Regulation of skeletal ryanodine receptors by dihydropyridine receptor II-III loop C-region peptides: Relief of Mg²⁺ inhibition

The aim of the present study was to explore interactions between surface-membrane DHPR (dihydropyridine receptor) Ca²⁺ channels and RyR (ryanodine receptor) Ca²⁺ channels in skeletal-muscle sarcoplasmic reticulum. The C region (⁷²⁵Phe-Pro⁷⁴²) of the linker between the 2nd and 3rd repeats (II-III loop) of the α₁ subunit of skeletal DHPRs is essential for skeletal excitation-contraction coupling, which requires a physical interaction between the DHPR and RyR and is independent of external Ca²⁺. Little is known about the regulatory processes that might take place when the two Ca ²⁺ channels interact. Indeed, interactions between C fragments of the DHPR (C peptides) and RyR have different reported effects on Ca²⁺ release from the sarcoplasmic reticulum and on RyR channels in lipid bilayers. To gain insight into functional interactions between the proteins and to explore different reported effects, we examined the actions of C peptides on RyR1 channels in lipid bilayers with three key RyR regulators, Ca²⁺, Mg²⁺ and ATP. We identified four discrete actions: two novel, low-affinity (> 10 μM), rapidly reversible effects (fast inhibition and decreased sensitivity to Mg²⁺ inhibition) and two slowly reversible effects (high-affinity activation and a slow-onset, low-affinity inhibition). Fast inhibition and high-affinity activation were decreased by ATP. Therefore peptide activation in the presence of ATP and Mg²⁺, used with Ca ²⁺ release assays, depends on a mechanism different from that seen when Ca²⁺ is the sole agonist. The relief of Mg²⁺ inhibition was particularly important since RyR activation during excitation-contraction coupling depends on a similar decrease in Mg²⁺ inhibition.