The effects of the immunophilins, FKBP12 and FKBP12.6, and phosphorylation on type II ryanodine receptor (RyR2) arrangement and function were examined using correlation microscopy (line scan confocal imaging of Ca 2+ sparks and dual-tilt electron tomography) and dSTORM imaging of permeabilized Wistar rat ventricular myocytes. Saturating concentrations (10 mmol/L) of either FKBP12 or 12.6 significantly reduced the frequency, spread, amplitude and Ca 2+ spark mass relative to control, while the

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... mograms revealed both proteins shifted the tetramers into a largely side-by-side configuration. Phosphorylation of immunophilin-saturated RyR2 resulted in structural and functional changes largely comparable to phosphorylation alone. dSTORM images of myocyte surfaces demonstrated that both FKBP12 and 12.6 significantly reduced RyR2 cluster sizes, while phosphorylation, even of immunophilin-saturated RyR2, increased them. We conclude that both RyR2 cluster size and the arrangement of tetramers within clusters is dynamic and respond to changes in the cellular environment. Further, these changes affect Ca 2+ spark formation.