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

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⁺-con-ducting 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.