title: Structural mapping of divergent regions in the type 1 ryanodine receptor using fluorescence resonance energy transfer.
abstract: Ryanodine receptors (RyRs) release Ca(2+) 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.
authors.affiliation: Department of Anesthesia, Perioperative and Pain Medicine, Brigham and Women's Hospital, Boston, MA 02115, USA.
authors.forename: Mohana
authors.initials: M
authors.lastname: Mahalingam
authors.affiliation: Department of Anesthesia, Perioperative and Pain Medicine, Brigham and Women's Hospital, Boston, MA 02115, USA.
authors.forename: Tanya
authors.initials: T
authors.lastname: Girgenrath
authors.affiliation: Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN 55455, USA.
authors.forename: Bengt
authors.initials: B
authors.lastname: Svensson
authors.affiliation: Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN 55455, USA.
authors.forename: David D
authors.initials: DD
authors.lastname: Thomas
authors.affiliation: Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN 55455, USA.
authors.forename: Razvan L
authors.initials: RL
authors.lastname: Cornea
authors.affiliation: Department of Anesthesia, Perioperative and Pain Medicine, Brigham and Women's Hospital, Boston, MA 02115, USA. Electronic address: jfessenden@partners.org.
authors.forename: James D
authors.initials: JD
authors.lastname: Fessenden
mesh_terms: D000494:Allosteric Regulation; D000818:Animals; D001665:Binding Sites; D002110:Caffeine / Q000494:pharmacology; D056966:Excitation Contraction Coupling; D031541:Fluorescence Resonance Energy Transfer; D057809:HEK293 Cells; D006801:Humans; D008958:Models, Molecular; D011485:Protein Binding; D011817:Rabbits; D019837:Ryanodine Receptor Calcium Release Channel / Q000737:chemistry / Q000502:physiology*; D022021:Tacrolimus Binding Proteins / Q000378:metabolism
chemical_list: D019837:Ryanodine Receptor Calcium Release Channel; D002110:Caffeine; D022021:Tacrolimus Binding Proteins