Ca2+ inactivation sites are located in the COOH-terminal quarter of recombinant rabbit skeletal muscle Ca2+ release channels (ryanodine receptors)

Abstract

Ca2+ activation of skeletal (RyR1) and cardiac (RyR2) muscle Ca2+ release channels (ryanodine receptors) occurs with EC50 values of about I μM. Ca2+ inactivation occurs with an IC50 value of about 3.7 mM for RyR1, but RyR2 shows little inactivation, even at >100 nM Ca2+. In an attempt to localize the low affinity Ca2+ binding sites responsible for Ca2+ inactivation in RyR1, chimeric RyR1/RyR2 molecules were constructed. Because [3H]ryanodine binds only to open channels, and because channel opening and closing are Ca2+-dependent, the Ca2+ dependence of [3H]ryanodine binding was used as an indirect measurement of Ca2+ release channel opening and closing. IC50 values for [3H]ryanodine binding suggested that Ca2+ affinity for the low affinity Ca2+ 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. Ca2+ affinity (IC50) for low affinity Ca2+ 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 Ca2+ binding sites or multiple components of a low affinity Ca2+ binding site are located between amino acids 3726 and 5037 and that their effects on Ca2+ inactivation of the release channel are cooperative. Measurement of Ca2+ activation of [3H]ryanodine binding showed that chimeras RF10, RF9/10, and RF9/10/11 were more sensitive to Ca2+ than was either RyR1 or RyR2. Measurement of caffeine activation of Ca2+ 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 Ca2+ and caffeine activation sites also involve COOH-terminal sequences in RyR1 and RyR2.