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 Ca 2+ pump protein-containing membrane, whereas the longitudinal cisternae contain only the Ca 2÷ pump protein-containing membrane. The junctional face membrane of the terminal cisternae contains feet structures that extend ~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 Ca 2+ pump protein are not observed between the feet at the junctional face membrane, either in thin section or by negative staining, even though the Ca 2÷ 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 Ca ~+ 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.The muscle fiber contains an intricate membraneous network that controls muscle contraction and relaxation by regulating the intracellular calcium concentration. The plasma membrane or plasmalemma invaginates transversely into the muscle sarcoplasm to form transverse tubules, which are connected to an internal reticular membrane system, the sarcoplasmic reticulum (SR). 1 The SR surrounds the sarcomere in Abbreviation used in this paper. SR, sarcoplasmic reticulum. a sleevelike manner, and is composed of two distinct portions: (a) the terminal cisternae which are junctionally associated with the transverse tubule, and (b) the longitudinal cisternae or longitudinal SR, which connect medially with the two term...

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.

The release of Ca2l from internal stores is requisite to muscle contraction. In skeletal muscle and heart, the Ca2' release channels (ryanodine receptor) of sarcoplasmic reticulum, involved in excitation-contraction coupling, have recently been isolated and characterized. In smooth muscle, inositol 1,4,5-trisphosphate (1P3) is believed to mobilize Ca2+ from internal stores and thereby modulate contraction. We describe the isolation of an IP3 receptor from smooth muscle. Bovine aorta smooth muscle microsomes were solubilized with 3-[(3-cholamidopropyl)dimethylammonioj-1-propanesulfonate, and the IP3 receptor was purified by sucrose gradient centrifugation and column chromatography with heparinagarose and wheat germ agglutinin-agarose. The purified receptor bound 2.7 ± 0.18 nmol of IP3 per mg of protein with a Kd of 2.4 ± 0.24 nM. That is, the purified receptor has been enriched about 1000-fold compared with the original microsomes, whereas the Kd for IP3 remains unchanged. The receptor is an oligomer of a single polypeptide with a Mr of 224,000 as determined by SDS/PAGE. Negative-staining electron microscopy reveals that the receptor is a large pinwheellike structure having surface dimensions of :250 x 250 A with fourfold symmetry. The IP3 receptor from smooth muscle is similar to the ryanodine receptor with regard to its large size and fourfold symmetry, albeit distinct with regard to appearance, protomer size, and ligand binding.Muscle contraction is triggered by a rise in myoplasmic free Ca2". Ca2l release in heart and skeletal muscle can be characteristically triggered by Ca2l/caffeine from the sarcoplasmic reticulum (SR) (1) by way of ryanodine-sensitive Ca2l release channels, which have been localized to the terminal cisternae of SR (1-3). Recently, the ryanodine receptors from SR of skeletal muscle and heart have been isolated and identified morphologically as the foot structures of the terminal cisternae (4-6). Reconstitution into planar lipid bilayers has allowed identification of the ryanodine receptors as the SR Ca2l release channels (7-10). The ryanodine receptor from skeletal muscle has been cloned (11,12). At present, the role of inositol 1,4,5-trisphosphate (IP3) in excitation-contraction coupling in heart and skeletal muscle remains controversial (1), whereas, in smooth muscle, two types of SR Ca2' release have been described (13,14). Ca2' release can be triggered by IP3 or by Ca2+/caffeine. The Ca2+/caffeine-sensitive Ca2' release pathway in smooth muscle, as in skeletal muscle and heart, is sensitive to ryanodine. In excitation-contraction coupling in smooth muscle, IP3 is produced at the level of the plasma membrane in response to hormone stimulation (1). Then IP3 binds to specific intracellular receptors on the SR membrane and mobilizes the release of Ca2+ (15) from Bio-Rad. Wheat germ agglutinin-Sepharose 4B (5 mg of protein per ml of gel) was prepared by using CNBr-activated Sepharose 4B according to the manufacturer's instructions (Pharmacia). Bovine aortae were quick-frozen with cr...

Abstract. 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 CaCI2 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 lxmol 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 Ca 2÷ release process in excitation-contraction coupling, is now available in the test tube.

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.