmembrane potential is at resting level (−90 mV inside cell vs outside), low concentrations of ryanodine 10 −10 M to 10 −8 M favor calcium influx from outside which in turn causes calcium release from the terminal cisternae via calcium operated calcium channels. The leak from the terminal cisternae is insufficient to cause contraction but does cause a large increase in aerobic energy utilization by the Ca-ATPase of the sarcoplasmic reticulum. When the transmembrane potential is made more positive (−40 mV) the transducer channel is opened to the terminal cisternae of sarcoplasmic reticulum and is maintained in an open state by ryanodine allowing calcium efflux from the terminal cisternae to the sarcoplasm. At higher concentrations of ryanodine the transducer-calcium channel becomes open to the terminal cisternae and its store of ionized calcium leaks from the terminal cisternae in sufficient quantities to cause a contracture. The ryanodine-sensitive calcium transducer calcium channel operates in a bimodal manner. At low concentrations < 10 −4 M the ryanodine-sensitive transducer calcium channel is open to the lumen of the T-tubule and allows calcium to flow in and trigger further calcium release. At higher concentrations the ryanodine-sensitive transducer channel opens to allow a calcium efflux from the terminal cisternae in sufficient quantities to cause contracture.

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.

Inhibition of Rapid Ca Release from Isolated Cardiac Sarcoplasmic Reticulum Cisternae

Inhibition of Rapid Ca Release from Isolated Cardiac Sarcoplasmic Reticulum Cisternae

The unraveling architecture of the junctional sarcoplasmic reticulum

The sarcoplasmic reticulum (SR) of skeletal muscle controls the contraction-relaxation cycle by raising and lowering the myoplasmic free-Ca2+ concentration. The coupling between excitation, i.e., depolarization of sarcolemma and transverse tubule (TT) and Ca2+ release from the terminal cisternae (TC) of SR takes place at the triad. The triad junction is formed by a specialized region of the TC, the junctional SR, and the TT. The molecular architecture and protein composition of the junctional SR are under active investigation. Since the junctional SR plays a central role in excitation-contraction coupling and Ca2+ release, some of its protein constituents are directly involved in these processes. The biochemical evidence supporting this contention is reviewed in this article.

Three-dimensional architecture of the calcium channel/foot structure of sarcoplasmic reticulum

The calcium channel responsible for the release of Ca2+ from the sarcoplasmic reticulum of skeletal muscle during excitation-contraction coupling has recently been identified and purified. The isolated calcium channel has been identified morphologically with the 'foot' structures which are associated with the junctional face membrane of the terminal cisternae of sarcoplasmic reticulum. In situ, the foot structure extends across the gap of the triad junction from the terminal cisternae of the reticulum to the transverse tubule. We describe here the three-dimensional architecture (3.7 nm resolution) of the calcium channel/foot structure from fast-twitch rabbit skeletal muscle, which we determined from electron micrographs of isolated, non-crystalline structures that had been tilted in the electron microscope. The reconstruction reveals two different faces and an internal structure in which stain accumulates at several interconnected locations, which could empty into the junctional gap of the triad junction. The detailed architecture of the channel complex is relevant to understanding both the physical path followed by calcium ions during excitation-contraction coupling and the association of the terminal cisternae and the transverse tubules in the triad junction.

Newly Synthesized Calsequestrin, Destined for the Sarcoplasmic Reticulum, Is Contained in Early/Intermediate Golgi-derived Clathrin-coated Vesicles

We have examined the possible role of clathrin-coated vesicles (CVs) in the genesis of the sarcoplasmic reticulum (SR) in developing chick skeletal myotubes. Calsequestrin (CSQ) a luminal Ca2+ binding protein of the terminal SR cisternae, is contained within the vesicle lumen of skeletal muscle CVs in substantial amounts, approximately four molecules/CV. Employing 3-day cultures of chick skeletal myotubes we demonstrate that after a 30-min labeling with [35S]methionine and cysteine, radioactivity in CSQ remains high in the CVs 45 min later and then declines, while labeled CSQ in the SR continues to rise. No CSQ appears to be secreted. All of the CSQ in both the CVs and SR is sensitive to the activity of endoglycosidase H, and a significant fraction also binds to wheat germ agglutinin. Based on these results, we discuss the hypothesis that a selective CV-mediated pathway exists in developing skeletal muscle cells for the transport of CSQ from the early/intermediate Golgi apparatus to the SR.

Modulation of the calcium release channel of sarcoplasmic reticulum by adriamycin and other drugs

The calcium release channel of sarcoplasmic reticulum mediates Ca 2+ release which triggers muscle contraction in excitation-contraction coupling. The channels have been identified morphologically with the feet structures, which are involved in junctional association of terminal cisternae of sarcoplasmic reticulum with the transverse tubules to form the triad junction. In this study, we further characterize the action of drugs on the calcium release channel from sarcoplasmic reticulum fused into planar bilayers. Adriamycin is an effective cancer chemotherapeutic drug, which is limited by its cardiotoxicity. The drug, when added to the myoplasmic side ( cis side), activates channel opening at μM concentrations in a dose dependent manner. Adriamycin together with ATP (mM) gives optimal activation, with an open probability ( P 0) of ≈1.0. Ruthenlum red added to the cis side, equivalent to the cytoplasmic (myoplasmic) domain, completely blocks channel opening. Qualitatively similar results are obtained with adriamycinol, the major metabollte of adriamycin. The inhibition by adriamycin is not reversed by reperfusion to wash out the drug. Silver ions are also found to activate the channel. The conductance of the channel activated by adriamycin, adriamycinol or Ag + is ≈100 ps, similar to that previously reported for activation of the channel with Ca 2+ and ATP. Ruthenium red has previously been observed to block channel activation from the cytoplasmic side. We now find that ruthenium red is also effective in blocking channel activity when added to the luminal ( trans) side. The block from the luminal side is different in that: (1) subconductance levels can prominantly be observed; and (2) the block of ryanodine activation is complete.

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.

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.