RYR1 and RYR3 have different roles in the assembly of calcium release units of skeletal muscle

Calcium release units (CRUs) are junctions between the sarcoplasmic reticul um (SR) and exterior membranes that mediates excitation contraction (e-c) c oupling in muscle cells. In skeletal muscle CRUs contain two isoforms of th e sarcoplasmic reticulum Ca2+ release channel: ryanodine receptors type 1 a nd type 3 (RyR1 and RyR3). 1B5s are a mouse skeletal muscle cell line that carries a null mutation for RyR1 and does not express either RyR1 or RyR3. These cells develop dyspedic SR/exterior membrane junctions (i.e., dyspedic calcium release units, dCRUs) that contain dihydropyridine receptors (DHPR s) and triadin, two essential components of CRUs, but no RyRs (or feet). La ck of RyRs in turn affects the disposition of DHPRs, which is normally dict ated by a linkage to RyR subunits. in the dCRUs of 1B5 cells, DHPRs are nei ther grouped into tetrads nor aligned in two orthogonal directions. We have explored the structural role of RyR3 in the assembly of CRUs in 1B5 cells independently expressing either RyR1 or RyR3. Either isoform colocalizes wi th DHPRs and triadin at the cell periphery. Electron microscopy shows that expression of either isoform results in CRUs containing arrays of feet, ind icating the ability of both isoforms to be targeted to dCRUs and to assembl e in ordered arrays in the absence of the other. However, a significant dif ference between RyR1- and RyR3-rescued junctions is revealed by freeze frac ture. While cells transfected with RyR1 show restoration of DHPR tetrads an d DHPR orthogonal alignment indicative of a link to RyRs, those transfected with RyR3 do not. This indicates that RyR3 fails to link to DHPRs in a spe cific manner. This morphological evidence supports the hypothesis that acti vation of RyR3 in skeletal muscle cells must be indirect and provides the b asis for failure of e-c coupling in muscle cells containing RyR3 but lackin g RyR1 (see the accompanying report, Fessenden et at., 2000).