Evidence for novel caffeine and Ca2+ binding sites on the lobster skeletalryanodine receptor

1 The effects of Ca2+. ATP and caffeine on the gating of lobster skeletal m uscle ryanodine receptors (RyR) was investigated after reconstitution of th e channels into planar phospholipid bilayers and by using [H-3]-ryanodine b inding studies. 2 The single channel studies reveal that the EC50 (60 mu M) for activation of the lobster skeletal RyR by Ca2+ as the sole ligand is higher than for a ny other isoform of RyR studied. 3 Inactivation of the channel by Ca2+ (EC50 = 1 mM) occurs at concentration s slightly higher than those required to inactivate mammalian skeletal RyR (RyR1) but lower than those required to inactivate mammalian cardiac RyR (R yR2). 4 Lifetime analysis demonstrates that cytosolic Ca2+, as the sole activatin g ligand, cannot fully open the lobster skeletal RyR (maximum Po approximat ely 0.2). The mechanism for the increase in open probability (Po) is an inc rease in both the frequency and the duration of the open events. 5 ATP is a very effective activator of the lobster RyR and can almost fully open the channel in the presence of activating cytosolic [Ca2+]. In the pr esence of 700 mu M Ca2+, 1 mM ATP increased Po to approximately 0.8. 6 Caffeine, often used as a tool to identify the presence of RyR channels, is relatively ineffective and cannot increase Po above the level that can b e attained with Ca2+ alone. 7 The results reveal that caffeine increases Po by a different mechanism to that of cytosolic Ca2+ demonstrating that the mechanism for channel activa tion by caffeine is not 'sensitization' to cytosolic Ca28 By studying the mechanisms involved in the activation of the lobster RyR we have demonstrated that the channel responds in a unique manner to Ca2+ a nd to caffeine. The results strongly indicate that these ligand binding sit es on the channel are different to those on mammalian isoforms of RyR.