Divergent functional properties of ryanodine receptor types 1 and 3 expressed in a myogenic cell line

Of the three known ryanodine receptor (RyR) isoforms expressed in muscle, R yR1 and RyR2 have well-defined roles in contraction. However, studies on ma mmalian RyR3 have been difficult because of low expression levels relative to RyR1 or RyR2. Using the herpes simplex virus 1 (HSV-1) helper-free ampli con system, we expressed either RyR1 or RyR3 in 1B5 RyR-deficient myotubes. Western blot analysis revealed that RyR1 - or RyR3-transduced cells expres sed the appropriate RyR isoform of the correct molecular mass. Although RyR 1 channels exhibited the expected unitary conductance for Csf in bilayer li pid membranes, 74 of 88 RyR3 channels exhibited pronounced subconductance b ehavior. Western blot analysis with an FKBP12/12.6-selective antibody revea ls that differences in gating behavior exhibited by RyR1 and RyR3 may be, i n part, the result of lower affinity of RyR3 for FKBP12. In calcium imaging studies, RyR1 restored skeletal-type excitation-contraction coupling, wher eas RyR3 did not. Although RyR3-expressing myotubes were more sensitive to caffeine than those expressing RyR1,they were much less sensitive to 4-chlo ro-m-cresol (CMC). In RyR1-expressing cells, regenerative calcium oscillati ons were observed in response to caffeine and CMC but were never seen in Ry R3-expressing 1B5 cells. In [H-3]ryanodine binding studies, only RyR1 exhib ited sensitivity to CMC, but both RyR isoforms responded to caffeine. These functional differences between RyR1 and RyR3 expressed in a mammalian musc le context may reflect differences in association with accessory proteins, especially FKBP12, as well as structural differences in modulator binding s ites.