CYTOPLASMIC CA2-MUSCLE( INHIBITS THE RYANODINE RECEPTOR FROM CARDIAC)

Ca2+-dependent inhibition of native and isolated ryanodine receptor (R yR) calcium release channels from sheep heart and rabbit skeletal musc le was investigated using the lipid bilayer technique. We found that c ytoplasmic Ca2+ inhibited cardiac RyRs with an average K-m = 15 mM, sk eletal RyRs with K-m = 0.7 mM and with Hill coefficients of 2 in both isoforms. This is consistent with measurements of Ca2+ release from th e sarcoplasmic reticulum (SR) in skinned fibers and with [H-3]-ryanodi ne binding to SR vesicles, but is contrary to previous bilayer studies which were unable to demonstrate Ca2+-inhibition in cardiac RyRs (Chu , Fill, Stefani & Entman (1993) J. Membrane Biol. 135, 49-59). Ryanodi ne prevented Ca2+ from inhibiting either cardiac or skeletal RyRs. Ca2 +-inhibiton in cardiac RyRs appeared to be the most fragile characteri stic of channel function, being irreversibly disrupted by 500 mM Cs+, but not by 500 mM K+, in the cis bath or by solublization with the det ergent CHAPS. These treatments had no effect on channel regulation by AMP-PNP, caffeine, ryanodine, ruthenium red, or Ca2+-activation. Ca2+- inhibition in skeletal RyRs was retained in the presence of 500 mM Cs. Our results provide an explanation for previous findings in which ca rdiac RyRs in bilayers with 250 nM Cs+ in the solutions fail to demons trate Ca2+-inhibition, while Ca2+-inhibition of Ca2+ release is observ ed in vesicle studies where K+ is the major cation. A comparison of op en and closed probability distributions from individual RyRs suggested that the same gating mechanism mediates Ca2+-inhibition in skeletal R yRs and cardiac RyRs, with different Ca2+ affinities for inhibition. W e conclude that differences in the Ca2+-inhibition in cardiac and skel etal channels depends on their Ca2+ binding properties.