Ryanodine receptors (RyRs) release Ca2+ to initiate striated muscle contraction. Three highly divergent regions (DRs) in the RyR protein sequence (DR1, DR2, and DR3) may confer isoform-specific functional properties to the RyRs. We used cell-based fluorescence resonance energy transfer (FRET) measurements to localize these DRs to the cryoelectron microscopic (cryo-EM) map of the skeletal muscle RyR isoform (RyR1). FRET donors were targeted to RyR1 using five different FKBP12.6 variants labeled with Alexa Fluor 488. FRET was then measured to the FRET acceptors, Cy3NTA or Cy5NTA, targeted to decahistidine tags introduced within the DRs. DR2 and DR3 were localized to separate positions within the "clamp" region of the RyR1 cryo-EM map, which is presumed to interface with Cav1.1. DR1 was localized to the "handle" region, near the regulatory calmodulin-binding site on the RyR. These localizations provide insights into the roles of DRs in RyR allosteric regulation during excitation contraction coupling.
Bibliographical noteFunding Information:
We are grateful to Dr. Florentin Nitu at the University of Minnesota, who produced each of the D-FKBPs used in this work. The rabbit RyR1 cDNA was initially cloned and provided by Dr. Paul D. Allen. This work was supported by NIH grants R01AR059126 (to J.D.F.) and R01HL092097 (to R.L.C.) and R01GM027906 (to D.D.T).