Abstract. The subcellular distribution of the 1,4-dihydropyridine receptor was determined in rabbit skeletal muscle in situ by immunofluorescence and immunoelectron microscopy. Longitudinal and transverse cryosections (5-8 #m) of rabbit gracilis muscle were labeled with monoclonal antibodies specific against either the otrsubunit (170,000-D polypeptide) or the ~-subunit (52,000-D polypeptide) of the 1,4-dihydropyridine receptor by immunofluorescence labeling. In longitudinal sections, specific labeling was present only near the interface between the A-and I-band regions of the sarcomeres. In transverse sections, specific labeling showed a hexagonal staining pattern within each myofiber however, the relative staining intensity of the type II (fast) fibers was judged to be three-to fourfold higher than that of the type I (slow) fibers. Specific immunofluorescence labeling of the sarcolemma was not observed in either longitudinal or transverse sections. These results are consistent with the idea that the t~rsubunit and the/3-subunit of the purified 1,4-dihydropyridine receptor are densely distributed in the transverse tubular membrane.Immunoelectron microscopical localization with a monoclonal antibody to the c~,-subunit of the 1,4-dihydropyridine receptor showed that the 1,4-dihydropyridine receptor is densely distributed in the transverse tubular membrane. Approximately half of these were distributed in close proximity to the junctional region between the transverse tubules and the terminal cisternae. Specific labeling was also present in discrete foci in the subsarcolemmal region of the myofibers. The size and the nonrandom distribution of these foci in the subsarcolemmal region support the possibility that they correspond to invaginations from the sarcolemma called caveolae. In conclusion, our results demonstrate that the 1,4-dihydropyridine receptor in skeletal muscle is localized to the transverse tubular membrane and discrete foci in the subsarcolemmal region, possibly caveolae but absent from the lateral portion of the sarcolemma. V OLTAGE-SENSITIVE Ca 2+ channels are present in smooth, cardiac, and skeletal muscle as well as in neuronal and endocrine cells (35,43). The 1,4-dihydropyridines are potent blockers of the L-type voltage-sensitive Ca 2+ channels (14). Electrophysiological studies have shown that 1,4-dihydropyridine-sensitive Ca 2+ channels are localized to the transverse tubule membrane in adult skeletal muscle (37). Binding studies have shown that high affinity receptors for the 1,4-dihydropyridines are enriched in isolated transverse tubular membranes (9) and isolated triads (25) from skeletal muscle, but constitute only 0.1-0.8 % of the total protein in purified transverse tubular membrane vesicles (4, 9). Recently, it has been shown that dihydropyridines also inhibit charge movement in the transverse tubular membrane and thus excitation-contraction coupling in skeletal muscle (36).The molecular properties of the dihydropyridine receptor from skeletal muscle has been extensively studied during...

The expression and subcellular distribution of the dystrophin-glycoprotein complex and laminin were examined in cardiac muscle by immunoblot and immunofluorescence analysis of rabbit and sheep papillary muscle. The five dystrophin-associated proteins (DAPs), 156-DAG, 59-DAP, 50-DAG, 43-DAG, and 35-DAG, were identified in rabbit ventricular muscle and found to codistribute with dystrophin in both papillary myofibers and Purkinje fibers. The DAPs and dystrophin codistributed not only in the free surface sarcolemma but also in interior regions of the myofibers where T tubules are present. Neither the DAPs nor dystrophin were detected in intercalated discs, a specialized region of cardiac sarcolemma where neighboring myocardial cells are physically joined by cell-cell junctions. Similarly, in bundles of Purkinje fibers, which lack T tubules, DAPs and dystrophin were also found to codistribute at the free surface sarcolemma but were not detected either in the region of surface sarcolemma closely apposed to a neighboring Purkinje fiber or in interior regions of these myofibers. Comparison between the distribution of the dystrophin-glycoprotein complex and laminin showed that laminin codistributes with the components of this complex in both papillary myofibers and Purkinje fibers. These results are consistent with previous findings demonstrating that the extracellularly exposed 156-DAG (dystroglycan) of the skeletal muscle dystrophin-glycoprotein complex binds laminin, a component of the basement membrane. Although we demonstrate that DAPs, dystrophin, and laminin colocalize to the sarcolemma in rabbit and sheep papillary myofibers as they do in skeletal myofibers, the most striking difference between the subcellular distribution of these proteins in cardiac and skeletal muscle is that the dystrophin-glycoprotein complex and laminin also localize to regions of the fibers where T tubules are distributed in cardiac but not in skeletal muscle. These results imply that the protein composition and thus possibly some functions of T tubules in cardiac muscle are distinct from those of skeletal muscle. (Circulation Research 1993;72:349-360

