Ca(2+) inactivation sites are located in the COOH-terminal quarter of recombinant rabbit skeletal muscle Ca(2+) release channels (ryanodine receptors).

Ca(2+) activation of skeletal (RyR1) and cardiac (RyR2) muscle Ca(2+) release channels (ryanodine receptors) occurs with EC(50) values of about 1 microM. Ca(2+) inactivation occurs with an IC(50) value of about 3.7 mM for RyR1, but RyR2 shows little inactivation, even at >100 mM Ca(2+). In an attempt to localize the low affinity Ca(2+) binding sites responsible for Ca(2+) inactivation in RyR1, chimeric RyR1/RyR2 molecules were constructed. Because [(3)H]ryanodine binds only to open channels, and because channel opening and closing are Ca(2+)-dependent, the Ca(2+) dependence of [(3)H]ryanodine binding was used as an indirect measurement of Ca(2+) release channel opening and closing. IC(50) values for [(3)H]ryanodine binding suggested that Ca(2+) affinity for the low affinity Ca(2+) inactivation sites was unchanged in a chimera in which a glutamate-rich sequence (amino acids 1743-1964) in RyR1 was replaced with the corresponding, less acidic sequence from RyR2. Ca(2+) affinity (IC(50)) for low affinity Ca(2+) inactivation sites was intermediate in RyR1/RyR2 chimeras containing RyR2 amino acids 3726-4186 (RF9), 4187-4628 (RF10), or 4629-5037 (RF11), was closer to RyR2 values in RyR1 chimeras with longer RyR2 replacements (RF9/10 or RF10/11), and was indistinguishable from RyR2 in RyR1 containing all three RyR2 replacements (RF9/10/11). These data suggest that multiple low affinity Ca(2+) binding sites or multiple components of a low affinity Ca(2+) binding site are located between amino acids 3726 and 5037 and that their effects on Ca(2+) inactivation of the release channel are cooperative. Measurement of Ca(2+) activation of [(3)H]ryanodine binding showed that chimeras RF10, RF9/10, and RF9/10/11 were more sensitive to Ca(2+) than was either RyR1 or RyR2. Measurement of caffeine activation of Ca(2+) release in vivo showed that chimeras RF9, RF10, RF9/10, RF10/11, and RF9/10/11 were more sensitive to caffeine than wild-type RyR1. These results suggest that Ca(2+) and caffeine activation sites also involve COOH-terminal sequences in RyR1 and RyR2.