ROLE OF RYANODINE RECEPTORS IN THE ASSEMBLY OF CALCIUM-RELEASE UNITS IN SKELETAL-MUSCLE

In muscle cells, excitation-contraction (e-c) coupling is mediated by ''calcium release units,'' junctions between the sarcoplasmic reticulu m (SR) and exterior membranes, Two proteins, which face each other, ar e known to functionally interact in those structures: the ryanodine re ceptors (RyRs), or SR calcium release channels. and the dihydropyridin e receptors (DHPRs), or L-type calcium channels of exterior membranes. In skeletal muscle, DHPRs form tetrads, groups of four receptors. and tetrads are organized in arrays that face arrays of feet (or RyRs), T riadin is a protein of the SR located at the SR-exterior membrane junc tions, whose role is not known. We have structurally characterized cal cium release units in a skeletal muscle cell line (1B5) lacking Ry(1)R . Using immunohistochemistry and freeze-fracture electron microscopy, we find that DHPR and triadin are clustered in foci in differentiating 1B5 cells. Thin section electron microscopy reveals numerous SR-exter ior membrane junctions lacking foot structures (dyspedic). These resul ts suggest that components other than Ry(1)Rs are responsible for targ eting DHPRs and triadin to junctional regions. However, DHPRs in 1B5 c ells are not grouped into tetrads as in normal skeletal muscle cells s uggesting that anchoring to Ry(1)Rs is necessary for positioning DHPRs into ordered arrays of tetrads. This hypothesis is confirmed by findi ng a ''restoration of tetrads'' in junctional domains of surface membr anes after transfection of 1B5 cells with cDNA encoding for Ry(1)R.