Abstract. The subcellular distribution of the Ca 2+-release channel/ryanodine receptor in adult rat papillary myofibers has been determined by immunofluorescence and immunoelectron microscopical studies using affinity purified antibodies against the ryanodine receptor. The receptor is confined to the sarcoplasmic retieulum (SR) where it is localized to interior and peripheral junctional SR and the corbular SR, but it is absent from the network SR where the SR-Ca2+-ATPase and phospholamban are densely distributed. Immunofluorescence labeling of sheep Purkinje fibers show that the ryanodine receptor is confined to discrete foci while the SR-Ca2+-ATPase is distributed in a continuous network-like structure present at the periphery as well as throughout interior regions of these myofibers. Because Purkinje fibers lack T-tubules, these results indicate that the ryanodine receptor is localized not only to the peripheral junctional SR but also to corbular SR densely distributed in interfibrillar spaces of the I-band regions. We have previously identified both corbular SR and junctional SR in cardiac muscle as potential Ca:+-storage/Ca~+-release sites by demonstrating that the Ca ~+ binding protein calsequestrin and calcium are very densely distributed in these two specialized domains of cardiac SR in situ. The results presented here provide strong evidence in support of the hypothesis that corbular SR is indeed a site of Ca:+-induced Ca 2+ release via the ryanodine receptor during excitation contraction coupling in cardiac muscle. Furthermore, these results indicate that the function of the cardiac Ca~+-release channel/ryanodine receptor is not confined to junctional complexes between SR and the sarcolemma.

Ca2+-ATPase of the sarcoplasmic reticulum was localized in cryostat sections from three different adult canine skeletal muscles (gracilis, extensor carpi radialis, and superficial digitalis flexor) by immunofluorescence labeling with monoclonal antibodies to the Ca2+-ATPase. Type I (slow) myofibers were strongly labeled for the Ca2+-ATPase with a monoclonal antibody (I1 DS) to the Ca2+-ATPase of canine cardiac sarcoplasmic reticulum; the type I1 (fast) myofibers were labeled at the level of the background with monoclonal antibody I1 DS. By contrast, type I1 (fast) myofibers were strongly labeled for Ca2+-ATPase of rabbit skeletal sarcoplasmic reticulum. The subcellular distribution of the immunolabeling in type I (slow) myofibers with monoclonal antibody I1 DS corresponded to that of the sarcoplasmic reticulum as previously determined by electron microscopy. The structuraI similarity between the canine cardiac Ca2+-ATPase present in the sarcoplasmic reticulum of the canine slow skeletal muscle fibers was demonstrated by immunoblotting . Monoclonal antibody (I1 DS) to the cardiac Ca2+-ATPase binds to only one protein band present in the extract from either cardiac or type I (slow) skeletal muscle tissue. By contrast, monoclonal antibody (I1 H11) to the skeletal type I1 (fast) Ca2+-ATPase binds only one protein band in the extract from type I1 (fast) skeletal muscle tissue. These immunopositive proteins coelectrophoresed with the Ca2+-ATPase of the canine cardiac sarcoplasmic reticulum and showed an apparent M, of 115,000. It is concluded that the Ca2+-ATPase of cardiac and type I (slow) skeletal sarcoplasmic reticulum have at least one epitope in common, which is not present on the Ca2+-ATPase of sarcoplasmic reticulum in type I1 (fast) skeletal myofibers. It is possible that this site is related to the assumed necessity of the Ca2+-ATPase of the sarcoplasmic reticulum in cardiac and type I (slow) skeletal myofibers to interact with phosphorylated phospholamban and thereby enhance the accumulation of Ca2+ in the lumen of the sarcoplasmic reticulum following 6 -adrenergic stimulation.

Abstract. Our previous immunofluorescence studies support the conclusion that the temporal appearance and subcellular distribution of TS28 (a marker of transverse (T) tubules and caveolae in adult skeletal muscle [Jorgensen, A. O., W. Arnold, A. C.-Y. Shen, S. Yuan, M. Gover, and K. P. . J. Cell Biol. 110:1173-1185), correspond very closely to those of T-tubules forming de novo in developing rabbit skeletal muscle (Yuan, S., W. Arnold, and A. O. . J. Ceil Biol. 110:1187-1198.To extend our morphological studies of the biogenesis of T-tubules and triads, the temporal appearance and subcellular distribution of the o~t-subunit of the 1,4-dihydropyridine receptor (a marker of the T-tubules and caveolae) was compared to (a) that of TS28; and (b) that of the ryanodine receptor (a marker of the junctional sarcoplasmic reticulum) in rabbit skeletal muscle cells developing in situ (day 19 of gestation to 10 d newborn) by double immunofluorescence labeling.The results presented show that the temporal appearance and relative subcellular distribution of the ct~-subunit of the 1,4-dihydropyridine receptor (otI-DHPR) are distinct from those of TS28 at the onset of the biogenesis of T-tubules. Thus, in a particular developing myotube the al-DHPR appeared before TS28 (secondary myotubes; day 19-24 of gestation). Furthermore, the otI-DHPR was distributed in discrete foci at the outer zone of the cytosol, while TS28 was confined to foci and rod-like structures at the cell periphery. As development proceeded (primary myotubes; day 24 of gestation) ,050% of the foci were positively labeled for both TS28 and the o~-DHPR, while ,020 and 30% of the foci were uniquely labeled for TS28 and the otl-DHPR, respectively. The foci labeled for both TS28 and the t~1-DHPR and the foci uniquely labeled for TS28 were generally confined to the cell periphery, while the foci uniquely labeled for the aI-DHPR were mostly confined to the outer zone of the cytosol.