Abstract
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.
Highlights
Ca2ϩ release channels from the sarcoplasmic reticulum of skeletal and cardiac muscle1 are modulated by endogenous and exogenous modulators such as ATP, Ca2ϩ, calmodulin, Mg2ϩ, ruthenium red, and ryanodine [1, 2]
We found that the low affinity Ca2ϩ inactivation site is not affected by exchange of the D3 sequence but that Ca2ϩ inactivation is affected to different degrees by multiple exchanges of fragments at the COOH terminus of RyR1
Transient Expression of Chimeric RyR cDNAs—In earlier studies, we showed that the curve of Ca2ϩ dependence for [3H]ryanodine binding to recombinant RyR1 is bell-shaped, indicating that [3H]ryanodine binding is sensitive to both Ca2ϩ activation and inactivation of Ca2ϩ release channel function
Summary
Ca2ϩ release channels from the sarcoplasmic reticulum of skeletal and cardiac muscle (ryanodine receptors, RyRs)1 are modulated by endogenous and exogenous modulators such as ATP, Ca2ϩ, calmodulin, Mg2ϩ, ruthenium red, and ryanodine [1, 2]. 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.
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