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

1. The effects of Ca2+, ATP and caffeine on the gating of lobster skeletal muscle ryanodine receptors (RyR) was investigated after reconstitution of the channels into planar phospholipid bilayers and by using [3H]-ryanodine binding studies. 2. The single channel studies reveal that the EC50 (60 microM) for activation of the lobster skeletal RyR by Ca2+ as the sole ligand is higher than for any other isoform of RyR studied. 3. Inactivation of the channel by Ca2+ (EC50 = 1 mM) occurs at concentrations slightly higher than those required to inactivate mammalian skeletal RyR (RyR1) but lower than those required to inactivate mammalian cardiac RyR (RyR2). 4. Lifetime analysis demonstrates that cytosolic Ca2+, as the sole activating ligand, cannot fully open the lobster skeletal RyR (maximum Po approximately 0.2). The mechanism for the increase in open probability (Po) is an increase 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 presence of 700 microM 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 be 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 activation by caffeine is not 'sensitization' to cytosolic Ca2+. 8. 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+ and to caffeine. The results strongly indicate that these ligand binding sites on the channel are different to those on mammalian isoforms of RyR.