A central core disease mutation in the Ca²⁺-binding site of skeletal muscle ryanodine receptor impairs single-channel regulation

Abstract

Cryoelectron microscopy and mutational analyses have shown that type 1 ryanodine receptor (RyR1) amino acid residues RyR1-E3893, -E3967, and -T5001 are critical for Ca2+-mediated activation of skeletal muscle Ca2+ release channel. De novo missense mutation RyR1-Q3970K in the secondary binding sphere of Ca2+ was reported in association with central core disease (CCD) in a 2-yr-old boy. Here, we characterized recombinant RyR1-Q3970K mutant by cellular Ca2+ release measurements, single-channel recordings, and computational methods. Caffeine-induced Ca2+ release studies indicated that RyR1-Q3970K formed caffeine-sensitive, Ca2+-conducting channel in HEK293 cells. However, in single-channel recordings, RyR1-Q3970K displayed low Ca2+-dependent channel activity and greatly reduced activation by caffeine or ATP. A RyR1-Q3970E mutant corresponds to missense mutation RyR2-Q3925E associated with arrhythmogenic syndrome in cardiac muscle. RyR1-Q3970E also formed caffeine-induced Ca2+ release in HEK293 cells and exhibited low activity in the presence of the activating ligand Ca2+ but, in contrast to RyR1-Q3970K, was activated by ATP and caffeine in single-channel recordings. Computational analyses suggested distinct structural rearrangements in the secondary binding sphere of Ca2+ of the two mutants, whereas the interaction of Ca2+ with directly interacting RyR1 amino acid residues Glu3893, Glu3967, and Thr5001 was only minimally affected. We conclude that RyR1-Q3970 has a critical role in Ca2+-dependent activation of RyR1 and that a missense RyR1-Q3970K mutant may give rise to myopathy in skeletal muscle.

RyR1-related myopathy mutations in ATP and calcium binding sites impair channel regulation

“…One possible mechanism to explain this phenomenon is that at low Ca 2+ concentration (from nM to µM), this Ca 2+ binding site was occupied by one Ca 2+ , which forms a brige between the CTD and CSol to stabilize the open state of the RyR1 channel, whereas at high Ca 2+ concentration (mM), CTD and CSol each bind to one Ca 2+ , which disrupts the CTD-CSol interface to make the channel close. A patient with a Q3970K mutation at this site exhibited the same impaired Ca 2+ -dependent activation and deactivation as the E3893A/D and E3967A/D mutant channels, which is consistent with a previous study showing that RyR1-Q3970K displayed low Ca 2+ dependent channel activity [ 11 ]. It is likely that the additional positive charge from the lysine substitution in the Q3970K mutant channel reduces Ca 2+ -binding at this site.…”

Ca2+ inactivation of the mammalian ryanodine receptor type 1 in a lipidic environment revealed by cryo-EM

“…However, on the CD-C' side, Gln3970 lost coordination to Ca 2+ , interacting with Ser4029 instead (Figure 2A, Figure 2-figure supplement 1). Mutations Q3970E/K in RyR1 are implicated in central core disease and equivalent RyR2 mutations in cardiac arrythmia (Chirasani et al, 2019), which highlights the important Ca 2+ sensing role for this residue.…”

A comprehensive overview of the complex world of the endo- and sarcoplasmic reticulum Ca2+-leak channels

“…Mutations causing RyR hypersensitization and leakage of Ca 2+ from the SR primarily correlate with a range of myopathies and cardiac disorders. Mutations in the RyR1 gene are associated with myopathies such as central core disease and malignant hyperthermia [65][66][67], while mutations in the gene encoding RyR2 are especially associated with arrhythmogenic syndromes, such as catecholaminergic polymorphic ventricular tachycardia and arrhythmogenic right ventricular cardiomyopathy type 2 [68][69][70].…”