Abstract. The subcellular distribution of sarcolemmal dihydropyridine receptor (DHPR) and sarcoplasmic reticular triadin and Ca 2÷ release channel/ryanodine receptor (RyR) was determined in adult rabbit ventricle and atrium by double labeling immunofluorescence and laser scanning confocal microscopy. In ventricular muscle cells the immunostaining was observed primarily as transversely oriented punctate bands spaced at approximately 2-/zm intervals along the whole length of the muscle fibers. Image analysis demonstrated a virtually complete overlap of the staining patterns of the three proteins, suggesting their close association at or near dyadic couplings that are formed where the sarcoplasmic reticulum (SR) is apposed to the surface membrane or its infoldings, the transverse (T-) tubules. In rabbit atrial cells, which lack an extensive T-tubular system, DHPR-specific staining was observed to form discrete spots along the sarcolemma but was absent from the interior of the fibers. In atrium, punctate triadin-and RyR-specific staining was also observed as spots at the cell periphery and image analysis indicated that the three proteins were co-localized at, or just below, the sarcolemma. In addition, in the atrial cells triadin-and RyR-specific staining was observed to form transverse bands in the interior cytoplasm at regularly spaced intervals of approximately 2 #m. Electron microscopy suggested that this cytoplasmic staining was occurring in regions where substantial amounts of extended junctional SR were present. These data indicate that the DHPR codistributes with triadin and the RyR in rabbit ventricle and atrium, and furthermore suggest that some of the SR Ca 2+ release channels in atrium may be activated in the absence of a close association with the DHPR. IN striated muscle, depolarization of the sarcolemma and transverse (T-) t tubular network induces release of Ca 2÷ from internal stores in the sarcoplasmic reticulum (SR) by a process commonly referred to as excitation-contraction (EC) coupling. In skeletal muscle it is generally accepted that the electrical signal is transduced to a release of Ca 2+ at specialized triad junctions formed between a central T-tubule flanked by two elements of closely apposed junctional SR (jSR). The junctional T-tubules contain DHPR which act as voltage sensors (36,45,(56)(57)(58) 1. Abbreviations used in this paper: DHP, dihydropyridine; DHPR, DHP receptor; jSR, junctional sarcoplasmic reticulum; RyR, ryanodine receptor; SR, sarcoplasmic reticulum; T-tubule, transverse tubule; TC, terminal cisternae.rows of "feet" (23) that have been identified as ryanodine receptors (RyR), the SR Ca 2÷ release channels (35,55,61). The exact mechanism of skeletal muscle EC coupling is not yet completely understood; however, it is thought that the release of Ca 2÷ from the SR is a depolarization-induced mechanism without the necessity of Ca 2÷ flow (46). In contrast, excitation contraction coupling in cardiac muscle requires an influx of Ca z÷ through L-type Ca ~÷ channels (the cardiac i...

The dihydropyridine receptors (DHPR) and the ryanodine receptors (RyR) are well-characterized proteins of the triad junctions of skeletal muscle fibers. Recently, a newly discovered 95-kDa protein, triadin, has been purified from rabbit skeletal muscle heavy sarcoplasmic reticulum (SR) vesicles. WE have used indirect immunogold EM to localize triadin to the junctional face of the SR in isolated triads. In addition, we have used indirect immunofluorescence to localize triadin in relation to the DHPR and the RyR in adult and developing rat skeletal muscle. In double immunolabelling experiments of longitudinally oriented adult rat skeletal muscle tissue, triadin-specific and RyR-specific antibodies resulted in a characteristic striated staining pattern. The staining arising from these antibodies completely overlapped when examined by computer analysis of digitized laser scanning confocal microscopy images. A similar result was obtained in double staining experiments using antibodies raised against the DHPR and the RyR suggesting that all three proteins are located in the triads in situ. The developmental expression of the three triad proteins was examined using double labeling of skeletal muscle tissue from several fetal and early postnatal ages. The staining patterns of triadin, RyR, and DHPR antibodies were overlapping throughout development, suggesting that from their earliest appearance the three proteins are components of the triads.

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