Rapid release of calcium from the sarcoplasmic reticulum (SR) of skeletal muscle fibers during excitation-contraction (e-c) coupling is initiated by the interaction of surface membrane calcium channels (dihydropyridine receptors; DHPRs) with the calcium release channels of the SR (ryanodine receptors; RyRs, or feet). We studied the early differentiation of calcium release units, which mediate this interaction, in BC3H1 cells. Immunofluorescence labelings of differentiating myocytes with antibodies against alpha1 and alpha2 subunits of DHPRs, RyRs, and triadin show that the skeletal isoforms of all four proteins are abundantly expressed upon differentiation, they appear concomitantly, and they are colocalized. The transverse tubular system is poorly organized, and thus clusters of e-c coupling proteins are predominantly located at the cell periphery. Freeze fracture analysis of the surface membrane reveals tetrads of large intramembrane particles, arranged in orderly arrays. These appear concomitantly with arrays of feet (RyRs) and with the appearance of DHPR/RyS clusters, confirming that the four components of the tetrads correspond to skeletal muscle DHPRs. The arrangement of tetrads and feet in developing junctions indicates that incorporation of DHPRs in junctional domains of the surface membrane proceeds gradually and is highly coordinated with the formation of RyR arrays. Within the arrays, tetrads are positioned at a spacing of twice the distance between the feet. The incorporation of individual DHPRs into 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 in the junction.
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