Abstract
The type 1 ryanodine receptor (RyR1) is an intracellular calcium (Ca2+) release channel on the sarcoplasmic/endoplasmic reticulum that is required for skeletal muscle contraction. RyR1 channel activity is modulated by ligands, including the activators Ca2+ and ATP. Patients with inherited mutations in RyR1 may exhibit muscle weakness as part of a heterogeneous, complex disorder known as RYR1-related myopathy (RYR1-RM) or more recently termed RYR1-related disorders (RYR1-RD). Guided by high-resolution structures of skeletal muscle RyR1, obtained using cryogenic electron microscopy, we introduced mutations into putative Ca2+ and ATP binding sites and studied the function of the resulting mutant channels. These mutations confirmed the functional significance of the Ca2+ and ATP binding sites identified by structural studies based on the effects on channel regulation. Under normal conditions, Ca2+ activates RyR1 at low concentrations (µM) and inhibits it at high concentrations (mM). Mutations in the Ca2+-binding site impaired both activating and inhibitory regulation of the channel, suggesting a single site for both high and low affinity Ca2+-dependent regulation of RyR1 function. Mutation of residues that interact with the adenine ring of ATP abrogated ATP binding to the channel, whereas mutating residues that interact with the triphosphate tail only affected the degree of activation. In addition, patients with mutations at the Ca2+ or ATP binding sites suffer from muscle weakness, therefore impaired RyR1 channel regulation by either Ca2+ or ATP may contribute to the pathophysiology of RYR1-RM in some patients.
Highlights
Calcium is a vital second messenger [6, 38] that regulates numerous cellular signaling pathways, including muscle contraction [38], hormone secretion [57], and synaptic transmission [64]
The RYR1-related disorders (RYR1-RD) linked mutation T4980M, which is located in the ATP binding site, impairs ATP binding and ATP-dependent activation of the RyR1 channel
The RyR1related disorders (RyR1-RD) linked mutation Q3969K, which is located in the C a2+ binding site, abolishes Ca2+-dependent activation
Summary
Calcium is a vital second messenger [6, 38] that regulates numerous cellular signaling pathways, including muscle contraction [38], hormone secretion [57], and synaptic transmission [64]. Ryanodine receptors (RyRs) are located on the sarcoplasmic/endoplasmic reticulum (SR/ER) and mediate the release of Ca2+ from. RyR1 and RyR2 are widely expressed and are the major SR Ca2+ release channels in skeletal and cardiac muscles, respectively [54, 77]. RyR1 is required for excitation–contraction (EC) coupling in skeletal muscle. Regulatory and targeting proteins for enzymes including protein kinase A (PKA) and CaM kinase II (CAMKII), are associated with the channel and regulate its function [41, 45]
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