COORDINATED INCORPORATION OF SKELETAL-MUSCLE DIHYDROPYRIDINE RECEPTORS AND RYANODINE RECEPTORS IN PERIPHERAL COUPLINGS OF BC(3)H1 CELLS

Rapid release of calcium from the sarcoplasmic reticulum (SR) of skele tal muscle fibers during excitation-contraction (e-c) coupling is init iated by the interaction of surface membrane calcium channels (dihydro pyridine receptors; DHPRs) with the calcium release channels of the SR (ryanodine receptors; RyRs, or feet). We studied the early differenti ation of calcium release units, which mediate this interaction, in BC( 3)H1 cells. Immunofluorescence labelings of differentiating myocytes w ith antibodies against alpha(1) and alpha(2) subunits of DHPRs, RyRs, and triadin show that the skeletal isoforms of all four proteins are a bundantly expressed upon differentiation, they appear concomitantly, a nd they are colocalized. The transverse tubular system is poorly organ ized, and thus clusters of e-c coupling proteins are predominantly loc ated at the cell periphery. Freeze fracture analysis of the surface me mbrane reveals tetrads of large intramembrane particles, arranged in o rderly arrays. These appear concomitantly with arrays of feet (RyRs) a nd with the appearance of DHPR/RyS clusters, confirming that the four components of the tetrads correspond to skeletal muscle DHPRs. The arr angement of tetrads and feet in developing junctions indicates that in corporation of DHPRs in junctional domains of the surface membrane pro ceeds gradually and is highly coordinated with the formation of RyR ar rays. Within the arrays, tetrads are positioned at a spacing of twice the distance between the feet, The incorporation of individual DHPRs i nto tetrads occurs exclusively at positions corresponding to alternate feet, suggesting that the assembly of RyR arrays not only guides the assembly of tetrads but also determines their characteristic spacing i n the junction.