Enhanced Cytosolic Ca2+ Activation Underlies a Common Defect of Central Domain Cardiac Ryanodine Receptor Mutations Linked to Arrhythmias

Abstract

Edited by Roger ColbranRecent three-dimensional structural studies reveal that the central domain of ryanodine receptor (RyR) serves as a transducer that converts long-range conformational changes into the gating of the channel pore. Interestingly, the central domain encompasses one of the mutation hotspots (corresponding to amino acid residues 3778 -4201) that contains a number of cardiac RyR (RyR2) mutations associated with catecholaminergic polymorphic ventricular tachycardia (CPVT) and atrial fibrillation (AF). However, the functional consequences of these central domain RyR2 mutations are not well understood. To gain insights into the impact of the mutation and the role of the central domain in channel function, we generated and characterized eight disease-associated RyR2 mutations in the central domain. We found that all eight central domain RyR2 mutations enhanced the Ca 2؉

Role of cardiac ryanodine receptor calmodulin‐binding domains in mediating the action of arrhythmogenic calmodulin N‐domain mutation N54I

“…Cytosolic 1 µ m [Ca 2+ ] free strongly activates RyR2 and consequently reduces the ER Ca 2+ load required for triggering SOICR, hence lowers the activation threshold. Under such conditions, RyR2 Ca 2+ release does not cause oscillations in ER Ca 2+ concentrations, but instead the ER Ca 2+ load reaches a steady‐state that reflects the opposing fluxes of RyR2 Ca 2+ release and the SR/ER Ca 2+ ATPase 2 (SERCA2b) Ca 2+ uptake — please see Ref. for a graphical overview of the process.…”

Diminished inhibition and facilitated activation of RyR2‐mediated Ca2+ release is a common defect of arrhythmogenic calmodulin mutations

“…The HEK293 cell endogenous SERCA2b operates at constant, maximum capacity under the perfusion conditions chosen, and CaM does not regulate SERCA2b as also evident from the a control experiment with RyR2‐ΔCaMBD (Fig. C) . The differences in steady‐state Ca 2+ load with no CaM, with CaM mutant and with CaM‐WT were evaluated using two‐way ANOVA (CaM presence vs. perfusion condition) with a Tukey's multiple comparisons correction, P < 0.05 considered significant.…”

“…Measurements of permeabilized HEK293 single cell steady‐state ER Ca 2+ load during perfusion with sustained 1 μ m [Ca 2+ ] free was done as previously described with modifications . Briefly, RyR2‐expressing HEK293 cells were permeabilized by perfusion with 0.25 g·L −1 saponin in Ca 2+ free intracellular‐like medium (ICM, 125 m m KCl, 19 m m NaCl, 10 m m HEPES, 2 m m ATP, 2 m m MgCl 2 , and 50 μ m EGTA at pH 7.4) for 1–2 min.…”

“…lowered the activation threshold. Under these conditions, RyR2 Ca 2+ release does not cause oscillations in ER Ca 2+ concentrations, and instead the ER Ca 2+ load reaches a steady-state that reflects an equilibrium between the opposing fluxes of RyR2 Ca 2+ release and the SR/ER Ca 2+ ATPase (SERCA2b) Ca 2+ uptake [27]. In this setting, any inhibition of RyR2 Ca 2+ release increases the steady-state ER Ca 2+ load because ER Ca 2+ efflux is reduced, while ER Ca 2+ Table 3).…”

“…Measurements of permeabilized HEK293 single cell steadystate ER Ca 2+ load during perfusion with sustained 1 lM [Ca 2+ ] free was done as previously described with modifications [27,65]. Briefly, RyR2-expressing HEK293 cells were permeabilized by perfusion with 0.25 gÁL À1 saponin in Ca 2+ .…”