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Triadin is an intrinsic membrane protein first identified in the skeletal muscle junctional sarcoplasmic reticulum and is considered to play an important role in excitation-contraction coupling. Using polyclonal antibodies to skeletal muscle triadin, we have identified and characterized three isoforms in rabbit cardiac muscle. The cDNAs encoding these three isoforms of triadin have been isolated by reverse transcription-polymerase chain reaction and cDNA library screening. The deduced amino acid sequences show that these proteins are identical in their N-terminal sequences, whereas the C-terminal sequences are distinct from each other and from that of skeletal muscle triadin. Based upon both the amino acid sequences and biochemical analysis, all three triadin isoforms share similar membrane topology with skeletal muscle triadin. Immunofluorescence staining of rabbit cardiac muscle with antibodies purified from the homologous region of triadin shows that cardiac triadin is primarily confined to the I-band region of cardiac myocytes, where the junctional and corbular sarcoplasmic reticulum is located. Furthermore, we demonstrate that the conserved region of the luminal domain of triadin is able to bind both the ryanodine receptor and calsequestrin in cardiac muscle. These results suggest that triadin colocalizes with and binds to the ryanodine receptor and calsequestrin and carries out a function in the lumen of the junctional sarcoplasmic reticulum that is important for both skeletal and cardiac muscle excitation-contraction coupling. Ca2ϩ release from the sarcoplasmic reticulum of skeletal and cardiac muscle is regulated by similar but distinct mechanisms (1-3). In cardiac muscle, depolarization leads to the opening of voltage-gated Ca 2ϩ channels. Ca 2ϩ influx through L-type Ca 2ϩ channels (the dihydropyridine receptor) triggers the opening of the ryanodine receptor/Ca 2ϩ release channel in the sarcoplasmic reticulum. However, in skeletal muscle, entry of external Ca 2ϩ is not needed for this signal transduction process. The skeletal muscle dihydropyridine receptor interacts either directly or indirectly with the ryanodine receptor, thereby activating the Ca 2ϩ release channel without a requirement for extracellular calcium. Despite this difference, many factors that modulate the channel properties of the skeletal muscle ryanodine receptor also affect the cardiac ryanodine receptor in a similar fashion (4, 5). Identification of protein components in the junctional sarcoplasmic reticulum is fundamental to our understanding of the mechanisms of Ca 2ϩ storage and release in muscle cells. So far, the major components of the excitationcontraction coupling, such as the ryanodine receptor, the dihydropyridine receptor, and calsequestrin, have been extensively studied in skeletal muscle, and their counterparts have later been identified and characterized in cardiac muscle.Triadin is an intrinsic membrane protein originally identified in skeletal muscle (6 -8). It is specifically enriched in the junctional sarcoplasmic ...

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...