Q3925E mutation causes strong RyR2 loss-of-function by increasing Ca2+ and Mg2+ inhibition, while W4646R and Q4937K mutations cause gain-of-function by enhancing Ca2+ activation.
Characterization of RyR2 variants reveals that Q3925E causes a loss-of-function phenotype by altering signal transmission, while W4646R and Q4937K cause gain-of-function phenotypes, providing mechanistic insights into RyR2-associated cardiac diseases.
Cardiac Ca 2+ release channels, type-2 ryanodine receptors (RyR2s) play a pivotal role in cardiac muscle contraction by releasing Ca 2+ 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 Ca 2+ and caffeine binding sites, respectively. Our molecular dynamics simulations predicted that the Q4937 residue in the carboxyl terminal domain forms a hydrogen bond interaction with the central domain where the Ca 2+ binding site is located. Three mutant RyR2s were expressed in heterologous cells, and activities of the recombinant mutant RyR2 channels were determined by 3 Hryanodine binding methods. As expected, Q3925E greatly reduced Ca 2+ -dependent activation, and W4646R abolished caffeine activation. Our novel finding is that Q3925E increased inhibitory effects by divalent cations, Ca 2+ and Mg 2+ , resulting in a strong loss-of-function phenotype. Both W4646R and Q4937K increased affinities for Ca 2+ activation, and reduced or unchanged Ca 2+ inhibitions, exhibiting typical gain-of-function phenotypes. Caffeine failed to activate the Q3925E mutant at resting Ca 2+ but restored its activation at ~20 µM Ca 2+ , where Q3925E mutant is at the sub-activated state. Computational predictions of the mutated structures suggested that the Q3925E mutation does not reduce Ca 2+ binding to its site but rearranges domain interface between the central domain involving Ca 2+ binding site and carboxyl terminal domain, which directly interacts with the channel pore. Thus, it is possible that Q3925E-RyR2 mutation alters signal transmission between activating Ca 2+ binding and pore opening.
Chirasani et al. (2026) studied this question. Q3925E mutation causes strong RyR2 loss-of-function by increasing Ca2+ and Mg2+ inhibition, while W4646R and Q4937K mutations cause gain-of-function by enhancing Ca2+ activation.