Characterization of cardiac disease-associated mutations in RyR2 Ca²⁺- and caffeine-binding sites

Cardiac Ca²⁺ release channels, type-2 ryanodine receptors (RyR2s), play a pivotal role in cardiac muscle contraction by releasing Ca²⁺ from the sarcoplasmic reticulum. Over 200 missense mutations in humans have been reported to be associated with cardiac diseases. Here, we characterize three RyR2 variants, Q3925E, W4646R, and Q4937K. Q3925E and W4646R mutations are in the Ca²⁺- and caffeine-binding sites, respectively. Our molecular dynamics simulations predicted that the Q4937 residue in the carboxyl terminal domain forms a hydrogen bond with the central domain where the Ca²⁺-binding site is located. Three mutant RyR2s were expressed in heterologous cells, and activities of the recombinant mutant RyR2 channels were determined by [³H]ryanodine binding methods. As expected, Q3925E greatly reduced Ca²⁺-dependent activation and W4646R abolished caffeine activation. Our novel finding is that Q3925E increased inhibitory effects by divalent cations, Ca²⁺ and Mg²⁺, resulting in a strong loss-of-function phenotype. Both W4646R and Q4937K increased affinities for Ca²⁺ activation, and reduced or unchanged Ca²⁺ inhibitions, exhibiting typical gain-of-function phenotypes. Caffeine failed to activate the Q3925E mutant at resting Ca²⁺ but restored its activation at ∼20 µM Ca²⁺, where the Q3925E mutant is in the subactivated state. Computational analysis of the mutated structures suggested that the Q3925E mutation does not reduce Ca²⁺ binding to its site but rearranges domain interface between the central domain involving Ca²⁺-binding site and carboxyl terminal domain, which directly interacts with the channel pore. Thus, it is possible that the Q3925E-RyR2 mutation alters signal transmission between activating Ca²⁺ binding and pore opening.NEW & NOTEWORTHY Q3925E in RyR2 is a part of Ca²⁺-binding site and is known to associate with cardiac sudden death. Our functional and structural modeling data suggested that the Q3925E mutation does not reduce Ca²⁺ binding but alter a domain interaction, causing an impaired Ca²⁺ activation of RyR2. We also found that the Q3925E mutation increases channel inhibition by Mg²⁺ and Ca²⁺, resulting in a strong loss-of-function phenotype.